Current knowledge and future perspectives on exosomes in the field of regenerative medicine: a bibliometric analysis

Exosomes are extracellular vesicles with a double lipid membrane structure. They are secreted by cells, are 30–130 nm in size, and contain proteins, lipids, DNA, mRNA and miRNA [1–3]. These were first discovered by Peter Wolf in 1967 and named ‘platelet dust’ [4]. Exosomes are vital for many biological processes, such as intercellular communication, immune system regulation and the life cycle of cell communities [5]. Exosomes have been extensively studied in many areas, such as osteoarthritis [6], cartilage regeneration and repair [7,8], brain neurovascular regeneration [9–11], cardiac regeneration [12,13], muscle regeneration [14], limb ischemia [15], kidney injury [16,17] and pulmonary hypertension [18,19]. Figure 1 shows a schematic diagram of exosome maturation and secretion mechanism and a functional diagram of exosomes.

Exosomes have many advantages in the diagnosis and treatment of diseases. First, their natural origin causes fewer immunogenicity problems, lower toxicity and higher safety than live cell therapy [20]. Second, they have a lipid bilayer structure, which is not affected by the external environment when transporting cargo, maintaining cargo integrity [21]. Third, in many cell therapies, large cell size prevents their passage through the capillary barrier, limiting drug delivery. However, exosomes are small, so they can easily pass through the capillary barriers [22]. Finally, the risks of uncontrolled growth or gene mutation associated with cell-based therapy can be avoided by using exosomes [23].

In addition, exosomes can play an important role as biomarkers in disease diagnosis. For example, Fu et al. [24] found that higher expression levels of circulating exosomal miR-17-5p and miR-92a-3p were significantly associated with pathologic stages and grades of colorectal cancer patients. PD-L1 levels on exosomes are associated with disease progression in head and neck squamous cell carcinoma patients [25]. Increased expression of serum exosomal miRNA-191, -21 and -451a of pancreatic neoplasm is considered to be an efficient diagnostic marker [26].

In order to assess the output of scientific research, research hotspots and research trends in a particular topic, bibliometric analysis analyzes published literature both qualitatively and quantitatively [27,28]. Many studies have been published recently on exosomes, most of which have shown that exosomes have a significant relationship with regenerative medicine. However, no bibliometric analysis of exosomes in the field of regenerative medicine has been published. Therefore, the authors of the present study performed this bibliometric analysis to evaluate the application of exosomes in regenerative medicine, with the aim of providing an update on the current developmental status of exosomes, identifying research hotspots and emerging trends in regenerative medicine and predicting future research priorities.

Materials & methods

Publications were retrieved from the Web of Science Core Collection (WOSCC) using the following search terms: Test Search = (exosomes) AND (regeneration). Only original, English-language articles and reviews were included. This study outlines the evolution of exosome research in the area of regenerative medicine in the 21st century. No relevant publication on exosome research in regenerative medicine had been published before 2010. Therefore, the authors searched and analyzed all publications between 2010 and 2021.

The information was gathered, the literature was reviewed and the references were cross-referenced independently by two researchers. In case of disagreement, a senior researcher resolved the issue. To assess the scientific impact of the country/region and the journal, the Hirsch index of the country/region, the impact factor (IF) of the journal and the quartile of the journal category (Journal Citation Reports) were gathered.

Using Microsoft Excel's features, all the data were entered into Excel 2016 (WA, USA) and shown in tables or charts. The coauthorship, co-occurrence and citation of countries/regions, journals, institutions, authors and hotspot keywords were evaluated using VOSviewer 1.6.18. The thickness of the lines between the nodes represents the degree of linkage, the size of the nodes represents the frequency of the pertinent parameters and the various colors represent the various modules in the visual mapping. The amount of cooperation with other items is indicated by an item's total link strength (TLS). The level of collaboration increases as the value rises.

Results

Publication output

A total of 1383 publications were identified from the WOSCC online database (July 2 2022) between 2010 and 2021. These included 846 (61.17%) original research articles and 537 (38.83%) review articles. The annual growth in publications is shown in Figure 2; 414 (29.93%) were published in 2021 and 330 (23.86%) in 2020. A total of 985 publications were open access. The number of articles increased rapidly, especially in the last 5 years; it was 10.35-times higher in 2021 than in 2015.

Analysis of countries/regions

All publications were from 68 countries/regions. Figure 3 displays the annual national production of publications in the ten most productive countries/regions. Annual publications worldwide in this field continue to increase. The USA studied this field earlier. Since 2015, the annual publication rate of China has increased rapidly, followed by the USA and Italy. Table 1 shows the ten most productive countries. China has the highest production, with 557 publications (40.27%), followed by the USA (349 publications, 25.67%) and Italy (129 publications, 9.33%). The collaboration network of countries/regions based on citation counts is visualized in Figure 4; the size of the nodes represents the quantity of articles published in that country/region, and the connecting lines represent the frequency of international cooperation. Seven clusters were seen, with China being the main driver of research in the field of exosomes in regenerative medicine and enjoying a high reputation in academia, followed by the USA, Italy, England and South Korea. The number of relevant research publications in other countries such as England, South Korea, Iran, Germany, Japan, Switzerland and Spain is relatively small, but the overall output of publications is still increasing, which is closely related to international cooperation.

Analysis of institutions

All publications were from 1629 institutions. Table 2 shows the ten most productive institutions, with Shanghai Jiao Tong University (4.41%, 61 articles) at the top, followed by Zhejiang University (2.53%, 35 publications) and Sichuan University (2.31%, 32 publications). Most of these ten most productive institutions are in China, demonstrating China's major academic influence in this field. Shanghai Jiao Tong University (China) had the highest number of citations per publication (69.11), indicating that this institution publishes a large number of high-quality articles and contributes to the promotion of research activities in the field. However, the circulation of publications and the number of citations per publication of other institutions are much lower than those of Shanghai Jiao Tong University. Therefore, emphasis should be placed on increasing academic exchanges between institutions. The network visualization map of institutions related to research on exosomes in regenerative medicine is show in Figure 5. Based on the calculation of five publications published by each institution, there are a total of 142 nodes. The connection between the nodes represents the cooperative relationship, which is divided into nine clusters. As can be seen from Figure 5, the main institutions that cooperate with Shanghai Jiao Tong University are Sichuan University, University of Turin, Tongji University, Huazhong University of Science and Technology and Sun Yat-sen University.

Analysis of authors & co-cited authors

Publications on the use of exosomes in the field of regeneration have been written by 7664 different writers. Supplementary Table lists the top ten co-cited authors as well as the top ten authors with the most publications. Marban (20 publications, 1.45%) published the highest number of articles, followed by Camussi (19 publications, 1.37%) and Hui (18 publications, 1.30%). Lai (495 citations, TLS: 18,241) is the author with the highest number of co-citations, followed by Théry (405 citations, TLS: 14,032) and Zhang (369 citations, TLS: 14,653). The network of co-cited authors is visualized in Figure 6. There are four clusters in the analysis diagram. The largest node indicates that it is most co-cited and includes Zhang (369 citations, TLS: 14,653, cluster 1), Théry (405 citations, TLS: 14,032, cluster 2), Lai (495 citations, TLS: 18,241, cluster 3) and Barile (198 citations, TLS: 7532, cluster 4).

Analysis of journals & co-cited journals

A total of 477 journals have published relevant publications. Supplementary Tables 2 & 3 show the ten most productive and most co-cited journals. Among them, 63 journals published more than five publications. Among the top ten most productive journals, the first three were Stem Cell Research and Therapy (79 publications, 5.71%), International Journal of Molecular Sciences (57 publications, 4.12%) and Cells (35 publications, 2.53%). Among the top ten, the IF of Stem Cell Research and Therapy was the highest (8.079), and Stem Cells Translational Medicine had the highest average number of citations per publication (96.39). Among the ten most co-cited journals, the top three were Stem Cell Research and Therapy (2432 citations, TLS: 228,734), PLOS ONE (2393 citations, TLS: 241,082) and Scientific Reports (2006 citations, TLS: 210,341). The IF of Circulation Research was the highest (23.213), and Procedures of the National Academy of Sciences of the United States of America had the highest Hirsch index (699). Supplementary Figure 1 displays the network visualization map of the co-cited journals. Stem Cell Research and Therapy (England) was the most productive and co-cited journal, providing a wealth of research resources on exosomes in regenerative medicine research.

Analysis of references

The visual network map of the most co-cited references in this field is shown in Supplementary Figure 2. The size of the node indicates the number of times the publication was cited. The map of the node increases with the number of citations. The top ten most frequently co-cited publications' journals, authors, journals and years of publication are listed in Supplementary Table 4. According to the ten most co-cited references, the title of “Exosome-mediated transfer of mRNAs and miRNAs is a novel mechanism of genetic exchange between cells”, published in Nature Cell Biology (IF: 28.213), was the most co-cited article and was authored by Valadi, with 273 citations. The second and third most co-cited articles were authored by Lai et al. (Stem Cell Research, 2010) and Raposo et al. (Journal of Cell Biology, 2013), respectively. Most publications were published after 2010. Based on the number of citations, the publications were divided into four different clusters (Supplementary Figure 2), with the size of the node indicating the number of times the publication was cited. The following four subjects were among the hotspots for research: exosome-mediated neurovascular regeneration (cluster 1, blue area), mechanism of exosomes (cluster 2, green area), exosome-mediated cartilage regeneration and repair (cluster 3, red area) and exosome-mediated cardiac regeneration (cluster 4, yellow area).

Analysis of keyword co-occurrence clusters

A total of 4764 different keywords were used in 1383 articles, with 500 keywords used at least five-times. Supplementary Figure 3 shows the network analysis map of keyword co-occurrence, with a total of 500 items and eight clusters. Each node represents the frequency of the corresponding keyword in the field of regenerative medicine. The larger the node, the more frequently the keyword was used. The thicker the connection between the two nodes, the greater the frequency of the two keywords in the subject area and the greater the relationship between the two keywords. Obvious keywords can help researchers and nonresearchers quickly identify hot topics in their literature research. It can be seen in Supplementary Figure 3 that the most commonly used keywords were "exosomes” (895 occurrences, TLS: 7497), “extracellular vesicles” (531 occurrences, TLS: 4857), “regeneration” (406 occurrences, TLS: 3421), “stromal cells” (230 occurrences, TLS: 2193), “angiogenesis” (224 occurrences, TLS: 2016), “mesenchymal stem cells” (MSCs; 211 occurrences, TLS: 1870), “microvesicles” (187 occurrences, TLS: 1787), “differentiation" (186 occurrences, TLS: 1566), "MSCs" (185 occurrences, TLS: 1697) and "in vitro" (183 occurrences, TLS: 1672).

Closely related keywords will relatively form a small group; then the keywords in this small group and a theme can be summarized, and the theme can be discussed in detail. In fact, the essence is cluster analysis. Supplementary Figure 3 shows that there were eight clusters, and different color groups correspond to different subject studies. Clusters of different colors are connected by lines, indicating that they are closely related. According to the color, the keywords are divided into eight related topics: injury repair, heart regeneration, bone regeneration and repair, secret expression of exosomes, action mechanism of exosomes, promotion and migration of exosomes, exosome mechanism in vitro and exosome transformation pathway. Cluster co-occurrence keyword analysis of exosomes in the field of regenerative medicine is shown in Supplementary Table 5.

Supplementary Figure 4 shows the timing analysis of keyword co-occurrence of exosomes in the field of regeneration research. The timing sequence map's various colors are displayed in different periods on the key in the figure's lower right corner. Nodes close to yellow indicate keywords with a high frequency in recent years and indicate the research trends in this field. Based on the research trends of exosome research in regenerative medicine, articles focused on the treatment of osteoarthritis and knee diseases and cartilage regeneration and repair. Supplementary Figure 5 shows the usage density of these keywords. The brighter the color, the more frequently the keywords were used, which indicates that they were the research hotspot.

Discussion

In-depth study of exosomes in many fields improves researchers understanding of exosomes. In the last decade, the number of articles on exosomes in regenerative medicine has increased rapidly, and researchers have realized the great potential of exosomes in regenerative medicine. Bibliometric analysis evaluates the global output of academic articles and research hotspots, enabling the prediction of trends in exosome research in the field of regenerative medicine. This is the first bibliometric analysis of exosomes in this field.

Quality & status of global publications

In recent years, research on exosomes has made breakthroughs. Since 2010, the research on exosomes has appeared in the field of regenerative medicine. A large number of animal experiments show that exosomes can be used to treat tissues and organs that were previously considered difficult to regenerate and repair. From 2015 to 2021, the number of global publications related to research increased rapidly. Bibliometric analysis shows that China is the main contributor in this field. The number of publications of its relevant research accounts for 40.27% of the total number of global publications (557 publications), and the number of publications in 2021 accounted for about half of the total number of global publications. However, with the exception of Shanghai Jiao Tong University, which had a high number of citations per publication, the number of citations per publication of other institutions was relatively low. Therefore, for China's research in this field, attention should be paid to improving the quality of publications, rather than the number of publications.

Stem Cell Research and Therapy, International Journal of Molecular Sciences, Cells and Stem Cells International published the most publications on exosome research in the field of regenerative medicine. Stem Cell Research and Therapy (England) was the journal with the largest number of publications and the most co-citations. It is recognized as a research resource for exosomes in the field of regenerative medicine and has had an important impact on the field.

Most contributing authors & most co-cited references

Marban (USA) from the Heart Institute of Cedars-Sinai Medical Center published the highest number of publications (20 publications, 1.45%). His most cited articles mainly reported that cardiac cell-derived exosomes inhibit apoptosis, promote cardiomyocyte proliferation and enhance angiogenesis, highlighting the utility of exosomes in cell-free therapy [29].

Lai from the Institute of Medical Biology (Singapore) was the most co-cited author. His most co-cited articles reported mainly that MSC-derived exosomes are well suited for mass-producing exosomes that are ideal for drug delivery. Exosomes can regulate their microenvironment and promote angiogenesis and metastasis. MSC-derived exosomes are a potential adjunct to reperfusion therapy for myocardial infection [30–32]. These articles are recognized as reliable references for follow-up research.

The article entitled “Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells” published in Nature Cell Biology is the most co-cited reference, and it describes a novel mechanism of exosome genetic exchange between cells. Exosomes contain mRNA and miRNA, which can be transmitted to other cells and regulate gene expression, according to the findings [33]. This study may provide insights for subsequent research on the mechanism of exosomes.

The article with the second most co-cited was titled “Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury” published in Stem Cell Research. Exosomes have been shown to decrease infarct size in a mouse model of cardiac ischemia/reperfusion injury, according to this article. Exosomes now play a new role in the intercellular mediation of tissue damage and regeneration [32]. Timmers et al. also used human MSC-derived exosome secretion in a pig myocardial ischemia/reperfusion injury model to effectively reduce the myocardial infarction area. These data indicate that human MSC-derived exosomes can be used to treat and reduce the myocardial infarction area of patients with acute myocardial infarction; the use of SC-derived exosomes to treat diseases has expanded the function of SCs themselves [34].

The third most co-cited article was titled “Extracellular vesicles: exosomes, microvesicles, and friends” published in the Journal of Cell Biology. In this review, Raposo et al. focused on the characterization of extracellular vesicles and on currently proposed mechanisms for their formation, targeting and function. Exosomes and microbubbles act as carriers for transferring molecules between the cell membrane and cytoplasmic proteins, lipids and RNA, representing their vital role in intercellular communication [35].

Research hotspots

The network map of co-cited references (Supplementary Figure 2) shows that research hotspots included exosome-mediated neurovascular regeneration (cluster 1, blue area), exosome mechanism research (cluster 2, green area), exosome-mediated cartilage regeneration and repair (cluster 3, red area) and exosome-mediated cardiac regeneration (cluster 4, yellow area).

Numerous studies focused on the ability of exosomes to induce neurovascular regeneration (group 1) [36–42]. Tassew et al. [43] found that fibroblast-derived exosomes promote the growth of neurites on inhibitory proteins of the central nervous system. The fibroblast-derived exocrine body mobilizes the autocrine Wnt10b-mTOR pathway to awaken the intrinsic regeneration ability of neurons, which is of great significance for the treatment of injury of the central nervous system. Zhang et al. [10] showed that exosomes produced by MSCs effectively improve the functional recovery of rats after traumatic brain injury by promoting endogenous angiogenesis and neurogenesis and reducing postinjury inflammation.

Group 2 contains the top ten most co-cited articles that focused on the mechanism of exosomes [44–49]. Valadi et al. [33] reported a novel mechanism of exosome genetic exchange between cells, which was their most co-cited article. Valadi et al. found that exosomes in mouse and human mast cells contain RNA and miRNA, and they verified that the RNA in exosomes is functional. Finally, the RNA that shuttles between cells mediated by exosomes is named esRNA. Lotvall et al. [50] showed that there may be multiple types of exosomes that can transmit information to neighboring cells by delivering RNA signals.

Guay et al. [51] first investigated and showed that miRNA transfer to neighboring β cells can be mediated by exosomes in a rat model of pancreatic β-cell dysfunction, prescribing a novel intercellular communication mechanism regulating islet β-cell activity. In addition, the role of exosomes in the communication between immune cells and tumors has also been extensively studied [52–56]. For example, Singh et al. [57] demonstrated that miR-10b promotes the invasion of breast tumor cells through exosome-mediated pathway.

Group 3 represents the most co-cited articles on exosome-mediated cartilage regeneration and repair. Zhang et al. [7] treated osteochondral defect of the distal femur of rats with human embryonic MSC-derived exosome injections for 12 weeks. The injured site was almost completely filled with new tissue, with good surface regularity and fully fused surrounding cartilage. However, fibrous tissue was found in the phosphate-buffered saline-treated control site. MSC-derived exosomes can repair and regenerate critical size osteochondral defects by enhancing proliferation, migration and matrix synthesis; reducing apoptosis; and regulating immune reactivity and other coordinated and multifaceted responses [8,58–61].

Exosome-mediated cardiac regeneration (group 4) is another branch of exosome research in regenerative medicine. Khan et al. [62] used mouse embryonic SC-derived exosomes to treat myocardial ischemia. The treatment increased neovascularization and myocardial cell survival and reduced fibrosis after infarction, improving myocardial cell regeneration. Mol et al. [63] reported that cardiac cell-derived exosomes inhibit apoptosis, promote cardiomyocyte proliferation and angiogenesis and reduce cardiac fibrosis after myocardial infarction. The potential utility of exosomes as candidates for cell-free therapy has been reported [29,64–69].

Research trends

According to the timing analysis of keyword co-occurrence (Supplementary Figure 4), the research trend of exosomes in the field of regenerative medicine focused on the treatment of osteoarthritis and knee diseases and cartilage regeneration and repair.

Adult articular cartilage cells have a limited ability to regenerate after damage. If the cartilage damage is not treated properly, secondary osteoarthritis can occur, followed by pain, movement disorders, deformities and loss of function [70–74]. Zhang et al. published an article in 2016 titled “Exosomes derived from human embryonic mesenchymal stem cells promote osteochondral regeneration”, which was the most frequently cited article related to cartilage regeneration and repair. Exosomes produced from human embryonic MSCs were shown to be effective in cartilage regeneration for the first time by Zhang et al. [7], and they can be used as “cell-free” therapeutic alternatives to cell-based MSC therapy. Cosenza et al. [75] reported that exosomes derived from bone marrow MSCs can re-induce the expression of chondrocyte markers, protect chondrocytes from apoptosis and inhibit macrophage activation, thereby protecting cartilage tissues from inflammatory damage. Liu et al. [76] proposed a novel method to integrate SC-derived exosomes and in situ hydrogels into acellular tissue patches for cartilage regeneration. The results showed that acellular tissue patches can naturally combine with cartilage matrix, enhance the deposition of SC-derived exosomes in cartilage defects and effectively promote the repair of cartilage defects. Chen et al. [77] reported that the implantation of 3D-printed extracellular matrix/gelatin methacrylate/exosome scaffold in a rabbit cartilage defect model effectively restored the mitochondrial dysfunction of chondrocytes, enhanced the migration of chondrocytes and promoted cartilage regeneration in the model. In addition, many studies have shown that exosomes can reduce inflammatory factors in bone and cartilage, increase cartilage matrix and promote bone and cartilage regeneration and repair [78–85].

Research on the therapeutic effect of exosomes on cartilage regeneration and repair is expected to grow. Research in this field is no longer limited to pikas, and large primates are also being used, making the findings more similar to clinical research. At the same time, the source of cells, treatment method, dosage and frequency of administration of exosomes, as a candidate for cell-free therapy, will need to be further studied.

Advantages & limitations

Through bibliometric analysis, the authors visually evaluated the situation, research hotspots and research trends of exosomes in the field of regenerative medicine. However, there are also some limitations. In recent years, the number of articles has increased rapidly, and some newly published high-quality articles that have been cited less frequently could have been ignored in this analysis. Moreover, the authors retrieved articles published only in the English language from the WOSCC, ignoring articles published in other languages. Moreover, this study omitted publications that were not included in the WOSCC database and could not cover all relevant publications.

Conclusion

The highest number of publications came from China, followed by the USA, Italy, England and South Korea. China was the main contributor to exosome research in the field of regeneration and enjoys a high academic reputation. The countries with the most cooperation with China were the USA, South Korea, Italy, Japan and Germany.

Shanghai Jiao Tong University was the institution with the highest output and the most citations of publications. Marban (20 publications, 1.45%) published the highest number of publications. Lai from the Institute of Medical Biology (Singapore) was the most co-cited author. The journal Stem Cell Research and Therapy (England) had the highest output and most co-citations.

Research hotspots included the following four topics: exosome-mediated neurovascular regeneration, exosome mechanism research, exosome-mediated cartilage regeneration and repair and exosome-mediated cardiac regeneration.

More articles are expected to be published in the next few years, and the application of exosomes in cartilage regeneration and repair may get more attention.