The Top 100 Most Cited Articles on Platelet-Rich Plasma Use in Regenerative Medicine—A Bibliometric Analysis—From the ESSKA Orthobiologic Initiative

Over the past few decades, more and more articles about platelet-rich plasma (PRP) use in regenerative medicine have been published. The aim of this study was to determine which articles have been most influential in this field by identifying and analyzing the characteristics of the 100 most cited articles. Articles on the use of PRP in regenerative medicine were identified via the Thomson ISI Web of Science database. A majority of the articles originated from the USA (36%). The top journal in terms of number of articles was American Journal of Sports Medicine (12%). Musculoskeletal system and orthopedics (54%) were the most popular fields of applications. Preclinical studies were the most represented study type, from which only 8 from 46 (17.4%) provided a complete numerical description of the injected product. Analysis showed a time-dependent trend of increasing quality of the clinical studies (p = 0.004), although none of them provided a complete biological characterization of the injected PRP. This study demonstrated that the use of PRP in regenerative medicine is a growing and popular area of research, mainly focused on orthopedic applications. Studies on PRP-derived exosomes, biological characterization, and correlation with clinical results might be areas of future trends.


Introduction
Regenerative medicine is the branch of medicine that develops methods to regrow, repair, or replace diseased and injured cells, tissues, or organs. This field has shown intense expanse and evolution in the past four decades [1]. These variable regenerative medicine therapies can be sorted according to their complexity of manufacturing process: on one hand, gene therapy [2], stem cells [3], or bioprinted tissues, which present more complex procedural steps [2][3][4], and on the other hand, "point of care" autologous therapies involving adipose tissue (nanofat [5], stromal vascular fraction [6]), bone marrow concentrate [7], and platelet-rich plasma (PRP) [8], which are easier to obtain and save time [6][7][8] to manufacture. This last one has emerged as a popular and promising treatment modality in regenerative medicine. PRP is an autologous biological product containing numerous bioactive proteins such as growth factors (IGF, TGF, VEGF, PDGF, etc.), cytokines, and chemokines, which have the potential to play therapeutic roles in a variety of treatments, including musculoskeletal conditions, gynecology, urology, plastic surgery, ophthalmology, and dermatology, while its production remains easy, fast, and quite cheap to set up [9][10][11]. Indeed, PRP is simply obtained by the centrifugation of anticoagulated whole blood, allowing separation of plasma and platelets and having the lowest densities compared to red blood cells (RBCs) and leukocytes. Different authors have classified PRP based on the presence or not of leukocytes, defining leukocytes as rich in PRP if their concentration is higher in PRP compared to whole blood or pure PRP in the opposite case [12][13][14][15]. This differs from Platelet-rich Fibrin (PRF), corresponding to a fibrin matrix whose structure traps platelets and their cytokines, which is obtained after centrifugation of non-anticoagulated blood [16]. Historically, PRP therapy was first described as a surgical adjuvant. Robert Marx is a maxillofacial surgeon who is considered the pioneer of this therapy and defined PRP as "a volume of autologous plasma that has a platelet concentration above baseline" [17].
Thanks to news media reporting the use of PRP products by elite athletes and celebrities [18], it has become a popular procedure over the last few years. A growing number of manual and mechanical procedures have been developed by manufacturers to produce PRP, with about 50 different kits currently available on the market [19]. Despite the popularity and massive use of PRP in regenerative medicine, medical practices remain heterogeneous, limiting its adoption as a standard of care in therapeutic strategies [20]. Several recommendations [21][22][23][24] and classifications [12][13][14][15]21,25,26] have emerged, encouraging both standardization and characterization of the injected biological products.
Due to the complexity of this field and the heterogeneity in clinical practice, the goal of this article is to identify the 100 most-cited articles published concerning the use of PRP in regenerative medicine that have made key contributions to the field, describing the characteristics of articles, providing a reference for better comprehending the worldwide research, and highlighting potential directions for use in regenerative medicine.

Collection and Allocation of Articles
We searched for all relevant articles on the use of PRP in regenerative medicine by using the Thomson ISI Web of Science database including Web of Science Core Collection, MEDLINE, KCI-Korean Journal Database, Russian Science Citation Index, BIOSIS Citation Index, and SciELO Citation Index. Two researchers (ANONYMIZED) independently identified articles for inclusion to enhance the search sensitivity. The keywords used were "platelet-rich plasma" OR "plasma rich in growth factors (PRGF)" AND "regenerative" AND "medicine". The search was performed on 9 January 2022, and yielded 9073 results in total, which contained all articles published since 1950. Filtering via "journal articles and reviews", the search resulted in 8311 documents.
The suitability for inclusion was assessed according to the following criteria: original articles (both preclinical and clinical), meta-analyses, systematic reviews, and classifications, whose subject was mainly the use of PRP or PRGF in regenerative medicine field. Articles concerning the use of platelets concentrate as culture media and the use of blood-derived products different from PRP and PRGF were excluded from the analysis.
Selected articles were ranked by the number of citations, with the exclusion of articles with <110 citations to reduce the workload. We sorted 561 results in descending order according to the total citations. The two independent investigators evaluated the articles for their relevance to the use of PRP within the regenerative medicine field, and articles were selected based on title and abstract. Articles whose subjects were the use of platelet-rich fibrin or the use of platelets for different purposes than regenerative medicine, systematic reviews about regenerative medicine, or when PRP was a minor topic were removed. Any disagreements were discussed between two authors until a consensus was reached. After the review of all included studies, 219 articles remained. These articles were arranged according to number of citations, and the top 100 most cited articles were included in the final analysis.
Since the most recent papers had less time to accumulate citations and to enter the top 100, a further search was performed to overcome this time limit and identify the most promising articles among the latest publications on PRP use in regenerative medicine. As the most recent article from the top 100 was published in 2017, articles published between 2013 and 2017 were analyzed. Among them, a threshold was defined as the minimum number of citations obtained in the first 5 years after their publication. Application of this threshold selected 11 promising articles among the 119 articles reaching inclusion criteria of the study and not yet listed as top 100 articles (Figure 1). The title and abstract were used by the investigators to categorize the themes and type of each article. The themes were (1) biology, (2) cosmetic/plastic surgery, (3) dentistry/maxillofacial surgery, (4) dermatology, (5) musculoskeletal system/orthopedics, and (6) manufacturing. The types were (1) preclinical studies, (2) clinical studies, (3) meta-analyses, (4) reviews, and (5) classifications that were relevant to any aspect of PRP use in regenerative medicine.

Data Extraction
All the selected articles were reviewed independently by the same two authors. The following information was listed for all articles: title, authors name, journal name, year of publication, impact factor of the journal in 2020 (Journal Citation Report or using internal software from an ANONYMIZED hospital when information was not available in the first database), total number of citations of the article, average citations per year (ACY), geographic origin and institutions/corporations of the first author, research theme, and article type. When the first author had more than one affiliation, the department, institution/corporation, and country of origin were reported for each of the affiliations. Subject of investigation was assessed for pre-clinical studies, whereas the following data were collected

Data Extraction
All the selected articles were reviewed independently by the same two authors. The following information was listed for all articles: title, authors name, journal name, year of publication, impact factor of the journal in 2020 (Journal Citation Report or using internal software from an ANONYMIZED hospital when information was not available in the first database), total number of citations of the article, average citations per year Bioengineering 2022, 9, 580 4 of 30 (ACY), geographic origin and institutions/corporations of the first author, research theme, and article type. When the first author had more than one affiliation, the department, institution/corporation, and country of origin were reported for each of the affiliations. Subject of investigation was assessed for pre-clinical studies, whereas the following data were collected for clinical studies: indications, number of patients treated in the included studies, mean follow-up, and study design. The level of evidence was first reported from the publishing journal or determined using the Oxford Centre for Evidence-Based Medicine (OCEBM) if it was not [27].
The biological characterization of whole blood and/or PRP was reported for preclinical and clinical studies excluding reviews, meta-analyses, and letters. Complete characterization was defined when authors provided biological data on platelets, red blood cells (RBC), and leukocytes concentration both in whole blood (WB) and PRP, and data for at least one growth factor within PRP. Incomplete characterization was defined at least by available data on platelets count in PRP. The absence of biological characterization was reported when none of the above parameters were mentioned in the publication.
An additional assessment of methodological quality of clinical studies was performed using a modified Coleman methodology score (CMS) [28]. Part A was reduced to five parameters for a total of 45 points, and Part B was reduced from 40 to 20 points (Table 1).

Statistical Analysis
The Shapiro-Wilk test was used to test the distribution of individual variables for normality. Data are presented as mean and standard deviation, followed by median and ranges (minimum-maximum), whereas figures are presented using median and ranges. The Kruskal-Wallis test was used to test for differences involving skewed data. The Mann-Kendall trend test was used to test for time-dependent trends. A p-value < 0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed via GraphPad Prism, version 9.3.1 (GraphPad Software, San Diego, CA, USA), and XLStat for Excel (Addinsoft, Paris, France/Okayama, Japan/New York, NY, USA).
The citation rank corresponds to the article's rank based on the total number of citations. The average citation per year (ACY) is the mean number of citations per year since the paper has been published. The mean and standard deviation of total number of citations and ACY do not include decimal value, whereas impact factor includes one decimal value.
The Shapiro-Wilk test and the Kolmogorov-Smirnov test both indicated an abnormal distribution of the citation data for all studied parameters (article type, article theme, institutions or corporations, level of evidence).

Characteristics of the Top 10 Most Cited Articles
The top ten most cited articles by number of total citations are listed in Appendix A, Table A2. The mean number of total citations was 853 ± 438 (median (min-max) = 741 (421-1879)). The article classified number ten had 421 citations. The ACY ranged from 20.05 to 75.16. Most of these articles (n = 6) were published between 2000 and 2009.
The first two articles were both published by Robert Marx et al. The article with most overall citations (n = 1879) involved the PRP use in mandibular continuity reconstructions due to oral cancer and was published in Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics Journal in 1998. This paper reported the results of a randomized controlled trial in which 88 patients received cancellous marrow bone graft with or without PRP.
The second top-cited article (n = 1255) was published in the Journal of Oral and Maxillofacial Surgery in 2004. In this review, Robert Marx provided a definition of PRP and collected data concerning its mechanism of action, safety, and therapeutic indications.
The third most cited article (n = 958) was published in Thrombosis and Hemostasis, in 2004 by Eduardo Anitua and colleagues in a review describing the beneficial therapeutic effects of autologous platelets in a wide range of clinical situation.
The article types were clinical studies (n = 3), reviews (n = 4), pre-clinical studies (n = 2), and one classification. Description and conclusions of the top ten most cited articles are presented in Appendix A, Table A2.

Characteristics of the Top 100 Most Cited Articles
The year of publication ranged from 1998 to 2017, and the majority of the articles were published in the 2000s (58%). The years with the greatest number of articles were 2012 (n = 13) and 2009 (n = 11), followed by 2011 (n = 9). The Mann-Kendall trend test showed an increasing trend between average citation per year and time (p < 0.001) ( Figure 2).

Characteristics of the Top 100 Most Cited Articles
The year of publication ranged from 1998 to 2017, and the majority of the articles were published in the 2000s (58%). The years with the greatest number of articles were 2012 (n = 13) and 2009 (n = 11), followed by 2011 (n = 9). The Mann-Kendall trend test showed an increasing trend between average citation per year and time (p < 0.001) ( Figure 2).  The first author of the top 100 most cited articles originated from 17 different countries. The majority of the articles were from Western Europe (n = 46) and North America (n = 38). The country with the greatest number of published articles was the United States of America (USA) (n = 36) from which first authors were mainly affiliated to universities or corporations located in the states of New York (n = 9; Cornell University: n = 6, University of New York: n = 3) and California (n = 7; Stanford University: n = 4, University of California: n = 2, The Orthobiologic Institute: n = 1) and followed by Massachusetts (n = 4) and Illinois (n = 4). In Europe, the first author affiliation was located in Italy for 13 articles, with the Rizzoli Orthopedic Institute (Bologna, Metropolitan City of Bologna, Italy) (n = 8) and the University of Milano (n = 2) as the most productive. Other productive European countries included Spain (n = 11) and Germany (n = 7), from which Biotechnology Institute located in Vitoria (n = 6) and Johannes Gutenberg University located in Mainz (n = 3) were highly represented, respectively. The first author was affiliated with Japan in eight articles, making this country the most represented in Asia. Geographic origins and affiliations of the first authors from the 100 most cited paper are detailed in Figures 3 and 4. A total of 11 researchers have published more than two publications as first author within the top 100 most cited articles ( Table 2).
Bioengineering 2022, 9, x FOR PEER REVIEW 7 The first author of the top 100 most cited articles originated from 17 different c tries. The majority of the articles were from Western Europe (n = 46) and North Ame (n = 38). The country with the greatest number of published articles was the United S of America (USA) (n = 36) from which first authors were mainly affiliated to univers or corporations located in the states of New York (n = 9; Cornell University: n = 6, Un sity of New York: n = 3) and California (n = 7; Stanford University: n = 4, Universi California: n = 2, The Orthobiologic Institute: n = 1) and followed by Massachusetts (n and Illinois (n = 4). In Europe, the first author affiliation was located in Italy for 13 arti with the Rizzoli Orthopedic Institute (Bologna, Metropolitan City of Bologna, Italy) 8) and the University of Milano (n = 2) as the most productive. Other productive Europ countries included Spain (n = 11) and Germany (n = 7), from which Biotechnology Inst located in Vitoria (n = 6) and Johannes Gutenberg University located in Mainz (n = 3) w highly represented, respectively. The first author was affiliated with Japan in eight cles, making this country the most represented in Asia. Geographic origins and affiliat of the first authors from the 100 most cited paper are detailed in Figures 3 and 4. A of 11 researchers have published more than two publications as first author within the 100 most cited articles (Table 2).      The most prolific authors were Eduardo Anitua from the Biotechnology Institute Corporation and Giuseppe Filardo from the Rizzoli Orthopedic Institute (n = 7 and n = 4, respectively). Isabel Andia from the Biotechnology Institute Corporation and PierMaria Fornasari from the Rizzoli Orthopedic Institute were the co-authors with the highest number of total publications (n = 10 and n = 8, respectively).

Most Recent and Promising Articles
The analysis of citations from the articles published from 2013 to 2017 among the top 100 cited articles allowed us to define a threshold of 80 citations in the five years following their publication to define the most promising articles. The second search, conducted on the 119 articles in accordance with inclusion criteria above the top 100 cited articles, allowed us to select a further 11 papers. These articles obtained from 81 to 160 citations in the first five years from publication (105 ± 23 (103 (81-160)).

Most Recent and Promising Articles
The analysis of citations from the articles published from 2013 to 2017 among the top 100 cited articles allowed us to define a threshold of 80 citations in the five years following their publication to define the most promising articles. The second search, conducted on the 119 articles in accordance with inclusion criteria above the top 100 cited articles, allowed us to select a further 11 papers. These articles obtained from 81 to 160 citations in the first five years from publication (105 ± 23 (103 (81-160)).

Discussion
In 1987, the first bibliometric article was published in Journal of American Medical Association (JAMA), launching a tradition for this type of article to be regularly published in scientific literature. Indeed, one way to measure the academic importance of an article is the rate at which the work is quoted or referenced by other authors' number of citations, which remains a valuable measure of the impact of an article has on a specific topic. Despite its development and clinical use for over thirty years, PRP use has become increasingly popular in the past decade, particularly for treating musculoskeletal injuries.
However, its clinical efficacy remains a matter of debate in the scientific community due to contradictory results that have been published which limit its widespread use. In the context of this innovative and debated treatment, we provide a large bibliometric analysis on PRP use in regenerative medicine by ranking the top 100 articles by number of citations. Article types published until 2007 (n = 42) were represented by preclinical studies (62%), clinical studies (21%), and reviews (17%), whereas from 2008 to 2017 (n = 58) articles were represented by preclinical studies (34%), clinical studies (29%), reviews (26%), meta-analyses, and classifications (5% each) confirming the growing popularity and interest of PRP therapy for clinical use. The results of our bibliometric analysis confirm the position of Robert Marx as a pioneer in the field of PRP use in regenerative medicine (total number of citations of 3134). The American nationality of Marx probably created a wide enthusiasm and investment of North American researchers in this field, as first authors originate from USA make up 36% of the top 100 most cited articles. This aspect may have been reinforced by the important representation of this topic in the American Journal of Sports Medicine (12 from 100 articles).
Browsing through the top 100 list also shows that most articles fall into the use of PRP in the musculoskeletal system (54 from 100 articles), far ahead of the other themes studied.
Surprisingly, preclinical study was the category of most cited articles among the top 100 (n = 46), with a mean impact factor of 4.8 ± 2.6 (median (min-max) = 3.6 (1.5-12.5)). They also represent the only type of study providing a complete biological characterization (n = 8).
Comparatively, the 26 clinical studies listed among the top 100 cited articles reached a mean IF of 5.9 ± 10.4 (4.3 (1.2-56.3)), of which 15 provided at least biological data on platelet count within PRP, whereas 10 did not provide any data related to biological characteristics of the injected product. This lack of characterization of PRP is systematically reported as a weakness in the field [29][30][31][32][33], although three classifications published in 2009, 2012, and 2014, respectively, are listed in the top 100 cited articles. Our top 100 most cited articles show that only 30% of clinical studies are level 1, according to the OCEBM classification. As this classification does not take into account the biological characterization of the PRP, 62.5% of these level 1 studies did not provide any data concerning the product injected. The interest in using the modified CMS was therefore to refine these levels of evidence analyses and to provide more accurate results for clinical studies using PRP. Our analysis showed an overall increasing quality of the clinical studies over time based on the modified CMS. As PRP is a new therapy, and its use remains controversial, it is crucial that future clinical studies strive to provide the highest level of evidence, including both classical applications of the concept of evidence-based medicine [34][35][36] and systematic detailed descriptions of the protocol for obtaining PRP and its related quality controls. In our opinion, researchers should systematically report at least the erythrocyte, leukocyte, and platelet counts of the blood sample and the PRP and, if possible, associated growth factors, which constitute the identity card of the injected product and would make it possible to classify the type of PRP produced. Moreover, this systematic characterization of the PRP would allow establishing correlations between the injected dose of each cellular element and growth factors and the clinical outcome.
The second level of research of this study allowed us to identify promising articles not yet referenced within the top 100, following the methodology recently used by Franceschini et al. [37]. Interestingly, the two first articles were high-level preclinical studies both published in Theranostics (impact factor in 2020: 11.6) on the use of exosomes derived from PRP in diabetic and osteonecrosis rat models, which could be a popular area of research in the future. Indeed, extracellular vesicles (EVs) are the subject of increasing interest due to their ability to transfer biological content between cells. EVs, emitted in the extracellular space, circulate via the different fluids of the organism, and modulate locally or remotely the responses of the cells with which they have interacted [38]. Three meta-analyses (two in musculoskeletal system and orthopedics and one in dermatology) were also listed as promising articles, confirming the growing clinical interest of PRP use in medicine. The presence of four preclinical studies (two in vitro and two with both in vitro-in vivo experiments) in this selection of articles highlights the need for continuing to explore some unrecognized effects of PRP. Topics studied in the three clinical studies (two in musculoskeletal system and orthopedics, one in dentistry and maxillofacial surgery) were comparable to the most popular topics found in the top 100 cited articles.
Although more recent expert recommendations and biological classifications published since 2014 were not listed as promising articles, it is likely they will also reach a high level of citations in the coming years, as three of them already reached more than 50 citations with ACY higher than 10 and were only published in 2015, 2018, and 2020 [15,21,25]. Interestingly, detailed and rigorous biological characterization of whole blood and PRP for each injection performed both in clinical studies or in routine care is part of the American Academy of Orthopedic Surgeons (AAOS), International Society on Thrombosis and Hemostasis (ISTH), American Medical Society for Sports Medicine (AMSSM), and European Society of Sports Traumatology Knee Surgery and Arthroscopy (ESSKA) guidelines. This point is reinforced by the fact that journals of high scientific quality now require the inclusion of a dedicated checklist as a supplemental file and detailing the Minimum Information for Studies Evaluating Biologics in Orthopedics (MIBO), which contains nine items referring to PRP preparation [39,40]. Recommendations are not limited to the musculoskeletal field, as the Academy of International Regenerative Medicine and Surgery Societies (AIRMESS) recently published recommendations about the use of PRP in androgenetic alopecia and wound healing, also encouraging researchers to standardize the use of PRP [41]. It will be interesting to follow the influence of these recent recommendations on the future landscape of publications in these fields, whether will it increase the level of the publications (8 from the 26 clinical studies are level 1 with only 5/54 journals with an impact factor higher than 6) or will participate to a broader diffusion of PRP therapy.
Concerning the 22 reviews listed in the top 100 cited articles, they represent a total of 8410 citations. They mainly describe the mechanisms of action of PRP, detail the role of platelet growth factors, and summarize PRP clinical applications either in a broad way or in a dedicated field. Comparison with a similar bibliometric analysis in a larger field like orthopedic knee research published in 2014 allows us to understand that PRP use remains a small area of research (2640 citations vs. 1879 citations for the first article of the top 100, 47,653 total citations for the top 100 cited articles vs. 31,000, and 29 articles vs. 8 articles with more than 500 citations, respectively) [42]. A recent bibliometric analysis published in 2022 reported the most-cited PRP-related articles in the field of knee osteoarthritis [43], confirming the USA and AJSM as the most productive country and journal on the topic. Interestingly, four of the top five most-cited articles in this bibliometric analysis were present in the 100 we reported in this article. This is consistent with the fact that the majority of the clinical studies on the musculoskeletal system category from our bibliometric analysis were focused on knee osteoarthritis (10/17).
This study has limitations, such as the use of the 2020 impact factor, which is questionable but necessary to classify the different journals. Furthermore, a total number of citations as the first criterion of classification is not appropriate to evaluate the quality of the most recent articles and is not systematically synonymous with quality. However, this parameter remains one of the best and is more often used to evaluate the impact of an article over time, whether or not it brings a positive opinion to the scientific community.

Conclusions
In conclusion, this bibliometric analysis provides a valuable snapshot of articles that have attracted citations regarding the use of PRP in regenerative medicine. Periodic updates will be necessary to include more recent articles and measure the impact of recent recommendations. The present study revealed that the majority of the top-cited articles were represented by preclinical and clinical studies in the musculoskeletal system and orthopedics. Studies on PRP-derived exosomes, biological characterization, and correlation with clinical results might be areas of future trends.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bioengineering9100580/s1, Table S1: Characteristics of the topcited meta-analyses (level of evidence), preclinical studies (study design and characterization of platelet-rich plasma), and clinical studies (study design, level of evidence, Coleman methodology score (CMS) and characterization of platelet-rich plasma); Table S2 Appendix A Table A1. List of the 100 most cited articles in platelets-rich plasma use in regenerative medicine.