CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).


Introduction
In inflammatory joint diseases, including certain phenotypes of osteoarthritis (OA), the synovium and IFP tissues serve as origins of pro-inflammatory and articular cartilage saline (DPBS; Sigma Aldrich, St. Louis, MO, USA), followed by enzymatic digestion using 235 U/mL collagenase I (Worthington Industries, Columbus, OH, USA) diluted in DPBS and 1% bovine serum albumin (Sigma) for 2 h at 37 • C with agitation. Enzymatic digestion was inactivated with complete media with DMEM low glucose (1 g/L) GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; VWR, Radnor, PA, USA), washed and seeded at a density of 1 × 10 6 cells/175 cm 2 flask in two different complete media: human platelet lysate (hPL) and chemically reinforced (Ch-R) media. Complete hPL medium was prepared by supplementing DMEM lowglucose GlutaMAX with 10% hPL solution (PL Bioscience, Aachen, Germany). Complete Ch-R medium was prepared by mixing mesenchymal stem cell growth medium 2 with supplement provided according to the manufacturer's instructions (PromoCell, Heidelberg, Germany). At 48 h post-seeding, non-adherent cells were removed by DPBS rinsing and fresh media were replenished accordingly.

Quantitative Real-Time PCR (qPCR)
RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD, USA) according to the manufacturer's instructions. Total RNA (1 µg) was used for reverse transcription with a SuperScript™ VILO™ cDNA synthesis kit (Invitrogen), and 10 ng of the resulting cDNA was analyzed by qPCR using a QuantiFast SYBR Green qPCR kit (Qiagen) and a StepOne real-time thermocycler (Applied Biosystems, Foster City, CA, USA). For each target, human transcript primers were selected using PrimerQuest (Integrated DNA Technologies, San Jose, CA, USA) (Supplementary Table S1). All samples were analyzed in triplicate. Mean values were normalized to GAPDH, and expression levels were calculated using the 2 −∆∆Ct method and represented as the relative fold change of the primed cohort to the naïve (=1).
A predesigned 90 gene Taqman-based MSC qPCR array (Stem Cell Technologies, Supplementary Table S1) was performed (n = 2) using 1000 ng cDNA per IFP-MSC sample and processed using a StepOne real-time thermocycler (Applied Biosystems). Data analysis was performed using a qPCR online analysis tool (Stem Cell Technologies). Sample and control Ct values were expressed as 2 −∆∆Ct (with 38-cycle cutoff point). The expression levels were represented in bar plots ranked by transcript expression levels on a logtransformed scale of the sample compared to control cohorts. Bar plots were color-coded by the functional class of genes (namely Stemness, MSC, MSC-related/angiogenic, chondrogenic/osteogenic, chondrogenic, osteogenic, adipogenic). CD10High and CD10Low IFP-MSC groups were compared and presented in bar plots.

Isolation and Validation of IFP-MSC-Derived sEVs
CD10High and CD10Low IFP-MSC-derived sEVs were isolated from IFP-MSCconditioned media by a stepwise ultracentrifugation method and CD63-immunomagnetic purification. Briefly, conditioned media from IFP-MSC groups cultured in sEV-depleted Ch-R or sEV-depleted hPL media [22] were filtered through a 0.22 µm filter to remove debris and large vesicles, and differentially centrifuged for 2000× g for 10 min, 10,000× g for 30 min, and ultracentrifuged for 120,000× g for 16 h [23]. Pre-enriched sEV preparations were incubated with the Dynabeads ® -based Exosome-Human CD63 Isolation/Detection Reagent (Invitrogen), and using a magnetic separator, sEV preparations were further purified. Samples from each group were assessed for biophysical and biochemical characterization [19].
A CD10 SimpleStep ELISA kit (Abcam, MA, USA) was used to quantify the CD10 protein cargo levels (pg/mL) in IFP-MSC sEVs, following the manufacturer's instructions. Levels were determined by measuring the fluorescence (450 nm) of individual samples in end-point mode (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA, USA). CD10 levels were normalized to total protein content per group.
The functional assessment of IFP-MSC sEVs was performed in a concentration corresponding to sEVs secreted from 1 × 10 6 IFP-MSC. For IFP-MSC sEV tracking, sEVs were stained with a PKH26 red fluorescent membrane staining kit (Fluorescent Cell Linker Kits, Sigma) according to the manufacturer's instructions and co-cultured with target cells in functional assessments.

miRNA Profile of IFP-MSC sEVs
miRNA was extracted from CD10High and CD10Low sEVs using a Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Total sEV miRNA (1 µg) was used for first-strand cDNA synthesis with an All-in-One miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA).
Predesigned human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using a StepOne real-time thermocycler (Applied Biosystems, LLC). Data analysis was performed using qPCR results with GeneCopoeia's online data analysis system (http://www.genecopoeia. com/product/qpcr/analyse/, accessed on 10 March 2023). Mean values were normalized to small nucleolar RNA, C/D box 48 (SNORD48), and expression levels were calculated using the 2 −∆Ct method. Putative miRNA interactomes were generated using an miRNetcentric network visual analytics platform (https://www.mirnet.ca/, accessed on 20 March 2023). The miRNA target gene data were collected from the well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with a 2.0 betweenness cutoff. Values (with a 34-cycle cutoff point) were represented in a topology miRNA-gene interactome network using a force atlas layout and hypergeometric test algorithm. The miRDB online database (http://mirdb.org, accessed on 2 April 2023) for prediction of functional miRNA targets was used to correlate highly expressed target genes in macrophages and synoviocytes with specific miRNAs identified by IFP-MSC sEV miRNA profiling. MirTarget prediction scores were in the range of 0-100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB [24].
CCK-8 cytotoxicity assay (Cell Counting Kit-8, Sigma) was performed in SYN TIC and IFP-MSC sEV/SYN TIC according to the manufacturer's instructions. CD10High and CD10Low sEV cytotoxicity was determined by measuring optical densities of individual wells at 450 nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA, USA).
RNA extraction from SYN TIC cultures was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD, USA) according to the manufacturer's instructions. Total RNA (1 µg) was used for reverse transcription with a SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). A predesigned 88-gene human synoviocyte array (GeneQuery™ Human Synoviocyte Cell Biology qPCR Array Kit, ScienCell) was obtained using 1000 ng cDNA per culture and processed using a StepOne real-time thermocycler (Applied Biosystems, LLC). Mean values were normalized to the ACTB housekeeping gene; expression levels were calculated using the 2 −∆Ct method with a 34-cycle cutoff. Values were represented in dot plots as the relative fold change of the CD10High sEVs/SYN TIC or CD10Low sEVs/SYN TIC to SYN TIC (reference sample, 2 −∆Ct = X sample/X reference sample).
The functional enrichment analysis was performed using g:Profiler (version e108_eg55_ p17_9f356ae) with the g:SCS multiple testing correction method, applying a significance threshold of 0.05 [25]. The colors for different evidence codes and for log scale are described in Supplementary Table S2.
On day 3, chondropellets were harvested for histology and molecular profiling. For histological analysis, chondropellets were cryosectioned and 6 µm frozen sections were stained with hematoxylin and eosin (Sigma), and 1% toluidine blue (Sigma) for semiquantitative assessment of pellet structure and chondrogenic differentiation, respectively. For molecular profile analysis, RNA extraction from chondropellets was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD, USA) according to the manufacturer's instructions. Total RNA (1 µg) was used for reverse transcription with a SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). A predesigned 88-gene human osteoarthritis array (GeneQuery™ Human Osteoarthritis and Cartilage repair qPCR Array Kit, ScienCell) was obtained using 1000 ng cDNA per culture and processed using a StepOne real-time thermocycler (Applied Biosystems, LLC). Mean values were normalized to the ACTB housekeeping gene; expression levels were calculated using the 2 −∆Ct method with a 34cycle cutoff. Values were represented in a stacked bar plot as the relative fold change of the chondropellets with CD10High sEVs to the chondropellets without CD10High sEV treatment (reference sample, 2 −∆Ct = X sample/X reference sample).
The functional enrichment analysis was performed using g:Profiler (version e108_eg55_ p17_9f356ae) with the g:SCS multiple testing correction method, applying a significance threshold of 0.05 [25]. The colors for different evidence codes and for log scale are described in Supplementary Table S2.

Mono-Iodoacetate Model of Acute Synovial/IFP Inflammation
All animal experiments were performed in accordance with relevant guidelines and regulations. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami, USA (approval no. 21-030 LF) and conducted in accordance to the ARRIVE guidelines [26]. Twelve (#12) 10-week-old male Sprague Dawley rats (mean weight 250 g) were used. The animals were housed to acclimate for 1 week before the experiment initiation. One rat was housed per cage in a sanitary, ventilated room with controlled temperature, humidity, and under a 12/12 h light/dark cycle with food and water provided ad libitum.
Acute synovial/IFP inflammation was generated by intra-articular injection of 1 mg of mono-iodoacetate (MIA) in 50 µL of saline into the right knee. Under isoflurane inhalation anesthesia, rat knees were flexed 90 • and MIA was injected into the medial side of the joint with a 27G needle using the patellar ligament and articular line as anatomical references. Four (4) days later, a single intra-articular injection of CD10High sEVs in 50 µL of Euro-Collins solution (MediaTech, Woodland, CA, USA) was performed, having as control (1) rat knees receiving MIA but not IFP-sEVs (Only MIA group) and (2) healthy rat knees. Animals were sacrificed at day 4 after IFP-sEV injection (d8 in total). This short exposure to MIA has been shown to induce inflammatory changes within the synovium and adjacent IFP [27].

Cytochemical Staining and Lubricin (PRG4) Immunolocalization In Situ
Rat knee joints were harvested by cutting the femur and tibia/fibula 1 cm above and below the joint line, muscles were dissected and removed, and joints were fixed with 10% neutral buffered formalin (Sigma-Aldrich) for 14 days at room temperature. Knee joints were decalcified, cut along the sagittal plane in half, embedded in paraffin, and serial 4 µm sections obtained. Toluidine blue staining was performed to evaluate the sulfated glycosaminoglycan content of articular cartilage. Masson's trichrome staining was performed to evaluate the collagen type and content of the articular cartilage. Microscope images of cytochemically stained tissues were acquired using a x10 and x20 objective Leica DMi8 microscope with Leica X software (Leica). Histochemical staining quantitative analysis was evaluated in 3 rat knees per condition and 4 microscopy fields per knee with Fiji/ImageJ 2.14.0 software.
For anti-lubricin (anti-PRG4) immunofluorescence staining, sections were permeabilized with 1x PBS + 0.2% Triton X-100 for 20 min at room temperature and incubated with blocking buffer (1x PBS + 0.1% Triton X-100 with 1% bovine serum albumin) for 1 h at room temperature. In between different treatments, sections were washed with 1X PBS. Mouse anti-rat PRG4 monoclonal antibody (Sigma, Burlington, MA, USA) was prepared in blocking buffer (1:500) and sections were incubated at 4 • C overnight. Sections were washed with 1X PBS + 0.01% Triton X-100 and incubated for 2 h with secondary antibody Alexa Fluor647 conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) at room temperature. Controls were incubated with secondary antibody only. All sections were rinsed with 1x PBS, mounted in prolong gold antifade reagent with DAPI (Invitrogen), and microscope images were acquired using a x20 objective Leica DMi8 microscope with Leica X software (Leica).

Statistical Analysis
Normal distribution of values was assessed by the Kolmogorov-Smirnov normality test. In the presence of a non-normal distribution of the data, one-way or two-way ANOVA tests were used for multiple comparisons. All tests were performed with GraphPad Prism v7.03 (GraphPad Software, San Diego, CA, USA). Level of significance was set at p < 0.05.

Distinct Molecular Profiles of IFP-MSC Based on CD10 Protein Expression Level
CD10 is a surface neutral endopeptidase expressed in multiple cell types including MSC [28,29], with enzymatic activity neutralizing various signaling substrates including substance P (SP) [30,31]. Specifically, SP is a neuropeptide associated with nociceptive pathways that is secreted by sensory nerve fibres in the synovium and IFP tissues. Upon its secretion, it actively affects local inflammatory/immune and fibrotic responses by modulation of cell proliferation, activation, and migration to sites of inflammation, and the expression of recruiting chemokines and adhesion molecules [3,4]. Importantly, for the onset and progression of human inflammatory joint diseases, SP-secreting sensory nerve fibers predominate over sympathetic fibers in anterior knee pain [7,8,32], whereas SP secretion is increased in synovial fluid during joint inflammation [3]. On this basis, we have previously reported that IFP-MSC can efficiently degrade substance P and suppress T cell proliferation in vitro, whereas upon intra-articular infusion in vivo, IFP-MSC can dramatically reverse signs of synovitis and IFP fibrosis [33,34]. Specifically, our in vivo animal studies revealed a correlation between CD10 expression magnitude in IFP-MSC and the reduction or even absence of SP+ fibers in areas of active inflammation and fibrosis for both the synovium and IFP tissues. Based on the correlation between CD10 positivity and therapeutic outcomes, levels around 70% or more should represents an effective therapeutic outcome [33].
In the present study, IFP-MSC cultured in regulation-compliant (Ch-R and hPL) media showed similar fibroblast-like morphology but importantly had different CD10 expression levels ( Figure 1A). Ch-R cultured IFP-MSC had 96.5 ± 5.6% expression whereas hPL cultured IFP-MSC had 59.2 ± 15.7% expression. According to the abovementioned 70% threshold for an effective therapeutic outcome in vivo, we named Ch-R cultures as CD10High (more therapeutic) and hPL cultures as CD10Low (less therapeutic).
Molecular profiling of CD10High versus CD10Low IFP-MSC revealed that 25 out of 90 genes tested were more highly expressed in CD10High IFP-MSC, with 8 genes being more than twofold higher (KDR, COL10A1, NGFR, PROM1, ALPL, EGF, BMP7, ITGAX) ( Figure 1B). Interestingly, genes tested were grouped in phenotype-related cohorts with MSC-associated, chondrogenic/osteogenic, and MSC cohorts showing overall the most prominent fold expression change between CD10High and CD10Low IFP-MSC cultures ( Figure 1C). Functionally, KDR (VEGFR2) and COL10A1 genes, which are more than 10-fold higher expressed in CD10High IFP-MSC, are strongly related to robust prochondrogenic and pro-angiogenic MSC actions [35,36]. Interestingly, studies show that the JNK-EGR1 signaling axis promotes TNF-α-induced endothelial differentiation of MSC via VEGFR2 expression [35]. COL10A1 is a specific marker of hypertrophic chondrocytes and is critical for endochondral bone formation, as mutation and altered COL10A1 expression is often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases [36]. Among the highly expressed genes (more than twofold), NGFR (CD271) and PROM1 (CD133) have important roles in pro-angiogenic, immunomodulatory, and chondrogenic MSC actions. In vivo studies showed that CD271 + MSC and CD133 + MSC infusion in ischemia animal models result in increased pro-angiogenic and decreased pro-inflammatory signaling [37,38]. Several studies have associated high NGFR gene and protein expressions in MSC with their increased chondrogenic capacity in vitro and osteochondral cartilage repair in vivo [39][40][41]. Pro-chondrogenic and pro-angiogenic actions can be also attributed to MSC with high expression levels of ALPL, EGF, BMP7, and ITGAX genes [42][43][44]. Of note, the MCAM (CD146) gene higher expressed in CD10High IFP-MSC is directly related to increased immunomodulatory MSC actions in vitro and in vivo [45].
In contrast, the CD10Low IFP-MSC molecular profile showed more than twofold higher expression of 45 genes compared to the CD10High IFP-MSC profile. Specifically, FGF9, FGF10, ACAN, and IGF-1 genes seem to be a characteristic molecular signature for CD10Low IFP-MSC as they are expressed more than 150-fold higher, respectively. These genes are involved in proliferation and differentiation of MSC signaling. Studies have shown that FGF9 and FGF10 expression acts as an important regulator of early chondrogenesis, partly through the AKT/GSK-3β signaling pathway [46,47]. High ACAN gene expression is related to pro-chondrogenic MSC action, as its protein is a key GAGcontaining proteoglycan in cartilage and plays an important role in stabilizing the ECM in articular cartilage [48]. Moreover, a recent study showed that IGF-1-transfected MSC have chondroprotective effects in an OA model [49].
Overall, CD10 protein levels in MSC strongly affected their molecular profile. CD10High IFP-MSC had a more distinct molecular profile consisting of only eight genes that were expressed significantly higher (more than twofold) compared to CD10Low IFP-MSC. Furthermore, the CD10High IFP-MSC molecular profile was related to robust pro-chondrogenic and pro-angiogenic MSC actions.
The CD10 protein levels in parental MSC were strongly correlated with the CD10 protein levels in their sEV cargo. Specifically, CD10 protein cargo was 610 ± 20.6 pg/mL for CD10High sEVs and 200 ± 20 pg/mL for CD10Low sEVs ( Figure 1D).

CD10High and CD10Low sEVs' Anti-Inflammatory Effects on Synoviocytes
Synovial lining hyperplasia is a main characteristic of IFP/synovium inflammation in OA. In general, there are two types of resident synoviocytes contributing to such pathological conditions; type A (macrophage-like synoviocytes) and type B (fibroblast-like synoviocytes) are mainly responsible for maintenance of synovial homeostasis [54,58]. Herein, we separately investigated the functional effects of CD10High and CD10Low sEVs on stimulated synoviocytes in vitro to identify the optimal therapeutic IFP-MSC sEV scheme for in vivo application.
In cocultures, both CD10High and CD10Low sEVs were internalized by TIC-primed SYNs (SYN TIC) and their proliferation was attenuated ( Figure 5A,B). This finding suggests that IFP-MSC sEVs, irrespective of the CD10 levels in their parental cells, could attenuate synovial lining hyperplasia upon their in vivo intra-articular infusion. Similarly, we and others have reported that SYN exposure to MSC sEVs results in suppressed healthy-SYN [19] and rheumatoid arthritis-SYN [59] activation, migration, and invasion in vitro.
At the molecular level, SYN and TIC culture gene expression was strongly affected by both CD10High and CD10Low sEVs. From 88 genes analyzed, most expression levels were downregulated upon exposure to IFP-MSC sEVs ( Figure 5C). Specifically, CD10High sEVs increased the expression level of 45 genes in SYN TIC, with only 13 genes being more than twofold higher (PTGS2, MDM2, EREG, KRT7, IGF1, ESM1, IL15, ITGA4, VIM, POSTN, CSNK1D, CD14, and ALB). Reactome analysis of highly expressed genes in CD10High sEVs/SYN TIC showed their involvement in the regulation of five molecular pathways related to the: regulation of cell population proliferation, cellular response to stimulus, prostaglandin biosynthetic process, regulation of neuroinflammatory response, and regulation of protein phosphorylation ( Figure 5D). Also, CD10Low sEVs increased the expression levels of 19 genes in SYN TIC, with only nine genes being more than twofold higher (PTGS2, EREG, MDM2, KRT7, NGF, POSTN, CD14, ESM1, and ITGA4). Reactome analysis of highly expressed genes in CD10Low sEVs/SYN TIC showed their involvement in the regulation of five molecular pathways related to the regulation of cell population proliferation, regulation of protein phosphorylation, cellular response to stimulus, prostaglandin biosynthetic process, and regulation of cellular senescence ( Figure 5D).
Among the commonly expressed genes, PTGS2 (COX2) was the highest expressed (more than 500-fold) after IFP-MSC sEV exposure compared to SYN TIC cultures alone. Importantly, PTGS2 plays a crucial role in the promotion of the prostaglandin E2 (PGE2) biosynthesis pathway in fibroblast-like cells (such as MSC), indicating its strong involvement in cell immunomodulatory capacity in vivo [60]. On this basis, via COX2-dependent PGE2 secretion, fibroblast-like cells can polarize macrophages to an alternative anti-inflammatory M2 phenotype, attenuate T cell proliferation and differentiation to Th17, and increase T cell differentiation to Tregs (reviewed in [60]). In parallel, a pioneering study showed a COX2-dependent PGE2-regulated suppression of fibroblast proliferation [61] that in concert with our data could explain the reduction of synoviocyte proliferation upon exposure to IFP-MSC sEVs. Among the highly expressed genes (more than 50-fold), EREG and MDM2 have important pro-survival and trophic roles in synoviocytes. Studies showed that EREG (epiregulin), a ligand for epidermal growth factor receptor (EGFR), can affect EGFR signaling, which is critical for maintaining the superficial layer of articular cartilage, and preventing osteoarthritis initiation [62]. Studies showed that higher MDM2 gene expression in fibroblast-like cells results in higher extracellular matrix accumulation (collagens and fibronectin) [63]. However, MDM2, which is a ubiquitin ligase and binds p53 transcriptional factor for the apoptotic pathway, has been associated with rheumatoid arthritis disease promotion [64]. Although these findings show only some of the pleiotropic IFP-MSC sEV actions on synoviocytes, they invite studies involving OA. rheumatoid arthritis disease promotion [64]. Although these findings show only some of the pleiotropic IFP-MSC sEV actions on synoviocytes, they invite studies involving OA. CD10High and CD10Low sEVs were internalized by TIC-primed SYN (blue, nucleus; red, sEVs) whereas they tended to attenuate SYN TIC proliferation. (C) At the molecular level, SYN TIC cultures gene expression was strongly affected by both CD10High and CD10Low sEVs. Specifically, CD10High sEVs increased the expression levels of 45 genes in SYN TIC, with 13 genes more than twofold higher. Moreover, CD10Low sEVs increased the expression levels of 19 genes in SYN TIC with 9 genes more than twofold higher. (D) Reactome analysis of highly expressed genes in CD10High sEVs/SYN TICs and CD10Low sEVs/SYN TICs showed their involvement in the regulation of five molecular pathways. Scale bar: 50 μm. CD10High and CD10Low sEVs were internalized by TIC-primed SYN (blue, nucleus; red, sEVs) whereas they tended to attenuate SYN TIC proliferation. (C) At the molecular level, SYN TIC cultures gene expression was strongly affected by both CD10High and CD10Low sEVs. Specifically, CD10High sEVs increased the expression levels of 45 genes in SYN TIC, with 13 genes more than twofold higher. Moreover, CD10Low sEVs increased the expression levels of 19 genes in SYN TIC with 9 genes more than twofold higher. (D) Reactome analysis of highly expressed genes in CD10High sEVs/SYN TICs and CD10Low sEVs/SYN TICs showed their involvement in the regulation of five molecular pathways. Scale bar: 50 µm. Excluding the commonly highly expressed genes, CD10High sEV-exposed SYN TICs showed additional expression of genes (such as IGF-1 and ALB) involved in synovial fluid enrichment with anabolic factors for cartilage homeostasis. IGF-1 gene expression was associated with insulin-like growth factor (IGF) signaling pathway activation that can promote chondrocyte proliferation, enhance matrix production, and inhibit chondrocyte apoptosis [65]. Also, high ALB gene expression is related to synoviocytes' increased production of albumin, the most common protein in synovial fluid, which plays an important role in the lubrication of the cartilage, thereby protecting the joint cartilage surface from degradation [66]. Therefore, due to more potent and pleiotropic effects of CD10High sEVs on synoviocytes at the molecular level, we used CD10High sEVs as our platform for further in vitro and in vivo experimentation.

CD10High sEVs-Treated Chondrocytes Show a Chondroprotective Molecular Profile under Inflammatory Conditions In Vitro
For inflammatory joint diseases, the synovium/IFP anatomical and functional unit [67] can serve as a site for immune cell infiltration, neovascularization, and the origin of proinflammatory and articular cartilage (AC) catabolic molecules [11,54]. In this pathological microenvironment, chondrocytes become responsive to pro-inflammatory factors, especially tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). These factors initiate the degradation of cartilage extracellular matrix mediated by matrix-metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs [11]. Herein, we simulated this pro-inflammatory microenvironment in vitro by co-culturing MSC-derived chondropellets with inflamed synoviocytes ( Figure 6A).
In the current experiments, IFP-MSC were successfully induced towards chondrogenesis in 3D micromass pellet cultures for 15 days. On this basis, we acknowledge that despite MSC ability to differentiate towards the chondrogenic lineage, their terminal phenotype is reminiscent of one characteristic of endochondral bone formation rather than articular cartilage chondrocytes. However, we and others have demonstrated that on day 15, MSC are adequately differentiated and possess most of the major characteristics of chondrocytes without entering hypertrophy and mineralization in vitro [33,68,69]. Subsequently, our generated chondropellets and SYN were co-cultured with and without CD10High sEVs under inflammatory/fibrotic TIC conditions for 3 days. Chondropellet histological analysis revealed that although CD10High sEVs, treated and non-treated, have similar structure, CD10High sEV treatment resulted in better chondrocyte differentiation and sulfated glycosaminoglycan production in vitro ( Figure 6B).

CD10High sEVs Show Chondroprotective and Anabolic Effects for Articular Cartilage In Vivo
We have previously reported that functionally, IFP-MSC sEVs can significantly reduce synovitis/IFP fibrosis, and also affect macrophage polarization towards an antiinflammatory therapeutic M2 phenotype in inflammatory conditions in vivo [19]. Herein, an acute synovial/IFP inflammation rat (MIA) model was used to test the chondroprotective capacity of CD10High sEVs upon intra-articular infusion to reverse cartilage degeneration in vivo ( Figure 7A).
Collagen type II and aggrecan are two major components of the extracellular matrix of healthy articular cartilage. Alterations in cartilage extracellular matrix composition and structure are characteristics of OA. Specifically, during OA progression, the presence of degrading cartilage proteins such as matrix metalloproteinases and aggrecanases result in decreased collagen type II/aggrecan content and loss of cartilage integrity [76,77]. In the current study, the diseased group (MIA only) demonstrated strong cartilage degeneration signs by reduced staining for sulfated proteoglycans compared to healthy rat knees ( Figure 7B,C, upper panels). According to the Udo et al. cartilage scoring system (0-5) for rat OA in the MIA model [27], only the diseased group developed grade 3 cartilage erosion (≤50% of joint surface). Importantly, CD10High sEV intra-articular infusion resulted in significantly (p < 0.05) reduced cartilage degeneration compared to the diseased group (average scores 0.4 ± 0.5 and 1.7 ± 1.1, respectively). Masson's trichrome staining has also been used as a suitable histochemical method [78] to evaluate collagen compositional changes. In general, the diseased group showed increased cartilage discoloration (stained as red) compared to other groups. In contrast, CD10High sEV intra-articular infusion resulted in significantly (p < 0.05) increased collagen composition, similar to the healthy control group ( Figure 7B,C, middle panels). Similar to our findings, previous studies using intra-articular infusions of MSC sEVs derived from various tissue sources, such as bone marrow, amniotic fluid, and umbilical cord, reported the anabolic effects of sEVs to enhance matrix synthesis of collagen type II and sulfated glycosaminoglycans (reviewed in [79]). However, in our study, for the first time, we demonstrate the chondroprotective effects of CD10High sEVs derived from IFP tissue.
The avascular nature of articular cartilage limits the capacity for perivascular MSC to migrate from the neighboring microenvironment (i.e., subchondral bone) to restore inflammatory and structural tissue imbalances. However, a pool of resident cartilagederived stem/progenitor cells (CSPCs) is present in the upper zone cartilage zone and is associated with the expression of proteoglycan 4 (PRG4) encoding for proteoglycan 4 or lubricin [80,81]. Interestingly, studies showed that CPSC is an intermediate cell population between MSC and differentiated chondrocytes, which can be stimulated to induce tissue healing and homeostasis at different stages of cartilage degradation in vivo [82,83]. On this basis, we performed in situ PRG4 protein expression evaluations to determine phenotypic cellular changes promoted in articular cartilage in disease and after intra-articular CD10High sEV infusion ( Figure 7B,C, bottom panels). Under these conditions, the healthy group showed a rigid distinct PRG4+ CSPC upper layer on articular cartilage. In the diseased group, PRG4 expression exhibited a reduction in the upper layer of articular cartilage, and simultaneously a significant (p < 0.05) increase in the intermediate zone randomly distributed within chondrocytes. This phenomenon can be attributed to the fact that intermediate zone chondrocytes increase the expression of PRG4 in order to replenish the significant loss from the upper cartilage surface during disease progression. Notably, CD10High sEV intra-articular infusion resulted in significant (p < 0.05) preservation of PRG4 expression on the upper cartilage surface and only minor expression from the intermediate zone chondrocytes. This effect can be attributed not only to the general CD10High sEVs' immunomodulatory/chondroprotective miRNA profile (Figure 2), but also more specifically to the presence of hsa-miR-369-3p and hsa-miR-374a-5p miRNA cargo that strongly affected PRG4 gene expression in target cells ( Figure 4B). Therefore, CD10High sEV treatment suggests robust chondroprotective and anabolic effects on articular cartilage in vivo.   group demonstrated strong cartilage degeneration findings exemplified by reduced staining for sulfated proteoglycans. In contrast, CD10High sEV intra-articular infusion resulted in significantly reduced cartilage degeneration with only minor cartilage depressions and significantly increased collagen composition. Compared to the diseased group, the CD10High sEV group showed preservation of PRG4 expression on the upper cartilage surface and only minor expression from the intermediate zone chondrocytes (indicated by white arrows and dotted lines, ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001). Toluidine blue scale bar: 200 µm, Masson's trichrome and PRG4 staining scale bars: 100 µm.

Conclusions
In summary, CD10High sEVs show potent molecular immunomodulatory and chondroprotective profiles that significantly affect synoviocyte and chondropellet functionality under inflammatory conditions in vitro. Notably, in an animal model of acute synovitis/IFP fibrosis, CD10High sEV intra-articular infusion resulted in robust chondroprotective and anabolic effects that retained articular cartilage structure/composition. However, the effects of CD10High sEVs in post-traumatic OA (PTOA) animal models with more advanced OA (structural/compositional alterations of articular cartilage) should be investigated in further experiments. Based on our findings, we propose a proof-of-concept viable cell-free CD10-based sEV alternative to MSC-based therapeutics in the treatment of inflammatory joint diseases, and perhaps OA phenotypes, where synovitis/IFP fibrosis and articular cartilage degradation are dominant.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12141824/s1, Table S1: Human mesenchymal stem cell qPCR array plate genes and their classification.; Table S2: The colors for different evidence codes and for log scale in the g: Profiler functional enrichment analysis. Informed Consent Statement: Written informed consent has been obtained from the patient(s) to publish this paper.
Data Availability Statement: Not applicable.