Comparative Efficacy of Exosomes Derived from Different Mesenchymal Stem Cell Sources in Osteoarthritis Models: An In Vitro and Ex Vivo Analysis

Abstract

Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their clinical application remains constrained by several inherent limitations. This study explores a cell-free alternative by investigating the therapeutic potential of exosomes derived from bone marrow (BMSCs), adipose tissue (ADSCs), and umbilical cord (UMSCs) MSCs in mitigating OA pathogenesis, utilizing both in vitro and ex vivo models. Exosomes from each MSC source were isolated and characterized through nanoparticle tracking analysis, transmission electron microscopy, and Western blotting to confirm their identity and purity. Subsequently, their chondroprotective, anti-inflammatory, and regenerative properties were systematically assessed through evaluations of cell viability, expression profiles of inflammatory and chondroprotective markers, and chondrocyte migration assays. The results demonstrate that all three types of MSC-derived exosomes (MSC-Exos) exhibit low cytotoxicity while significantly suppressing proinflammatory markers and enhancing the expression of chondroprotective genes. Notably, BMSC-Exos and UMSC-Exos displayed superior efficacy in attenuating inflammation, promoting cartilage protection, and inhibiting chondrocyte apoptosis. Furthermore, all MSC-Exos markedly enhanced chondrocyte motility, a critical component of cartilage repair. Collectively, these findings support the therapeutic promise of MSC-Exos, particularly those derived from BMSCs and UMSCs, as a targeted, cell-free approach for the treatment of OA compared to ADSCs. By modulating inflammation, promoting cartilage regeneration, and preventing chondrocyte apoptosis, MSC-Exos may serve as a viable and scalable alternative to current MSC-based therapies for this widespread degenerative disease.

Keywords: MSC-derived exosomes; anti-inflammation; ex vivo OA model; osteoarthritis; regenerative medicine.

Figure 1

(A) Aqueous two-phase system (visualized by adding Coomassie Brilliant Blue R-250). (B–D) The size distribution of BMSC-Exos, ADSC-Exos, and UMSC-Exos, as determined by nanoparticle tracking analysis. (E) Phase diagram of PEG/DEX ATPS. The two-phase forms when system concentration is above the binodal curve. (F–H) Transmission electron microscopy images of BMSC-Exos, ADSC-Exos, and UMSC-Exos. (I) Western blot expression of the exosomal markers CD81, CD63, and ALIX.

Figure 2

(A) Cell viability as a function of BMSC-Exos. (B) Cell viability as a function of ADSC-Exos. (C) Cell viability as a function of UMSC-Exos—all in decreasing concentrations from 1000 μg/mL to 1.0 μg/mL. Data are shown as the mean ± standard deviation (n = 3). The data depicted represent significance at p < 0.05 in all data sets.

Figure 3

Effects of BMSC-Exos, ADSC-Exos, and UMSC-Exos on MAPK pathways (p38, pERK, and pJNK) and NF-κB (pp65) in chondrocytes. (A) Protein expression levels of BMSC-Exos, ADSC-Exos, and UMSC-Exos on IL-1β treated chondrocytes and (B) quantitative analysis. Data are presented as the mean ± standard deviation (n = 3). ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the control group.

Figure 4

Effects of BMSC-Exos, ADSC-Exos, and UMSC-Exos on inflammation mediators (A) Protein expression levels of MMP-3, MMP-13, IL-6, TNF-α, and COX-2 detected via Western blot analysis and (B) quantitative analysis. Data are presented as the mean ± standard deviation (n = 3). * p < 0.01, ** p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to the control group.

Figure 5

Effects of BMSC-Exos, ADSC-Exos, and UMSC-Exos on chondroprotective markers. (A) Protein expression levels and quantitative analysis of ACAN and COL-2 were found through Western blot analysis and (B) quantitative analysis. Data are presented as the mean ± standard deviation (n = 3). ns = Non significant, * p < 0.01, ** p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to the control group.

Figure 6

Effects of BMSC-Exos, ADSC-Exos, and UMSC-Exos on apoptotic markers (A) Protein expression levels and quantitative analysis of Cas-9 were found through Western blot analysis and (B) quantitative analysis. Data are presented as the mean ± standard deviation (n = 3). *** p < 0.001 and **** p < 0.0001 compared to the control group.

Figure 7

(A) Migration rate in an in vitro model of osteoarthritis. The scratch wound assay demonstrates the migration rates of osteoarthritic chondrocytes treated with IL-1β (10 ng/mL) and various exosomes (BMSC-Exos, ADSC-Exos, and UMSC-Exos). Scale bar = 100 μm. (B) followed by migration rate % quantitative analysis. BMSC-Exos (5 μg/mL) significantly promoted the migration of osteoarthritis chondrocytes induced by IL-1β compared with control group at both 24 h and 36 h. Data are presented as the mean ± standard deviation (n = 3). * p < 0.01, ** p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to the control group.

Figure 8

H&E staining of ex vivo model osteoarthritis. Tissues were pretreated with IL-1β (10 ng/mL) and various exosomes (BMSC-Exos, ADSC-Exos, and UMSC-Exos), depicting irregularity in the cartilage structure.

Figure 9

Safranin O staining of ex vivo model of osteoarthritis. Tissues were pretreated with IL-1β (10 ng/mL) and various exosomes (BMSC-Exos, ADSC-Exos, and UMSC-Exos), depicting the proteoglycan GAG content in the tissues. Red color represents the GAG distribution and green stains the cytoplasm.

Figure 10

Collagen level of cartilage tissue evaluated by Picrosirius Red assay. Tissues were pretreated with IL-1β (10 ng/mL) and various exosomes (BMSC-Exos, ADSC-Exos, and UMSC-Exos). Collagen distribution is depicted in red, with pink areas indicating reduced collagen content. Yellow regions highlight cytoplasm and muscle fibers. Scale bar = 100 μm.

Figure 11

Schematic representation of the in vitro and ex vivo models of osteoarthritis.

Figure 12

Ex vivo inflammatory OA model using human knee osteochondral specimens. (A) Initial cartilage diameter measurement (6–8 mm) with a cartilage thickness of (1–3 mm) of day 0 specimens. (B) Experimental groups: control, IL-1β-stimulated, and various exosome treatments. (C) Timeline of the ex vivo inflammatory OA model experiment.