Why Use Adipose-Derived Mesenchymal Stem Cells in Tendinopathic Patients: A Systematic Review

The aim of the present systematic review was to provide a clear overview of the clinical current research progress in the use of adipose-derived mesenchymal stem cells (ASCs) as an effective therapeutic option for the management of tendinopathies, pathologies clinically characterized by persistent mechanical pain and structural alteration of the tendons. The review was carried out using three databases (Scopus, ISI Web of Science and PubMed) and analyzed records from 2013 to 2021. Only English-language papers describing the isolation and manipulation of adipose tissue as source of ASCs and presenting ASCs as treatment for clinical tendinopathies were included. Overall, seven clinical studies met the inclusion criteria and met the minimum quality inclusion threshold. Data extraction and quality assessment were performed by groups of three reviewers. The available evidence showed the efficacy and safety of ASCs treatment for tendinopathies, although it lacked a clear description of the biomolecular mechanisms underlying the beneficial properties of ASCs.


Introduction
Tendinopathies is an umbrella term that includes several different clinical entities characterized by persistent mechanical pain and structural alteration of the tendons [1,2], a tough band of bright white fibro-elastic tissue connecting the muscle to the bone [3].
Tendon disorders cause an invalidating musculoskeletal pain that represents a common reason for physician consultation [4]. They severely impact the patient's daily life, thus being associated with an increase in both direct and indirect social costs [5][6][7]. However, the prevalence of tendinopathies is probably underestimated, considering the increase in sport activities. It represents approximately 30-50% of sports injuries [4].
The pathogenesis of tendinopathies is clearly multifactorial [6]. A genetic component was reported in Achilles, elbow and rotator cuff tendinopathies [8,9], although genetic modifications alone are not able to justify the insurgence of the disease [8]. Indeed, the possibility of developing a tendinopathy seems to be increased by the interaction between intrinsic (age, body structure, nutrition, metabolic, genetic) and extrinsic factors (fatigue, improper loading, disuse, exogenous damage) [9,10]. Among them, tendon overload in sports or in specific work activities has been reported to be a crucial factor promoting

Eligibility Criteria
The inclusion criteria for the studies were: (1) English as language of publication; (2) tendinopathy as target disease; (3) clinical studies involving patients with tendinopathies; (4) evaluation of tendinopathy with magnetic resonance imaging (MRI), ultrasonography (US) and/or clinical scores; (5) description of autologous or allogeneic adipose tissue isolation and manipulation for ASCs isolation; (6) description of ASCs isolation or characterization in other cell fractions from adipose tissue; (7) evaluation of ASCs as treatment for tendinopathy; (8) long-term follow-up (at least 6 months). The authors only considered research articles, excluding editorials, book chapters and reviews. The primary outcome considered was the measure of ASCs therapeutic efficiency in tendinopathy, while the secondary outcome was related to the adverse events of ASCs treatment.

Information Sources and Search Strategy
The literature search was carried out by using 3 electronic databases as information sources (ISI Web of Science, Scopus and PubMed) and was based on a pre-determined series of keywords related to tendinopathies and ASCs application. The keywords used in PubMed were: "tendon" OR "tendon pain" OR "tendinopathy" OR "tendinopathies" AND "Adipose-derived mesenchymal stem cells", by adapting them to subject headings or syntax of Scopus and ISI Web of Science. The literature search had no language or study design limits. It considered the period between 2013 and 2021. The date of last search was 29 December 2021.

Study Selection and Data Collection Process
During the selection of the studies, the search results were uploaded to Zotero, excluding duplicates. The results were then shared in a OneDrive folder, available to all the reviewers. The selection was composed of four stages. During the first stage, the eligibility criteria were applied to title, abstract and keywords, not excluding the studies of uncertain eligibility. During the second stage, eligible and uncertain studies were analyzed and screened in their full text, according to the inclusion criteria previously described. During the third stage, reviewers were randomized in groups of 3 in order to identify the suitable studies by consensus between at least 2 out of 3 researchers. Data extraction was then performed in the last phase. This was performed by discussing the relevant findings in a meeting with the whole research group. In case of any disagreement between the reviewers, findings were accepted when a consensus was expressed by all the reviewers for at least 8 of the 11 data items subsequently described.

Data Items and Quality Assessment
From each study included, the following data were extracted: (1) the type of clinical study; (2) the type of tendinopathy and its diagnosis; (3) the type of adipose tissue and its manipulation for ASCs isolation; (4) the type of cell fraction isolated from adipose tissue; (5) the ASCs treatment for tendinopathy; (6) the final follow-up; (7) the primary (scores evaluating tendinopathy) and secondary outcomes (adverse reactions reported) for ASCs treatment; (8) the number of treated and control patients at baseline and at final followup; (9) the characteristic of the patients (at least age and sex; body mass index-BMI-if reported); (10) the inclusion and the exclusion criteria; (11) the significant changes in scores at final and intermediate follow-up, confirmed by MRI or US.
Following the full-text selection, each of the 3 reviewers constituting a group independently assessed the methodological quality by using the criteria from the QualSyst tool for quantitative/qualitative studies [54] adapted to a systematic review (Table S2).

Results
Seven clinical studies were included in the current systematic review. The search performed in the three databases (PubMed, Scopus, Isi Web of Science) retrieved a total of 522 records. They became 366 after 186 duplicate eliminations. Of these, 240 articles were rejected, since they did not meet the eligibility criteria during the evaluation of their title, abstract and keywords. Specifically, two studies were not included, since they were not published in English. The other rejected records included: 53 reviews and four book chapters, not considered in order to avoid bias derived from the analysis of the original published forms; eight editorials, which did not specify all the data related to the outcomes selected for the present review; 111 studies for which tendinopathy was not the target disease and 62 studies analyzing non-adipose-tissue-derived stem cells. Then, the remaining 96 full-text records were carefully evaluated with a screening and eligibility process. This rejected 89 studies. Of these 89, 54 described in vitro or in vivo experiences; 5 reported only a qualitative description of ASCs isolation methodology for their use in tendinopathy; 12 did not report full data about ASCs isolation process or characterization; 5 did not provide a complete description of ASCs volume or concentration; 13 did not consider a follow-up of at least 6 months. The resulting seven studies met the inclusion criteria and were considered eligible to be included in the present review ( Figure 1). All the studies included met the minimum quality inclusion threshold (65%, see Table S2).
Following the full-text selection, each of the 3 reviewers constituting a group independently assessed the methodological quality by using the criteria from the QualSyst tool for quantitative/qualitative studies [54] adapted to a systematic review (Table S2).

Results
Seven clinical studies were included in the current systematic review. The search performed in the three databases (PubMed, Scopus, Isi Web of Science) retrieved a total of 522 records. They became 366 after 186 duplicate eliminations. Of these, 240 articles were rejected, since they did not meet the eligibility criteria during the evaluation of their title, abstract and keywords. Specifically, two studies were not included, since they were not published in English. The other rejected records included: 53 reviews and four book chapters, not considered in order to avoid bias derived from the analysis of the original published forms; eight editorials, which did not specify all the data related to the outcomes selected for the present review; 111 studies for which tendinopathy was not the target disease and 62 studies analyzing non-adipose-tissue-derived stem cells. Then, the remaining 96 full-text records were carefully evaluated with a screening and eligibility process. This rejected 89 studies. Of these 89, 54 described in vitro or in vivo experiences; 5 reported only a qualitative description of ASCs isolation methodology for their use in tendinopathy; 12 did not report full data about ASCs isolation process or characterization; 5 did not provide a complete description of ASCs volume or concentration; 13 did not consider a follow-up of at least 6 months. The resulting seven studies met the inclusion criteria and were considered eligible to be included in the present review ( Figure 1). All the studies included met the minimum quality inclusion threshold (65%, see Table S2).

Study Design and Methods
The design and methods of the seven studies included in the present review are summarized in Table 1.

Characteristics of Tendinopathic Patients
The description of tendinopathic patients is reported in Table 2, along with the inclusion/exclusion criteria considered in the seven studies.   All the patients enrolled provided written informed consent [55][56][57][58][59][60][61]. Except for the case report describing one male patient [61], the number of patients with tendinopathies described in the remaining six studies ranged from 12 [57] to 35 [55]. The mean age of the patients ranged from 24.4 [57] to 59.2 [55] years; at least 61% of the cases were males in three studies [57,59,60], while female cases accounted for more than 57% in the remaining three studies [55,56,58]. BMI was not reported in three studies [58,60,61], while it ranged from 23.4 to 51.8 in the other records [55][56][57]59]. The most frequent inclusion criteria for these patients were tendon pain and disability for at least 3 months [55][56][57][58][59][60][61], together with failure of conventional treatments [55][56][57][58][59][60][61]. The case report was the only study describing a successful treatment with autologous ASCs although not for tendinopathy but for symptomatic bilateral knee osteoarthritis [61]. Patient compliance with the protocol at follow-up was reported in 57% of the studies [55,57,58,61]. In the remaining three studies, a total of four patients withdrew consent during the study [56,59,60].

Therapeutic Efficacy and Adverse Reactions to ASCs Intratendinous Injections
The significantly modified scores following intratendinous injections of ASCs are summarized in Table 3, along with the adverse reactions reported following ASCs treatment in tendinopathic patients.  Among the seven studies included in the present systematic review, only one record compared a group of tendinopathic patients receiving ASCs intratendinous injections during arthroscopic rotator cuff repair with a different control group of tendinopathic patients treated with arthroscopic rotator cuff repair alone [55]. The other six studies compared scores between patients at baseline prior to intratendinous injections of ASCs at different intermediate time points and at the follow-up [56][57][58][59][60][61]. In only one case, the ASCs in SVF were also compared with the PRP group [58]. All the studies used a single dose of ASCs [55,[58][59][60][61], except for two records [56,57]. In particular, the study comparing two ASCs doses showed no significant differences between the two groups [57]. In contrast, the study analyzing three ASCs doses showed the highest dose as the most effective one at all the time points considered [56].
Except for one study reporting only an improvement of the rate of tear recurrence after 28 months [55], the therapeutic efficacy of ASCs in the tendinopathy was evident in almost all studies at the final follow-up, as demonstrated by the improvement of the different scores and by MRI/US [56][57][58][59][60][61]. All the scores also significantly improved at the intermediate time points analyzed [56][57][58][59][60][61], starting from 15 days after ASCs treatment [58] and lasting up to 13 months [57]. Improvement of scores up to 30 months was shown in only one case [61].
Adverse reactions after intratendinous injections of ASCs were reported in three studies [56,59,60]. The most common were pain or subcutaneous hematoma [56,59,60]. However, none of the adverse reactions reported were serious, and all were resolved.

Discussion
To date, a wide array of treatments have been available for tendon disorders. However, their effectiveness remains ambiguous. Therefore, one of the main controversies of the clinical management of tendinopathies is to determine the real effectiveness of both standard medical treatments and innovative therapeutic approaches in order to assure an improvement in tendon pain and dysfunction.
Currently, some specific treatment strategies are applied for a specific tendon injury, with physical therapy representing one of the most important options, considering its long-term effectiveness [23]. However, since the traditional treatments may not be the most effective options, clinical regenerative therapies for tendinopathies have been developed over the past decade and continue to evolve. The use of ASCs has emerged as a good candidate for tendon healing due to their properties promoting proliferation and tenogenic differentiation [43][44][45]. These properties have been mostly investigated in musculoskeletal disorders [62], while the application in tendon disorders is mainly supported by pre-clinical evidence [45,[48][49][50][51][52]. Nevertheless, the efficacy of intratendinous ASCs injections was reported by all seven studies analyzed in the present review. ASCs acted in terms of tendon pain recovery, starting from the second week after treatment and lasting up to 30 months. This was evident not only in the improvement of the VAS score but also by MRI or US evaluations. In this regard, tendon pain recovery induced by ASCs was in line with previous evidence, reporting a rapid and long-lasting pain relief after the application of ASCs in pre-clinical and clinical osteoarthritis, musculoskeletal and neuropathic pain [63][64][65][66]. From the mechanistic point of view, it may be that the therapeutic efficacy of ASCs in tendon pain could be due to ASCs paracrine anti-inflammatory and anti-oxidative activities [67]. Similar hypothesis could be formulated for the effects of ASCs on the tendon structure. It may be that ASCs preserve tendons by reducing the burden of inflammatory damaging mediators released into the injured tissue and promoting local release of new pro-resolutive and trophic factors. A hypothesis of the latter needs confirmation, although the study conducted by Usuelli et al. on human lymphocyte cell culture exposed to ASCs from the autologous SVF fraction paved the way for this. In this study, human lymphocyte cell culture exposed to ASCs showed reduction in the expression levels of the pro-inflammatory interleukin 6 (IL-6) [58].

Conclusions
The use of ASCs has emerged as a good candidate for tendon healing over the past decade. ASCs isolated from the autologous adipose tissue seem the best candidate, since they lack immune rejection after intratendinous application, while allogeneic ASCs from allogeneic adipose tissue could be immunogenic. By adding paracrine anti-inflammatory and anti-oxidative activities [67] to the well-known pro-proliferation and tenogenic differentiation [43][44][45], ASCs will widen the choice of clinical tools for tendinopathies.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pharmaceutics14061151/s1, Table S1: PRISMA (Preferred Reporting Items for Systematic review and Meta-Analysis) checklist: recommended items to address in a systematic review; Table S2: Checklist for assessing the quality of the study.

Data Availability Statement:
The data presented in this study are contained within the article and its supplementary material.