Secretome and Extracellular Vesicles as New Biological Therapies for Knee Osteoarthritis: A Systematic Review

Secretome and extracellular vesicles (EVs) are considered a promising option to exploit mesenchymal stem cells’ (MSCs) properties to address knee osteoarthritis (OA). The aim of this systematic review was to analyze both the in vitro and in vivo literature, in order to understand the potential of secretome and EVs as a minimally invasive injective biological approach. A systematic review of the literature was performed on PubMed, Embase, and Web of Science databases up to 31 August 2019. Twenty studies were analyzed; nine in vitro, nine in vitro and in vivo, and two in vivo. The analysis showed an increasing interest in this emerging field, with overall positive findings. Promising in vitro results were documented in terms of enhanced cell proliferation, reduction of inflammation, and down-regulation of catabolic pathways while promoting anabolic processes. The positive in vitro findings were confirmed in vivo, with studies showing positive effects on cartilage, subchondral bone, and synovial tissues in both OA and osteochondral models. However, several aspects remain to be clarified, such as the different effects induced by EVs and secretome, which is the most suitable cell source and production protocol, and the identification of patients who may benefit more from this new biological approach for knee OA treatment.


Introduction
Osteoarthritis (OA) is a degenerative disease with progressive degradation of articular cartilage and subchondral bone leading to loss of joint function and pain which significantly impairs patient quality of life [1,2]. Worldwide estimates indicate that 9.6% of men and 18.0% of women over 60 years old suffer from symptoms of OA, with knee OA representing one of the most disabling conditions, with a huge social impact [3][4][5]. This high prevalence of OA is further increasing due to the augmented risk of OA due both to non-modifiable risk factors, such as the aging population and the gender, and to local risk factors, such as physical activity [6]. The classic clinical approaches in the treatment of OA offer mainly temporary symptom relief without disease modifying effects [7]. The limitations of available treatments fostered the development of new strategies, with cell-based procedures being proposed, such as minimally invasive injective approaches with the aim of modulating the inflammation process as well as stimulating and supporting the regeneration of articular tissues, thus re-establishing joint homeostasis. Mesenchymal stem cells (MSCs) represent the most promising cell population [8,9] showing, in several clinical studies, the possibility to increase joint function and reduce pain in knee OA patients [10][11][12]. However, the efficacy of this cell injection approach may be impaired by cell manipulation, and its wide application is strongly limited by regulatory issues [13,14].
To overcome these limitations, in the past 15 years researchers focused on the secretome of MSCs. In fact, it has been demonstrated that the therapeutic ability of MSCs is mainly related to their secretion of biologically active factors, rather than their differentiation properties [15]. These soluble factors belong to different biochemical classes and include growth factors, cytokines, chemokines, lipids, and other molecules with immunomodulatory effects [15]. All these paracrine factors, with the addition of a broad variety of acid nucleic and different lipids, can also be found within cell-secreted vesicles (extracellular vesicles (EVs)), a key part of the secretome which is gaining increasing attention by the scientific community. EVs (either microvesicles (MVs) or Exosomes (Exo)) represent important mediators between articular cell types [16]. Once secreted in the extracellular space, they can interact and be internalized by target cells, ultimately influencing and modifying their phenotype. Preclinical in vitro studies suggested a wide range of potential benefits with immunomodulatory, regenerative, anti-catabolic, and chondro-protective properties of secretome and EVs, which could overcome the limits of cell therapies while offering comparable biological effects. However, while secretome and EVs appear to be very promising, it is important to confirm their role and effects in the complex in vivo environment of knee OA joints [17].
Therefore, the aim of this systematic review was to analyze the available literature on both in vitro and in vivo settings, in order to understand the potential of secretome and EVs as a minimally invasive injective biological approach for the treatment of knee OA.

Data Source
A systematic review of the literature was performed on the use of secretome and EVs in both in vitro and in vivo studies for the treatment of OA affecting the knee joint. This search was performed on PubMed, Embase, and Web of Science databases up to the 31 August 2019 using the following string: (exosom* OR microvesicle* OR vesicle* OR ectosom* OR secretome) AND (mesenchymal stem cell* OR MSC* OR mesenchymal stromal cell* OR ASC* OR ADSC* OR BMC OR BMSC* OR stem cell*) AND (cartilage OR synovi* OR menisc* OR chondrocyte OR chondral OR osteoarthritis OR OA).

Study Selection Process
Two independent reviewers (A.R. and D.D.) conducted the screening process and the analysis of the papers according to PRISMA guidelines. First, the reviewers screened the resulting records by title and abstract, then the full text of selected manuscripts was screened entirely according to the following inclusion criteria: in vitro and in vivo studies of any level of evidence, written in English language, on the use of secretome and EVs for the treatment of cartilage lesions and OA with focus on the knee joint. Exclusion criteria were articles written in other languages, reviews, studies not analyzing the effect of secretome and EVs or exploiting their potential effect not in the knee. The reviewers also screened the reference lists of the selected papers. The flowchart reported in Figure 1 graphically describes the systematic review process.

Data Extraction and Synthesis
From the included studies, relevant data were extracted, summarized, and analyzed according to the purpose of the present work. In particular, the following data were evaluated: cell source of secretome and EVs, target cell types, type of the secreted products (divided in secretome, Exo, and MVs, Figure 2), production method, storage, and study design; for the in vivo studies, the animal model was also considered together with the method of OA induction; in vitro effects were evaluated in terms of EVs internalization, effect on viability, proliferation and migration, effect on chondrocyte phenotype, production of cartilaginous ECM, anti-catabolic effect, anti-inflammatory and immunomodulatory effect, effect on apoptosis, autophagy, and senescence; the in vivo effects were evaluated in terms of effect on cartilage tissue and ECM deposition, effect on synovial inflammation and cytokines, effect on bone tissue, effect on pain, and gait.

Data Extraction and Synthesis
From the included studies, relevant data were extracted, summarized, and analyzed according to the purpose of the present work. In particular, the following data were evaluated: cell source of secretome and EVs, target cell types, type of the secreted products (divided in secretome, Exo, and MVs, Figure 2), production method, storage, and study design; for the in vivo studies, the animal model was also considered together with the method of OA induction; in vitro effects were evaluated in terms of EVs internalization, effect on viability, proliferation and migration, effect on chondrocyte phenotype, production of cartilaginous ECM, anti-catabolic effect, anti-inflammatory and immunomodulatory effect, effect on apoptosis, autophagy, and senescence; the in vivo effects were evaluated in terms of effect on cartilage tissue and ECM deposition, effect on synovial inflammation and cytokines, effect on bone tissue, effect on pain, and gait.

Results
According to the search strategy, 154 papers were found from PubMed, 148 from Embase, 206 from Web of science. After duplicates removal, 20 papers were analyzed, nine of those were in vitro studies, nine were in vitro and in vivo, and two were in vivo studies. The in vitro studies have been described in detail in Table 1 while Table 2 pools together studies performed both in vitro and in vivo and those in vivo only. All these studies have been summarized in the following paragraphs.

Results
According to the search strategy, 154 papers were found from PubMed, 148 from Embase, 206 from Web of science. After duplicates removal, 20 papers were analyzed, nine of those were in vitro studies, nine were in vitro and in vivo, and two were in vivo studies. The in vitro studies have been described in detail in Table 1 while Table 2 pools together studies performed both in vitro and in vivo and those in vivo only. All these studies have been summarized in the following paragraphs.

In Vitro Studies
In vitro studies were published from 2017 with a rapidly increasing trend of publications ( Figure 3). Among the 18 in vitro studies, six articles used bone marrow-derived MSCs (BMSCs), four adipose-derived stem cells (ASCs), two embryonic stem cell-derived MSCs (EMSCs), two commercial (not otherwise specified) MSCs, one synovial-derived MSCs (SMSCs), one chondrocytes, one infra patellar fat pad (IPFP)-derived MSCs, and one compared SMSCs with induced pluripotent stem cell line (iPSC)-derived MSCs. Furthermore, one study compared the effects of secretome, MVs, and Exo compared to BMSCs. Twelve articles investigated the effect of Exo, four evaluated Exo with MVs, one EVs (without other details), and one secretome. The most selected method to isolate EVs was differential centrifugation (five), followed by precipitation-based commercial kits (four), differential centrifugation coupled with a filtration step (three), filtration (three), differential centrifugation with sucrose density centrifugation (one), filtration and sucrose density centrifugation (one), while one paper did not report the detailed isolation protocol (one). Results of in vitro studies were summarized according to:

In Vivo Studies
In vivo studies were published from 2016 with a rapidly increasing trend of publications ( Figure  3). Among 11 in vivo studies, nine included both an in vitro investigation and an animal model study. Six studies have been performed in mouse, four in rat, and four in rabbit. Three studies created an osteochondral defect model and eight an OA model. Eight articles investigated the effect of Exo, 1 of secretome, one of MVs, and one compared Exo with MVs. Regarding the cell source, four used EVs or secretome from BMSCs, three from EMSCs, two from SMSCs, one from IPFP, and one commercial not better specified MSCs. The most selected method to isolate EVs was differential centrifugation (four), followed by ultrafiltration (two), filtration (two), precipitation-based commercial kits (one), and sucrose density centrifugation (one). All studies showed positive effects after the administration of secretome, Exo, or MVs in both osteochondral [21,22,35] and OA defect models [23,26,28,30,31,33,34,37]. The results of in vivo studies have been summarized according to: Effect on cartilage tissue and ECM deposition: Animal studies showed that Exo was effective in cartilage surface restoration and ECM deposition [21,26,31,34,35], regenerating a hyaline-like cartilage completely integrated with the adjacent tissues [31,35]. Zhang et al. [21] demonstrated that this repair and the deposition of ECM started 2 weeks post-injection and increased over time for up to 12 weeks. Similar results after Exo and MVs injections were reported, both providing protection from OA development [33] and showing that both vesicles are equally effective in counteracting tissues degeneration and promoting cartilage regeneration. Positive effects on cartilage repair and ECM deposition have also been described for Exo derived from cells over-expressing microRNA [23,30] or engineered to silence specific genes [28]. These results were superior to those induced by normal Exo. Finally, Khatab et al. [37] and Xiang et al. [22] demonstrated that the effect of secretome and MVs injections on cartilage and ECM were the same as those exerted by MSC injection. EVs internalization: EVs [18][19][20][21][22][23] can be internalized very quickly, already after 30 min from their administration [20]. Moreover, the kinetic of their uptake reached a maximum after 12-18 h [19,21] when cells appeared to be saturated, and continued up to the last evaluation performed at 24 h after EVs addition [19,21]. The intracytoplasmic localization of internalized Exo was identified in the perinuclear region [18,19,23].
Effect on apoptosis: As increased chondrocyte apoptosis represents another feature of OA cartilage, six papers investigated the impact of EVs on this cell process [18,22,26,28,32,33], all reporting a significant decrease in apoptosis rate. Among these, three also demonstrated a dose dependent reduction of OA chondrocyte apoptosis [22,28,33], with superior results for Exo versus MVs [33]. One paper studied the effect of Exo overexpressing a long non-coding RNA (KLF3-AS1) [32], showing that not transfected MSC-Exo significantly reversed IL-1β-mediated chondrocyte apoptosis, and that KLF3-AS1-Exo consolidated this inhibition.
Autophagy and senescence: Another cell process important for cartilage biology during OA progression is autophagy, assessed by Wu et al. [26], showing that Exo significantly increased autophagy in IL-1β-treated chondrocytes. Finally, Tofiño-Viann et al. [36] demonstrated that Exo, MVs, and secretome significantly reverted mitochondrial membrane increase and oxidative stress induced by IL-1β, thus causing a reduction in DNA damage and resulting in inhibition of the senescence process.

In Vivo Studies
In vivo studies were published from 2016 with a rapidly increasing trend of publications (Figure 3). Among 11 in vivo studies, nine included both an in vitro investigation and an animal model study. Six studies have been performed in mouse, four in rat, and four in rabbit. Three studies created an osteochondral defect model and eight an OA model. Eight articles investigated the effect of Exo, 1 of secretome, one of MVs, and one compared Exo with MVs. Regarding the cell source, four used EVs or secretome from BMSCs, three from EMSCs, two from SMSCs, one from IPFP, and one commercial not better specified MSCs. The most selected method to isolate EVs was differential centrifugation (four), followed by ultrafiltration (two), filtration (two), precipitation-based commercial kits (one), and sucrose density centrifugation (one). All studies showed positive effects after the administration of secretome, Exo, or MVs in both osteochondral [21,22,35] and OA defect models [23,26,28,30,31,33,34,37]. The results of in vivo studies have been summarized according to: Effect on cartilage tissue and ECM deposition: Animal studies showed that Exo was effective in cartilage surface restoration and ECM deposition [21,26,31,34,35], regenerating a hyaline-like cartilage completely integrated with the adjacent tissues [31,35]. Zhang et al. [21] demonstrated that this repair and the deposition of ECM started 2 weeks post-injection and increased over time for up to 12 weeks. Similar results after Exo and MVs injections were reported, both providing protection from OA development [33] and showing that both vesicles are equally effective in counteracting tissues degeneration and promoting cartilage regeneration. Positive effects on cartilage repair and ECM deposition have also been described for Exo derived from cells over-expressing microRNA [23,30] or engineered to silence specific genes [28]. These results were superior to those induced by normal Exo. Finally, Khatab et al. [37] and Xiang et al. [22] demonstrated that the effect of secretome and MVs injections on cartilage and ECM were the same as those exerted by MSC injection.
Effect on synovial inflammation and cytokines: Two studies addressed this issue, one showing that Exo increased M2 macrophage infiltration while decreased M1 and inflammatory cytokines [21], while the other study was unable to demonstrate any effect on synovial inflammation for both secretome and MSCs [37].
Effect on bone tissue: Regarding subchondral bone, both Exo and MVs were effective in terms of regeneration: Zhang et al. [21,35] showed complete subchondral bone restoration; Cosenza et al. [33] described higher bone volume and lower bone degradation at epiphyseal and subchondral level following MVs or MSCs injections with respect to controls. Conversely, no effect on bone remodeling was reported by Khatab et al. for both secretome and MSCs [37].
Effect on pain and gait: Another interesting aspect is that Exo injections were able to partially ameliorate gait abnormality patterns in the OA mouse model [26]. Moreover, Khatab et al. [37] demonstrated that both secretome and MSCs provided early (day 7) pain reduction in the treated animals.

Discussion
The main finding of this systematic review is that the use of secretome and EVs for the treatment of cartilage pathology and knee OA had pleiotropic effects and overall positive results. In vitro, both secretome and EVs showed anticatabolic, immunomodulatory, and regenerative properties, and in vivo studies confirmed the effectiveness as minimally invasive treatment, with positive effects on the whole joint.
The literature analysis supports the use of secretome and EVs with an increasing number of preclinical studies. The overall successful results, coupled with the same low immunogenicity of MSCs, and potentially fewer legal issues compared to therapies based on cell transplantation [38], make this biological approach a good candidate for human translatability. The use of secretome and EVs as a minimally invasive treatment for OA in an in vivo preclinical model showed that it was equally effective as MSCs in terms of pain improvement and morphological changes [37], and even proved the superiority of MVs and Exo over BMSCs in terms of joint protection from OA [33]. On the other hand, the literature analysis also underlined that, despite the increasing interest with many recent publications, this field is still in its infancy, with several approaches proposed but lacking the underlying understanding of biological roles and functions. In addition, standardizations and indications on the most suitable strategies for exploiting the potential of this biological approach are also still lacking.
With the aim to evaluate the potential of secretome and EVs as new cell derived approaches for the treatment of knee OA, the available literature was screened for both in vitro and in vivo studies assessing the role of these biological products in the different physiologic processes involved in cartilage lesions and OA progression and treatment. Three different cell derived products were considered: secretome, Exo, and MVs. For this analysis, the secretome group included all studies that specifically referred to the secretome. Regarding the EVs, they are a heterogeneous population which has been classified into three classes according to their biogenesis and size: apoptotic bodies, MVs, and Exo [39]. The apoptotic bodies, the largest EVs population, range from 200 nm to 5000 nm, and they are secreted by the shedding of the plasma membrane of apoptotic dying cells. The MVs, also called ectosomes or microparticles, are 200-800 nm sized EVs that are shed from the plasma membrane of viable cells. Exo, which are 30-200 nm in size, are formed intracellularly and then released within the multivesicular bodies pathway. However, this classic EVs nomenclature results overburdened and sometimes confusing [40]. For the purpose of this systematic review EVs have been subdivided in two different population as small (below 200 nm) and medium-sized EVs (larger vesicles), following the statement of the of the International Society for Extracellular Vesicles [41], but maintaining the nomenclature used in all the papers analyzed, Exo and MVs respectively.
The literature analysis showed a great heterogeneity among studies in terms of EVs used, size, and isolation procedures. The most investigated EVs type is Exo, with a different size range (from 50 to 200 nm), making it difficult to compare among studies or correlate EVs characteristics and in vitro and in vivo results. Only one study [33] investigated the effect of different EVs types, comparing MVs and Exo on a chondrocytes culture and an in vivo OA model. The study showed that both EVs exert similar chondroprotective and anti-inflammatory effects, delaying OA development, leaving the question on the most suitable approach still open. The isolation procedures represent a critical aspect, since there is no standardized method to isolate EVs, resulting in different protocols and therefore different products to be used. The main methods used are differential centrifugation, filtration, and precipitation-based reagent, but there is a lack of standardized methods to obtain them, possibly contributing to EVs variability. Moreover, secretome and EVs can be obtained from different cell sources.
This systematic review showed that the most used cell source are currently BMSCs, followed by ASCs, EMSCs, SMSCs, but there is lack of information available about the difference between vesicles derived from different cells and thus the optimal cell source to address OA remains elusive. Only one study [31] compared the effects of Exo secreted by iPS-derived MSCs and SMSCs in vitro. This showed that they both stimulated chondrocyte proliferation in a dose-dependent manner, but results depended on the cell source, with superior effect of Exo from iPS-derived MSCs on cell proliferation, at high concentration, and superior therapeutic effect in attenuating OA in a mouse model.
The proper selection of EVs cell source and also the stage of cell differentiation are actually critical aspects, since they can determine the characteristics and properties of EVs to fit specific applications (such as reducing inflammation, promoting cartilage regeneration and protection from OA features) [38]. Furthermore, the surrounding microenvironment seems to play an active role in determining the composition of both secretome and EVs cargo, ultimately affecting their action on target cells [42]. Analogously, the type of media and substrate used for cell culture, as well as the use of primary or immortalized cells can also independently affect secretome and EVs composition [38]. For the translational potential of secretome and EVs into a clinically available therapeutic option, another key factor to be considered is the proper dosage [21,22,29,30,33]. In this regard, the literature presents concordant findings, with all papers that compared different amounts reporting a dose dependent effect and superior results at the higher quantities. However, no effects of different dosages were described in the analyzed animal models. In addition, the lack of standardization, also in terms of unit of measurement employed to express the used amount of EVs and thus the presence of heterogeneous products, prevents the possibility to identify the best EVs concentration for an optimal effect in terms of OA treatment. Further efforts should investigate the protocols to optimize secretome and EVs production toward OA treatment.
While studies focusing on the most suitable cell source and dosage could foster the clinical translatability of this biological approach, research efforts are already invested into the investigation on how to further develop this field by optimizing secretome and EVS potential. In this light, among the beneficial effects mediated by EVs, one aspect remains critical: Tao et al. [23] reported that chondrocytes treated with normal Exo decreased ECM proteins expression. On the contrary, the treatment with miR-140-5p-Exo, expressed during the development and homeostasis of cartilage and lowered in OA [43], did not affect ECM protein secretion. miRNAs are important Exo components and their role has been demonstrated in repressing chondrocytes inflammation, promoting chondrogenesis, and inhibiting cartilage degeneration. Considering these effects on chondrocytes, five studies [23,25,26,29,30] investigated the overexpression of different miRNAs in Exo, describing in general better results compared to the normal Exo in terms of cell proliferation, gene expression, ECM components' production in vitro and inhibition of cartilage degradation in vivo. Furthermore, Liu et al. [32] described the effect of over expressing a long non-coding RNA KLF3-AS1, a competitive endogenous RNA which was able to inhibit miR-206, a miRNA that resulted overexpressed in OA.
Another feature of OA is synovial inflammation, notably characterized by activation of monocytes and macrophages. One major immunosuppressive effect of BM-MSCs is to inhibit macrophage activation and to induce a shift from M1 pro-inflammatory to M2 anti-inflammatory phenotype [44]. In this light, Cosenza et al. [33] demonstrated that both MVs, Exo, and BM-MSCs inhibited in vitro macrophage activation to a similar extent. On the contrary, Khatab et al. [37] did not report any significant change on synovial thickness or synovial macrophages phenotypes using secretome injection in an OA mouse model, although several significant moderate correlations between macrophage phenotypes and OA characteristics were found. Another aspect was investigated: different types of stress can lead to a premature cellular senescence. Among these, chronic inflammation can increases oxidative stress driving to cellular senescence, a process that can contribute to the development and progression of OA [45]. In this context, Exo was able to revert the oxidative stress induced by IL-1β, thus causing a reduction in DNA damage and resulting in inhibition of the senescence process [36].
All these investigated targets confirmed the pleiotropic effects of secretome and EVs, which led to positive effects also in vivo. Exo injections were able to partially ameliorate the gait abnormality patterns in the OA mouse model [26], and secretome injections provided early (day seven) pain reduction in treated animals, similar to MSCs [37], further supporting the translational potential of this biological approach. On the other hand, this systematic review also underlined several critical aspects needing additional investigation to further develop and optimize this biological treatment strategy. The promising in vitro and in vivo results support the potential of this new treatment approach, opening new perspectives for cell-based therapies. Secretome and EVs could require less complex regulation procedures than treatments based on cell transplantation, while providing similar results of MSCs. The standardization of protocols could further facilitate clinical translatability. In this light, research efforts are required for the identification of the proper cell source, the best preparation protocol and the most suitable target and, in the end, for the translation of the preclinical promising findings into clinical trials to confirm the potential of secretome and EVs as a minimally invasive biological treatment to address knee OA.

Conclusions
This systematic review of the literature underlined an increasing interest towards this emerging field, with overall positive findings. Promising in vitro results have been documented in terms of enhanced cell proliferation, reduction of inflammation, and down-regulation of catabolic pathways while promoting anabolic processes. The positive in vitro findings were confirmed in vivo, with studies showing positive effects on cartilage, subchondral bone, and synovial tissues in both OA and osteochondral models. However, several aspects remain to be clarified, like the different effects induced by EVs and secretome, the most suitable cell source and production protocol, as well as the identification of patients that may benefit more from this new biological approach for the treatment of knee OA.