Anti-Inflammatory and Angiogenic Effects of Stem Cell Secretome

Abstract

Mesenchymal stem cells (MSCs) exert biological effects in part through their secretome which includes extracellular vesicles. In this study, we isolated and characterized the secretome from clinically relevant stem cell lines: human embryonic stem cell–derived mesenchymal stem cell line (ES-MSCs) and Infrapatellar fat pad derived MSC (IPFP-MSC) cultured in xeno-free medium. We assessed the biological activity of concentrated cell secretome or isolated fractions of extracellular vesicles (EVs) on cell proliferation, microvascular formation, and cartilage degradation in a human osteoarthritic (OA) ex vivo model. Serum-free conditioned medium from ES-MSC (N = 1) or IPFP-MSC (N = 2) monolayer cultures were concentrated by ultrafiltration to generate concentrated conditioned medium (CCM). Size exclusion chromatography was used to fractionate extracellular vesicles (EVs). Vesicle size, concentration, morphology, and surface markers were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. Biological activity was evaluated by treating human umbilical vein endothelial cells (HUVECs), IPFP-MSCs, and ES-MSCs with CCM and EVs at defined particle concentrations. Endothelial network formation was tested in fibrin gels with different cell and secretome combinations. For analysis of cartilage degradation, human cartilage explants (N = 4; 3.5 mm in diameter) were harvested from patients undergoing total knee arthroplasty and subjected to IL-1β stimulation to induce an OA phenotype. Explants were treated with varying doses from CCM or EVs. Release of glycosaminoglycan in the medium and RNA analysis of catabolic genes were used as readouts. Secretome preparations yielded on average approximately 50 billion vesicles per mL with a similar particle size distribution between 50–200 nm in ES-MSC and IPFP-MSC cultures. Transmission electron microscopy confirmed vesicle morphology and flow cytometry confirmed expression of exosomal surface markers (CD9, CD63, CD81). Functionally, CCM and EVs enhanced proliferation in a dose-dependent manner. Endothelial networks formed by HUVECs in fibrin were stabilized over 7 days by CCMs, most notably by hypoxic ES-MSC CCM relative to no CCM treatment (control). In the OA cartilage model, IL-1β stimulation increased glycosaminoglycan release, whereas ES-MSC CCM treatment and EV treatment reduced glycosaminoglycan release and ES-MSC CCM reduced gene expression of IL-1β, MMP-1, and MMP-3. We isolated and characterized the concentrated secretome and the isolated vesicle-enriched fractions from xeno-free ES-MSC and IPFP-MSC and demonstrated bioactivity in promoting cell proliferation, modulating endothelial network formation, and mitigating cartilage degradation in osteoarthritic tissue. These findings support the bioactivity and therapeutic potential of stem cell–derived secretomes for OA.

1. Introduction

Cells secrete a complex repertoire of bioactive molecules collectively known as the secretome, which comprises soluble factors, matrix proteins, and extracellular vesicles (EVs). The soluble fraction includes cytokines, chemokines, growth factors, hormones, and enzymes that regulate cell behavior, angiogenesis, immune responses, and extracellular matrix remodeling [1,2]. Matrix proteins are structural and regulatory proteins secreted into the extracellular matrix (ECM) that contribute to tissue homeostasis and remodeling [3,4]. EVs are membrane-bound vesicles such as exosomes (30–150 nm), microvesicles (100–1000 nm), and larger apoptotic bodies. EVs can contain proteins, lipids, mRNAs, miRNAs, DNA, and mitochondria, acting as cargo delivery systems to target cells [2,4,5,6,7,8].

In the context of regenerative medicine, the stem cell secretome plays a pivotal role in intercellular communication and tissue repair [2,3,4,5]. Secretome factors regulate cellular activities including survival, proliferation, migration, and immunomodulation [5,9]. Collectively, these processes can support tissue homeostasis and promote endogenous tissue repair and regeneration by enhancing angiogenesis and modulating immune responses. To leverage these biological activities, an increasing array of studies have demonstrated the therapeutic use of the secretome from several stem cell sources, including bone marrow (BM-MSCs), adipose tissue (ADSCs), and umbilical cord (UC-MSCs).

The regenerative activity of MSC-derived secretomes is mediated by pro-angiogenic and trophic factors which promote endothelial cell activation, proliferation, and capillary network formation [8,10]. Furthermore, conditioning of MSCs under stress-inducing microenvironments (e.g., hypoxia) amplifies the release of pro-regenerative mediators, enhancing wound repair and vascularization in preclinical models [11,12]. Incorporation of secretomes into engineered scaffolds also improved neovascularization, attenuated inflammation, and accelerated tissue restoration [13,14,15,16].

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage loss, synovial inflammation, and subchondral bone remodeling that collectively led to chronic pain and functional disability. Current surgical and pharmacological treatments alleviate symptoms but do not halt or reverse disease progression [17], which highlights the need for disease-modifying and regenerative therapeutic options. MSC-derived secretomes exhibit potent immunomodulatory properties relevant to OA pathophysiology. These secretomes downregulate pro-inflammatory cytokines while concurrently upregulating anti-inflammatory mediators [18,19,20]. In vitro and ex vivo OA models have demonstrated that MSC secretomes enhance chondrocyte survival, preserve extracellular matrix integrity, and stimulate autophagy mechanisms, resulting in reduced cartilage degeneration and delayed disease progression [8,21,22]. Similar outcomes have been observed in vivo, where secretome administration attenuates synovial inflammation, decreases joint fibrosis, and supports structural and functional recovery. Compared to direct cell transplantation, cell-free secretomes have a lower risk of immunogenicity and tumorigenicity [13,23,24]. Collectively, these findings underscore the translational potential of MSC-derived secretomes as an effective, cell-free therapeutic strategy for the management of osteoarthritis.

Several methods have been utilized to process the secretome and isolate EVs. For clinical translation, reproducible and scalable isolation methods are essential. Ultracentrifugation (UC) is still considered the “gold standard,” separating EVs based on density and sedimentation velocity, but is time-intensive, challenging to scale, and may compromise vesicle integrity [13,25]. Bulk concentration by ultrafiltration or tangential flow filtration (TFF) enables scalable, GMP-compatible processing for large clinical batches and can be used in conjunction with size-exclusion chromatography (SEC), to yield functionally intact EVs with high-purity [13,26,27,28]. Further refinement by immunoaffinity capture allows for isolation of targeted EV subpopulations (e.g., CD9, CD63 + EVs), and innovative microfluidic platforms offer promise for high-throughput, automated workflows [13,29]. To minimize batch-to-batch variability and immunogenic risk, xeno- and serum-free chemically-defined media are needed for MSC and EV production [13,30].

This study leverages clinically relevant stem cell lines expanded in xeno- and serum-free media to produce a therapeutic secretome. We combined a scalable ultrafiltration approach with size exclusion chromatography for EV enrichment. We evaluated secretome fractions for bioactivity and modulation of cell proliferation and microvascular formation with human cell cultures. We assessed therapeutic potential in a human ex vivo osteoarthritis model as proof-of-concept to support translation for clinical applications.

2. Results

2.1. Characterization of Extracellular Vesicles (EVs)

CCM and EVs from ES-MSC and IPFP-MSC typically yielded on average 49.2 ± 12.7 billion particles per mL (NTA analysis). CCM and EVs preparations from ES-MSC and IPFP-MSC yielded EV concentrations in the same order of magnitude but with differences between sources and fractions: ES-MSC-CCM averaged 42.4 ± 16.8 × 10⁹ particles/mL, ES-MSC-EV 45.0 ± 6.2 × 10⁹ particles/mL, IPFP-MSC-CCM 41.3 ± 17.2 × 10⁹ particles/mL, and IPFP-MSC-EV 68.1 ± 14.9 × 10⁹ particles/mL. Particle sizes ranged between 50–300 nm in the CCM from ES-MSC and IPFP-MSC (NTA, Figure 1A). The EVs contained particles ranging from 50–180 nm in size (Figure 1B). An overview of the concentrations and sizes is provided in Supplemental Data.

TEM confirmed the typical morphology and size corresponding to that of EVs (Figure 1C–F) [31,32,33]. Flow cytometry analysis confirmed the presence of exosome-specific surface molecules (CD9, CD63, CD81) in EVs from ES-MSC (Figure 1G), and CD63 in EVs from IPFP-MSC (Figure 1H).

2.2. ES-MSC Derived CCM and EVs Modulate Cell Proliferation

Three cell types (ES-MSC, IPFP-MSC, and HUVEC) were subjected to CCM or EVs derived from ES-MSC. CCM had a consistently greater and dose-dependent effect compared to untreated controls (Figure 2A–C). CCM increased proliferation of ES-MSC and IPFP-MSC (p < 0.002) but no significant change in HUVECs was observed. EV treatment significantly increased ES-MSC proliferation (p < 0.003) with decreasing EV dose (Figure 2A). EV treatment also significantly enhanced cell proliferation of IPFP-MSC compared to controls (p < 0.001), but without a clear dose response (Figure 2B). The proliferation of HUVEC cells was highly variable with no significant change compared to controls (Figure 2C).

2.3. Secretome Treatment Stabilizes Endothelial Networks

The formation of microvascular networks has been studied with endothelial cells in collagen and fibrin hydrogels [34]. In our preliminary experiments, HUVECs encapsulated in fibrin formed rudimentary networks within 3 days, which subsequently disintegrated by 7 days (Figure 3), consistent with other reports [35]. However, endothelial cells when co-cultured with stem cells form more complex and stable microvascular networks [35]. In this study, we sought to establish whether the secretome from ES-MSC or IPFP-MSC was sufficient to induce and sustain microvascular formation with HUVECs cultured without MSC in fibrin gels. To assess whether oxygen concentration affected the microvascular forming potential of the secretome, we also harvested CCM from cells (N = 1; IPFP-MSC 61M and N = 1 ES-MSC) cultured in a hypoxic chamber at 2% oxygen concentration. By day 3 timepoint (Figure 3), control gels (HUVEC without CCM) exhibited a network with high numbers of nodes, junctions, and meshes. However, by day 7, the networks in control gels disintegrated with substantial regression of network indices, marked decreases in node count, junctions, mesh area, and total segment length (p < 0.05 day 7 vs. day 3). On day 7, CCM harvested from hypoxic ES-MSC maintained greater network integrity as measured by total network length, total segment length, total branch length, and mesh index, relative to day 7 control gels (p < 0.05, Figure 3 and Figure 4, and Supplemental Data Angiogenesis Analysis). Whereas the other CCM treatments did not result in a statistically significant difference relative to the day 7 control condition. This increase in network complexity and continuity indicates that secretome treatment supports both the extension of vessel-like structures and the maintenance of interconnected loops within the endothelial networks.

These results establish that while initial networks form in the absence of CCM, hypoxic ES-MSC–derived conditioned media preserves vascular architecture over time. These data support a protective effect of the hypoxic secretome in preserving endothelial networks within fibrin matrices.

2.4. CCM Treatment Reverses Catabolism in Human Osteoarthritic Cartilage Explants

We assessed the anti-inflammatory potential of the stem cell secretome in our previously reported human OA explant model [36]. We chose to analyze ES-MSC secretomes because of the greater reproducibility and efficacy of our results. In our model, IL-1β treatment induces a catabolic response in human OA cartilage explants and resulted in increased release of GAGs by 3–4 days. The conditions and timing of treatment are outlined in Figure 5. Cartilage explants (N = 36) from femoral condyles and patella from three human OA donors (70M, 71M and 76F) were cultured with or without IL-1β for 3 days. We noted an increase in GAG release from explants exposed to IL-1β. After media change, explants were divided into groups for treatment with CCM or EVs (10B particles). After 3 days of subsequent exposure to either CCM or EVs, we noted a significant decrease in GAG levels (Figure 6A,B). GAG release levels remained higher in the IL-1β pretreated explants not subjected to secretome treatment (Figure 6A,B). GAG release levels in control explants (not exposed to IL-1β) remained low (Figure 6). A significant 2.9-fold reduction of GAGs were observed in the CCM treated cartilage samples pre-treated with IL-1β (p < 0.004), while sustained GAG release was observed in the IL-1β treated samples (Figure 6A). Treatments with EVs also showed a significant 1.6-fold (p < 0.02) reduced GAG release profile (Figure 6B) and EV treatment of control explants (without pre-treatment with IL-1β) also reduced GAG release (p < 0.05).

In separate experiments, we measured GAG release and gene expression in explants harvested from femoral condylar and patellar cartilage (Figure 7). GAG release was increased in both condyle and patella samples after IL-1β treatment (Figure 7A,B) and after subsequent CCM treatment, a significant reduction (p < 0.05) in GAG release was observed after 3 days (Figure 7A,B). IL-1β pre-treatment significantly (p < 0.05) increased expression of IL-1β, IL-6, MMP-1 and MMP-3 compared to controls (Figure 7 C–F). CCM treatment after IL-1β exposure significantly reduced (by 2- to 6-fold) gene expression of IL-1β, MMP-1 and MMP-3 (Figure 7, p < 0.05), but not IL-6 gene expression levels. CCM treatment without IL-1β pre-treatment did not induce significant changes in any gene expression (Figure 7). These data indicate that treatment with a single dose of CCM (10B particles) effectively reduced degradation of cartilage.

3. Discussion

Stem cell secretomes act as potent cell-free therapeutic platforms capable of orchestrating cellular responses such as proliferation, angiogenesis, and inflammation modulation [2,37]. This study demonstrates the feasibility and bioactivity of ES-MSC and IPFP-MSC secretomes that were produced using scalable ultrafiltration and size-exclusion workflows in chemically defined, xeno- and serum-free conditions, supporting their translational potential.

Both concentrated conditioned medium (CCM) and EV-enriched fractions (EVs) significantly enhanced cell proliferation, with a more pronounced, dose-dependent effect in ES-MSC and IPFP-MSC compared to HUVECs. CCM was generated by concentrating serum-free conditioned medium on 100 kDa ultrafiltration devices, enriching for vesicle-associated cargo and providing an EV-rich input for SEC fractionation and particle-normalized dosing, while unconcentrated conditioned media remain widely used for broader secretome formats. These results align with prior studies, showing stem cell secretomes stimulate proliferation and regeneration in wound repair and musculoskeletal models [1,8]. Enhancement of cell proliferation by MSC secretomes supports tissue regeneration by supplying bioactive molecules, growth factors, and extracellular vesicles that stimulate resident cells to proliferate, survive, and repair damaged areas [2]. This paracrine activity can accelerate healing in many tissues (e.g., bone, muscle and skin), by boosting angiogenesis, reducing fibrosis, and activating endogenous stem cell populations, without the immunogenic and tumorigenic risk of cell-based therapies [38,39,40].

Hypoxia enriches the secretome for growth factors such as VEGF-A and bFGF, via HIF-1 signaling, which are important for neovascularization [27,41,42]. In our model, endothelial networks rapidly regressed after initial network formation. However, the secretome from stem cells, especially when preconditioned with hypoxia, stabilized and preserved endothelial networks in engineered fibrin gels. Our results are consistent with other studies reporting that hypoxia induces secretomes that enhance angiogenic network preservation and stability in vitro and in vivo [43,44,45]. These secretomes also enhance tissue regeneration by promoting angiogenesis, cell proliferation, and migration in skeletal muscle [39] and bone [46]. In skin, for example, conditioned medium from hypoxia-cultured MSCs markedly boosts cell proliferation and angiogenesis, enhances migration of keratinocytes, fibroblasts, and endothelial cells, to improve wound closure, collagen synthesis and remodeling [11].

Treatment with ES-MSC-derived CCM had a significant chondroprotective and anti-inflammatory effect on osteoarthritic human cartilage explants, as evidenced by reduced glycosaminoglycan (GAG) release and downregulation of catabolic genes (IL-1β, MMP-1, MMP-3), respectively. These findings indicate that ES-MSC secretome counteracts key inflammatory and catabolic pathways in human OA cartilage, supporting its potential as a disease-modifying, cell-free therapeutic [47].

Our findings are consistent with other ex vivo explant studies using human and animal tissue, which have shown that the MSC secretome or EVs can preserve tissue morphology, reduce catabolic marker expression, and attenuate damage induced by inflammatory cytokines [48]. For example, adipose- and bone marrow–derived MSC secretomes or EVs reduce IL-1β–induced MMPs and ADAMTS5, preserve proteoglycan content, and maintain collagen II in cartilage explants [49,50,51], which parallels the reduced GAG loss and catabolic gene expression observed in our human OA explants. By demonstrating similar chondroprotective responses in clinically relevant human tissue, this study strengthens the translational bridge from preclinical explant data to potential intra-articular use in OA patients. Comparative analyses of EVs derived from bone marrow (BMSC), adipose tissue (ADSC), and umbilical cord (UMSC) MSCs indicate MSC secretomes exert anti-inflammatory and chondroprotective effects in both explant and in vivo models. BMSC- and UMSC-derived EVs were effective in suppressing inflammation (reduced MMP-13, IL-6, TNF-α, and COX-2) and preserving cartilage integrity [52,53,54]. Collectively, these data suggest that modulation of common OA effectors (IL-1β, TNF-α, MMPs, ADAMTS5) is a shared mechanism across MSC sources, and our ES-MSC-derived CCM similarly targets this network in human OA cartilage, implying that ES-MSC secretome could provide efficacy comparable to adult-tissue–derived products [49,55,56]. Notably, ES-MSC exosomes have been shown in a mouse OA model to maintain chondrocyte phenotype, increase collagen II, and reduce ADAMTS5, closely aligning with the anti-catabolic profile seen in our explant system and reinforcing the relevance of ES-MSC derived therapies for OA [51].

Intra-articular injections of EVs reduced some of the arthritic changes in mouse joints after medial meniscus destabilization, presumably via decreased expression of ADAMTS5 [51]. Across multiple OA models, MSC-EVs lessen synovial inflammation, cartilage erosion, and pain behaviors while restoring anabolic–catabolic balance, supporting their potential as candidate disease-modifying OA drugs [51,53,57].

Bringing together our observations that stem cell–derived secretomes and EVs enhance various cellular activities, future work will focus on comparative analyses of secretomes from different MSC sources and culture conditions on the therapeutic potential for cartilage and bone regeneration [46]. There is a need for more comprehensive characterization of EVs, optimization of dosing, and in vivo validation. Future research should address these limitations by conducting systematic, direct comparisons across diverse MSC secretome sources and conditions, and rigorously designed dose-response studies. Larger multi-donor, multi-tissue trials that integrate advanced imaging, “omics” characterization, and functional in vivo models are needed to optimize formulation, dosing, and delivery strategies. Mechanistic studies should further dissect the contributions of specific EV cargo, soluble mediators, and recipient cell interactions. Advancing the development of animal transplantation and engineered scaffold systems will also bridge the gap between in vitro discovery and clinical applications [58].

In summary, this study demonstrates that secretomes from ES-MSC and IPFP-MSC can be produced at scale and exert significant pro-regenerative, angiogenic and anti-catabolic actions in human cell and tissue models under xeno- and serum-free conditions. In particular, hypoxic conditioning generated EVs that preserved microvascular networks. Collectively, these findings support secretome therapies as promising, cell-free platforms for musculoskeletal and inflammatory disorders, although ongoing work is required to standardize production and validate efficacy in translational settings.

4. Methods

4.1. Differentiation and Cultivation of Human Embryonic Derived Mesenchymal Stem Cells (ES-MSC)

Xeno-free human ES-MSC were derived as previously described [30,59]. Briefly, ESC (HADC-100 ESC) were placed in suspension culture as cell clusters for 5 days with an ALK-5 inhibitor (10 mM SB525334; Selleckchem, Houston, TX, USA) and then cultured in monolayer on fibronectin-coated flasks in serum free medium (StemPro-34, ThermoFisher Scientific, Carlsbad, CA, USA) and supplemented with bFGF (20 ng/mL; ThermoFisher Scientific). Cells emerging from the clusters exhibited a typical MSC surface marker profile (CD34 and CD45 negative; CD73, CD90 and CD105 positive), and capacity for chondrogenic and osteogenic differentiation [30,60,61].

4.2. Isolation and Culture of Infrapatellar Fat Pad Mesenchymal Stem Cells (IPFP-MSC)

Infrapatellar fat pad tissues were obtained from OA patients (N = 2) undergoing total knee arthroplasty (TKA) (approved by Scripps Institutional Review Board) within 2–6 h after surgery. IPFP-MSC were isolated from the tissues as previously described [62]. Briefly, the tissue was sectioned into pieces (5–6 mm³) via scalpel and placed into 6-well plates pre-coated with human collagen type I (Cell Adhere, StemCell Technologies, Cambridge, MA, USA) and cultured in MSC media (StemPro MSC SFM XenoFree, ThermoFisher Scientific, Carlsbad, CA, USA) for 7–10 days to allow the IPFP-MSCs to migrate out of the tissue. Upon confluence, the IPFP-MSCs were detached using Accutase (StemCell Technologies, Cambridge, MA, USA) and expanded cultured in collagen type I coated T75cm² flasks.

4.3. Secretome Isolation

Conditioned medium (CM) was initially centrifuged at 300× g for 15 min to remove cells and large debris and subjected to ultra filtration (UF) to concentrate medium (between 30× to 300×) using Amicon^® Ultra-15 Centrifugal Filter filters having 100K nominal MW cutoff (UFC9100, Millipore Sigma, Burlington, MA, USA). The CM was centrifuged at 4000 rpm for 20 min (Legend XTR, Thermo Scientific Sorvall, ThermoFisher Scientific, Carlsbad, CA, USA) to make concentrated conditioned medium (CCM). CCM was used rather than unconcentrated supernatant to enable exosome-enriched fractionation by SEC from a defined, EV-rich starting material and to achieve particle doses in the 10⁹ range per treatment in practical culture volumes. To generate EVs, CCM was subjected to size exclusion chromatography (SEC) using EV SEC COLUMNS (NBP3-11763, Novus Biologicals LLC, Centennial, CO, USA) and specific fractions were collected according to manufacturer instructions. The SEC fractions enriched with extracellular vesicles (1.5–2 mL) were concentrated again with Amicon^® Ultra-2 Centrifugal Filter (10 kDa; Thermo, ThermoFisher Scientific, Carlsbad, CA, USA); each 2 mL of SEC eluate concentrated to approximately 200 uL. Both CCM and EV samples were maintained at 4 °C for immediate use or stored at −80 °C.

4.4. Nano Tracking Analysis (NTA)

CCM and EV samples were prepared for NTA by diluting the sample between 1:5000 to 1:10,000 in phosphate-buffered saline (PBS). Diluted samples were mixed by vortexing at low speed to ensure homogeneity. NTA (ViewSizer 3000 instrument, HORIBA Instruments Incorporated, Irvine, CA, USA) was used to determine particle size distribution and concentration. A total of 300 µL of diluted sample was loaded into a clean quartz cuvette for measurement. Measurements were acquired under the following settings: blue laser, 70 mW; green laser, 12 mW; red laser, 8 mW; gain, 0 dB; exposure time, 5 ms; and frame rate, 30 frames per second. For each sample, ten videos of 200 frames each were captured. Particle size distributions and concentrations were analyzed using ViewSizer software (HORIBA Instruments Incorporated, Irvine, CA, USA) and reported as particles per milliliter (particles/mL). Density of particle size distributions (PSD) was measured as particles/mL/nm.

4.5. Detection of EVs via Immuno-Capture and Flow Cytometry

Streptavidin-coated paramagnetic beads with biotinylated antibodies specific to tetraspanins CD9, CD63 and CD81 were used to immunocapture exosomal EVs from CCM and EVs, following manufacturer instructions (EXOFLOW, System Biosciences, Palo Alto, CA, USA). Briefly, separate tubes containing each individual tetraspanin target were incubated with either CCM or EVs and placed on a rotating rack at 4 °C for 18–22 h. Beads were then washed using the magnet and resuspended into 200uL of wash buffer and placed at 4 °C for storage until further testing. For the detection of captured EVs, each sample was stained with PKH67 (4 mM; Sigma, MilliporeSigma, Carlsbad, CA, USA) for 1–2 min and then quenched using PBS with 1% BSA and washed. Streptavidin beads not exposed to CCM or EVs were also stained with PKH67 to monitor non-specific PHK67 staining. The beads and captured EVs were observed using flow cytometry (Novocyte, Santa Clara, CA, USA). FlowJo (Version 10.1.0.0, Ashland, OR, USA) was used to gate fluorescence signal beads specific for CD9, CD63, CD81, or negative control beads.

4.6. Negative Stain Transmission Electron Microscopy (TEM)

EV samples were prepared for negative stain (Scripps Research Core Microscopy Facility, La Jolla, CA, USA). Briefly, samples were fixed by mixing the suspension 1:1 with 4% paraformaldehyde in 0.1 M phosphate buffer and incubated for 10 min at room temperature to preserve vesicular morphology. Carbon-coated copper grids (400 mesh) were glow-discharged and 10 µL of each sample was adsorbed for 2 min. Excess sample was wicked away and grids were negatively stained with 2% uranyl acetate for 2 min. Excess stains were wicked away and the grids were allowed to dry. Samples were analyzed at 120 kV with a ThermoFisher Scientific Talos L120C transmission electron microscope (ThermoFisher Scientific, Carlsbad, CA, USA) and images were acquired with a CETA 16M CMOS camera (ThermoFisher Scientific, Carlsbad, CA, USA).

4.7. MTT Assays

Cells (5 × 10³ cells per well) were seeded into pre-coated 96-well plates. ES-MSC were plated onto fibronectin and HUVECs, and IPFP-MSC onto collagen type 1 and allowed to adhere for 24 h. Cells were subjected to a range of particle concentrations from CCM or EVs. Control wells were subjected to concentrated medium that was never exposed to cells. All cells were cultured for 2–3 days before the addition of MTT reagent (5 mg/mL; ThermoFisher Scientific, Carlsbad, CA, USA) for 3 h. The medium was removed, and 100 μL of DMSO added and triturated to fully lyse cells before reading on a plate reader (Sunrise, Tecan, Männedorf, Switzerland) at 540 nm. Control OD measurements were used as a basis of comparison to the treatments for each cell type to detect the change in proliferation rate between each treatment.

4.8. Hypoxic Conditioning of Stem Cells

IPFP-MSC and ES-MSC were cultivated on collagen type I or FN coated T75cm² tissue culture flasks, respectively until 50% confluence before being placed into a hypoxic chamber (Stemcell Technologies, Cambridge, MA, USA). A gas mix consisting of 2% O₂, 5% CO₂ and 93% N₂ was perfused through the hypoxic chamber for 2 min to displace normal air and then sealed (following manufacturer’s instructions) and placed into at 37 °C incubator for 72 h. Following hypoxic treatment, the medium was changed, and the cells were cultured for another 3 days post hypoxia treatment where the medium was collected and considered as hypoxia conditioned medium. Cells of the same passage were cultured in parallel under normoxia for direct comparisons. The medium from both conditions was processed to produce CCM and stored at 80 °C until use.

4.9. Endothelial Network Formation in Fibrin Gels

HUVEC were cultured in flasks precoated with human collagen type I (Cell Adhere, StemCell Technologies, Cambridge, MA, USA) with endothelial medium (Cell Applications, San Diego, CA, USA). For subculture and use in experiments, the cells were detached using Trypsin-EDTA (Gibco). HUVECs (passage 3 to 5) were suspended in 2% fibrinogen (Bovine, Sigma) at a density of 4 × 10⁶ cells per mL and plated in 24 wells and crosslinked in thrombin (100 U/mL, Sigma) for 10 min and cultured in endothelial medium (Cell Applications, San Diego, CA, USA). After 3 or 7 days of culture, the encapsulated cells were incubated with Calcein-AM (1 mM; ThermoFisher Scientific, Carlsbad, CA, USA) to visualize live cells and cellular distributions and network formation. Several images (N = 4–6) from each condition were captured via fluorescence microscopy at a 10× magnification (CKX53, Evident Corporation, Tokyo, Japan). Quantitative analysis of endothelial network formation was performed using the Angiogenesis Analyzer plugin [63] in Fiji/ImageJ (version 1.54P) [64]. All output metrics generated by image analysis are reported in pixel units.

4.10. Human Cartilage and Tissue Explant Cultures

Osteochondral tissues (femoral condyles and patellar tissue) were acquired from patients (N = 4) undergoing total knee arthroplasty (The study was conducted in accordance with the Declaration of Helsinki and approved by Scripps Institutional Review Board). As described previously, cartilage was removed from subchondral bone using a scalpel. A dermal punch was used to harvest uniform 3.5 mm tissue explant discs which were equilibrated in DMEM + 1% CS in 24-well culture plates for 2–3 days. Selected wells were exposed to IL-1β (10 ng/mL; Peprotech, ThermoFisher Scientific, Carlsbad, CA, USA) for 3 days. DMMB assay was performed to measure GAG release in media to quantify the effects of IL-1β on cells. Media was changed and CCM or EVs (10 billion particles per well) were added to selected wells for 3 more days. Media was tested again using DMMB assay to monitor GAG release over time. The tissue was processed for RNA preservation (RNAlater, ThermoFisher Scientific, Carlsbad, CA, USA) and stored at −20 °C until processed for RNA extractions. Figure 5 shows the overview of treatments and their timing.

4.11. DMMB Assays

The release of glycosaminoglycans (GAGs) into the cell culture medium was measured using an adapted protocol of a dimethylmethylene blue (DMMB) assay [65]. Briefly, DMMB (Sigma-Aldrich, Burlington, MA, USA) was dissolved in ethanol then mixed with glycine (Sigma-Aldrich, Burlington, MA, USA), sodium chloride, 1M HCl, and distilled water to create the DMMB reagent. Bovine chondroitin sulfate (Sigma-Aldrich, Burlington, MA, USA) was used to generate a standard curve ranging between 0.5 mg/mL to 75 mg/mL. Medium from explant cultures was mixed into the DMMB reagent at a ratio of 1:19 (10 μL of medium with 190 mL of the DMMB reagent). The standard curve was also prepared using the same ratio. Samples were pipetted into a 96-well plate and immediately read on a plate reader (Tecan) at 540 nm. The standard curve and observed OD reading were used to calculate the concentration of GAGs in the medium. All readings were performed in triplicate.

4.12. Tissue RNA Extraction and Gene Expression Assays

Ex vivo cartilage samples were finely chopped with a scalpel and preserved in RNAlater (ThermoFisher Scientific, Carlsbad, CA, USA) following the manufacturer’s instructions by placing in 10 volumes of solution and stored at −20 °C. For RNA isolation, between 10 mg and 20 mg of cartilage fragments were weighed and suspended in 1 mL of Phenol/Qiazol (Qiagen, Germantown, MD, USA) on ice in a 15 mL polypropylene tube for tissue homogenization using a PowerGen Sawtooth device (ThermoFisher Scientific, Carlsbad, CA, USA). Following homogenization, the supernatant was removed and mixed with chloroform (Sigma-Aldrich, Burlington, MA, USA) at a ratio of 1 part chloroform to 5 parts Qiazol. The solution was mixed well and centrifuged at 12,500 rpm at 4 °C to perform a phase extraction of the RNA. The top layer containing RNA was removed and added to a fresh tube. To remove residual phenol, the RNA Clean & Concentrator kit (Zymo, Irvine, CA, USA) was used. RNA quality and concentration was measured on the Nanodrop One, then stored at −80 °C.

For gene expression analysis, High-Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to create cDNA. Pre-validated primer/probes were used (Applied Biosystems, Foster City, CA, USA) to detect GAPDH, IL-1β, IL-6, MMP-1, and MMP-3, using the Roche Light Cycler 96 Instrument (Roche, Basel, Switzerland). Gene expression was normalized to GAPDH and the ΔCt method was used as previously described [66].

4.13. Statistical Analysis

Comparisons between cell proliferation (MTT), angiogenic parameters (Fiji/ImageJ), glycosaminoglycan (DMMB) and gene expression (qPCR) were made using ANOVA or paired t-tests; p-values less than 0.05 were considered significant.