This study offered a bibliometric analysis of stem cells for cartilage regeneration from 2010 to 2020. With the development of bibliometric software, bibliometric analysis is now widely used. It can help beginners to understand the development process and trends of a specific field intuitively and systematically. It is also beneficial to find milestone achievements and new research hotspots.
Regenerative therapy uses multidisciplinary knowledge and technology to achieve regeneration of damaged tissues or organs. Regenerative therapy integrates knowledge and technology from multiple disciplines, aiming to regenerate damaged tissues or organs12,13. The number of publications and RRI in stem cells for cartilage regeneration have been on the rise, especially in the past 5 years, suggesting that the proportion of this field among all academic fields is increasing (Fig. 2A). In addition, more than half of the top 5 journals in this field have an impact factor of 5 or more (Fig. 4), which means that the topic of stem cells for cartilage regeneration has drawn widespread attention according to the method used to calculate the impact factor. Therefore, more in-depth studies will be published in the future and prove the feasibility and application prospects of stem cells for cartilage.
In terms of country and regional distribution, the total number of publications and citations of China ranks first in the world. However, in terms of the H-index, the USA still ranked first, indicating its great influence (Fig. 2B, Fig. 2D). When we further analyse the average citation rate (only compare countries with more than 100 publications), the average citation rate of China is 16.88, while those of the United States and South Korea are 25.03 and 28.49, respectively (Table 1). Although China’s contribution in this field is nonignorable, the centrality needs to be further improved. The influence and contribution of scientific institutions are an important part of that of a country or region. Nine of the top 10 institutions are located in China, and the top 10 institutions in China account for 70% of the total publication number. However, among the top 5 institutions in China in terms of publication volume, none of them rank among the top 5 in the world in terms of total citations (Table 2, Fig. 3). Although the citation rate of the article is not completely equal to the importance of academic achievements, it can still reflect some practical problems when a low citation rate becomes a common phenomenon. From the perspective of time distribution, the concentrated years of article output of China were approximately 2018 (Fig. 2D). However, there were no keywords or references with a sudden change in the number of citations in the past 3 years (Fig. 5D, Fig. 6D). This discrepancy suggests that scientific achievements in China have not formed a hot topic in this field. These results indicate that most academic achievements are the completion and supplementation of previous breakthrough results instead of original creative discoveries and techniques. The National Natural Science Foundation of China (NSFC) is one of the main channels for supporting basic research in China, and a series of changes in the NSFC application rules have taken place in recent years, aiming to improve the quality and innovativeness of publications. We believe that the centrality of China will gradually increase in the future.
As far as authors are concerned, SEKIYA I (Tokyo Medical & Dental University, JAPAN) and YANG F (Stanford University, USA) are the most productive. However, in terms of the number of citations, the data of Toh WS (National University of Singapore, Singapore) are particularly attractive (Table 3). He has published 10 papers on stem cells for cartilage regeneration, but the total number of citations has reached 740. The total citations of Toh WS accounted for approximately 60% of Singapore and 70% of National University of Singapore (Table 1, Table 2). In 2007, Toh WS successfully constructed an experimental system in which human embryoid body (EB)-derived cells could directly differentiate into chondrocytes under specific culture conditions14. In 2009, Toh WS and his team improved this high-density micromass model system. Through using a more effective combination of growth factors and extracellular matrix substrate, they isolated a highly expandable and homogenous chondrogenic cell population named TC1, providing a potential source of chondrogenic cells for scientific research and clinical application15. In 2010, Toh WS and his team constructed hESC-derived chondrogenic cell-engineered cartilage (HCCEC) from the abovementioned cell population in the culture of hyaluronic acid (HA)-based hydrogel and verified its long-term viability and safety in a rat model, providing a practical strategy of applying hESCs for cartilage regeneration4. In 2014, Toh WS and his team made a systematic description of the various tools and techniques of the culture system, tissue engineering protocol, and analytical methods, as well as the improvements16. Afterwards, Toh WS and his team shifted the focus of research to mesenchymal stem cell-derived exosomes and illustrated the underlying mechanisms of MSC exosomes in the biological behaviour of chondrocytes, extracellular matrix homeostasis, and immune reactivity in cartilage regeneration17. In addition, the author and his team published 3 reviews and summarized the potential and perspective of human embryonic stem cells (ESCs) in cartilage tissue engineering and regenerative medicine, the interactions between stem cells and extracellular matrix for cartilage regeneration, and the potential and perspective of MSC exosomes in cartilage regeneration. Among publications of Toh WS, 3 articles had more than 100 citations, and the topics of these 3 articles were “Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells” (DOI: 10.1016/j.biomaterials.2010.05.064, PMID: 20619789), “MSC exosomes as a cell-free MSC therapy for cartilage regeneration: implications for osteoarthritis treatment” (DOI: 10.1016/j.semcdb.2016.11.008, PMID: 27871993), and “MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity” (DOI: 10.1016/j.biomaterials.2017.11.028, PMID: 29182933).
As a key node in the co-citation network, Jo Ch's article (doi:10.1002/stem.1634) not only had the largest number of citations (Table 4) but also had the highest impact (Fig. 5A, Fig. 5B). Jo Ch is the first author, and the corresponding author of this article is Kang Sup Yoon. Kang Sup Yoon and his team substantiated the safety and efficacy of intra-articular injection of autologous AD-MSCs through clinical index, radiological evaluation, arthroscopic evaluation and histological evaluations. More importantly, Kang Sup Yoon and his team evaluated the effects of different doses of AD-MSCs injected into articular tissue. It is currently known that a sufficient number of MSCs can inhibit articular cartilage degradation and promote its regeneration18. However, when evaluating efficacy and safety comprehensively, the optimal cell dose needs to be clarified. Even if it is not the optimal dose, Kang Sup Yoon and his team suggested that at least 1.0x108 MSCs per injection would be a prerequisite for consistently good results, preliminarily exploring the therapeutic dose19. In conclusion, this proof-of-concept clinical trial confirmed the effectiveness of intra-articular injection of MSCs and gave a fair opinion on the injection dose, promising to encourage larger clinical research and application.
In the “Clinical study”, the latest word was “double-blind” (AAY 2018.75), with 8 times (Fig. 6C), which heralds future trends in the biological study of stem cells for cartilage regeneration. A double-blind trial is a test where neither the tester nor the testees know the exact group of testees, which helps to avoid cognitive bias20. Due to its higher cost, there are very few double-blind trials in this field (table 5). A double-blind trial from South Korea explored the effectiveness and safety of genetically engineered autologous chondrocytes, which were named TissueGene-C (TG-C), for knee osteoarthritis. TG-C was administered by a single intra-articular injection, and then subjective and objective assessments were performed by a clinical rating scale and radiographic evaluation21. Although subjective evaluation shows the effectiveness of this treatment, changes in joint space widths, bone area and cartilage thicknesses only show a trend that is not statistically significant. This research has laid the foundation for the development of double-blind trials in this field. On the basis of reality and possibility, double-blind experiments in stem cells for cartilage regeneration should also be attempted.
In tissue engineering, the latest word was “3D printing” (AAY 2019.625), with 9 times (Fig. 6C). 3D printing was proposed by Chuck Hull in 1983 and includes two main processes: computer-aided design (CAD) and 3D printing to form products. In the 2000s, 3D printing was used in the production of surgical models and later developed to be used in the production of live cell structures, including articular cartilage. Regarding the product formation process, there are also different strategies: inkjet printing, laser-assisted printing, and bioextrusion22,23. The materials used for 3D printing (bioink) are a hot and difficult point. Bioink mainly consists of two parts: biomaterials as scaffolds (2nd highest frequency keywords in tissue engineering clusters) and embedded cells24. Biomaterials used for tissue engineering need to have biocompatibility, biodegradability, and porosity, and they are mainly divided into natural polymers (alginate, gelatine, chitosan, and hyaluronan) or synthetic polymers (PCL, PGA, PEG) at present25. After being modified, natural polymers can be made into a more stable form, hydrogels, which have been widely used in 3D printing26. Synthetic polymers could be combined or coated with hydrogels to enhance their biocompatibility. Biomaterials can play a variety of roles depending on the embedded cell types, including chondrogenic differentiation (highest frequency keywords in tissue engineering clusters).
As mentioned earlier, chondrocytes have limited expansion efficiency and potential for dedifferentiation in vivo. In contrast, the application of stem cells in tissue engineering is more promising. According to current research, the mechanism underlying cartilage regeneration in stem cells is mainly attributed to direct differentiation of chondrocytes and paracrine secretion27,28. BM-MSCs are one of the most commonly used cell types in tissue engineering (table 5), and they can be induced into multiple cell types, including chondrocytes. AD-MSCs are easily accessible via minimally invasive procedures, and they have the characteristics of synthesizing more collagen than other MSC types. SF-MSCs, which can be isolated from synovial fluid and synovial membrane, have higher chondrogenic potential among osteoarticular cell types. Dental pulp MSCs have already been used in cartilage tissue engineering and have shown potential for hyaline-like cartilage formation with the synthesis of ECM components, such as aggrecan or collagen 29,30. In addition to biological materials and stem cell types, environmental conditions and differentiation-promoting factors are also key points for 3D printing technology. For example, the surrounding medium’s oxygen content affects the chondrogenic differentiation of MSCs31,32, and TGF-β1 can promote and stabilize the chondrogenic phenotype33. Overall, 3D printing is a microcosm of tissue engineering. Whether it can be promoted clinically depends on many factors, and 3D printing is an important direction of future research.
In the biological study, the latest word was “extracellular vesicles” (AAY 2018.9091), with 12 times (Fig. 6C). Extracellular vesicles are released from the cell surface to body fluid, and they can transfer multiple types of cargo, such as lipids, nucleic acids and proteins, contributing to intercellular communication34. Exosomes, microvesicles (MVs) and apoptotic bodies are currently the most attractive categories of extracellular vesicles, and they are different in generation mechanism and size35. According to publication numbers, exosomes have attracted more attention among researchers. Currently, exosomes have been identified from multiple MSCs, including those derived from AD-MSCs, BM-MSCs, US-MSCs, hESCs, and iPSCs36,37.When MSCs sense changes in the surrounding microenvironment, they can secrete exosomes to interact with other cells. Exosomes have been proven to regulate the biological behaviour of chondrocytes, to modulate immune responses and to restore homeostasis to the ECM, thereby promoting cartilage regeneration38,39. Although the therapeutic efficacy, biosafety, kinetics and biodistribution of MSC exosomes need to be studied in depth, the regenerative potency of MSC exosomes provides new perspectives for the development of a cell-free MSC therapy strategy for cartilage regeneration.
The purpose of clinical trials is to repair damaged articular cartilage. Without undergoing such a major operation as joint replacement, the application of stem cells is expected to replace traditional medical treatment and achieve the goal of radical cure. According to current clinical trials, except for CARTISTEM, which uses commercial hUCB-MSCs40, all other clinical trials use autologous stem cells. The entire process includes collection, in vitro expansion, and intra-articular injection41. Subjective clinical scores, imaging evidence, and histological evaluation are currently the most important evaluation methods. The effectiveness, feasibility and safety of the concept of using mesenchymal stem cells in cartilage repair have been verified (Table 5). However, there are still some issues that need to be resolved. First, the most suitable cell source needs further exploration. Except for CARTISTEM, all other clinical trials use autologous stem cells, and the most commonly used type is BM-MSCs. The horizontal comparison of different types of cells is still missing. Second, it is unclear whether MSCs mainly differentiate into chondrocytes directly or more through paracrine forms to promote cartilage formation. Third, the optimal therapeutic course and dose need to be further explored. NCT01207661 indicated that the effect of a single injection was improved in the first 12 months and decreased from the 12th month42. NCT01873625 also explored the number of cells per injection and provided a reference value of 40 million43. However, the best course of treatment and therapeutic dose need more exploration. In addition, current clinical trials are mainly short-term follow-ups, typically only 24 months or less. Long-term efficacy and safety evaluations need further verification.
This study investigated publications on stem cells for cartilage regeneration extracted from the Web of Science database and discussed hot issues in this comprehensive and objective field. However, limitations are inevitable. First, we only enrolled publications in the language of English, which may miss some important non-English studies in this field. In addition, although keywords can reflect the subject of the article to a certain extent, the preference of different authors when using keywords and the shortness of keywords will lead to bias in trend analysis.