BMSC‐Exosomes Combined With TGF‐β1 Enhance Meniscal Fibrochondrocyte Function: Implications for Cartilage Repair

TL;DR

Combining exosomes from bone marrow stem cells with TGF-β1 improves the function of meniscal cells, offering a new treatment for cartilage injuries.

Contribution

The novel contribution is demonstrating the synergistic effect of BMSC-Exos and TGF-β1 on enhancing meniscal fibrochondrocyte function.

Findings

BMSC-Exos are crescent-shaped with an average size of 118 nm and express TSG101 protein.

BMSC-Exos combined with TGF-β1 significantly enhance proliferation, migration, and DNA content in meniscal fibrochondrocytes.

Fluorescently labeled BMSC-Exos are aggregated in meniscus fibrocartilage cells.

Abstract

Meniscal healing is often limited because adult meniscal fibrochondrocytes (MFCs) possess inherently low proliferative and reparative capacities. Bone marrow mesenchymal stem cell‐derived exosomes (BMSC‐Exos) have recently emerged as promising cell‐free therapeutics with regenerative potential, whereas transforming growth factor‐β1 (TGF‐β1) is a well‐established chondrogenic factor. In this study, we investigated the potential synergistic effects of BMSC‐Exos and TGF‐β1 on MFC proliferation, migration, and extracellular matrix synthesis in vitro.

To explore the effects of BMSC‐Exos combined with TGF‐β1 on MFCs and to investigate new approaches for treating meniscus injuries.

BMSC‐Exos were extracted by differential centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. The meniscus fibrochondrocytes were treated with BMSC‐Exos, TGF‐β1, and BMSC‐Exos + TGF‐β1 for 24 h. The distribution of fluorescently labeled BMSC‐Exos in meniscus fibrochondrocytes was observed by fluorescence microscopy. The effects of BMSC‐Exos, TGF‐β1, and BMSC‐Exos + TGF‐β1 on the proliferation and migration of meniscus fibrochondrocytes were evaluated by CCK‐8 assay, DNA quantification, cell migration assay, and cell scratch assay.

(1) BMSC‐Exos are crescent‐shaped, with an average particle size of approximately 118 nm, and express the specific protein TSG101. (2) The results of immunofluorescence staining showed that BMSC‐Exos were aggregated in the fibrocartilage cells of the meniscus. (3) Compared with the blank control group (CON group), the proliferation and migration abilities of the fibrocartilage cells of the meniscus in the three experimental groups were all enhanced, among which the BMSC‐Exos + TGF‐β1 group had the most significant effect. (4) The DNA content of the cells in the three experimental groups was all higher than that of the CON group, and the DNA content of the cells in the BMSC‐Exos + TGF‐β1 group was the highest (p < 0.001).

The combined application of BMSC‐Exos and TGF‐β1 can more effectively promote the proliferation and migration of meniscus fibrocartilage cells, and holds promise as a new approach for the treatment of meniscus injuries.

Figures

Graphical workflow of the experimental procedures described in section 2 (cell culture → exosome isolation/characterization → uptake imaging → treatments → functional assays → statistics).

Representative fluorescence images showing uptake of PKH26‐labeled BMSC‐Exos (red) by meniscal fibrochondrocytes stained with phalloidin (green, F‐actin) and DAPI (blue, nuclei). Merged images indicate predominant cytoplasmic localization of exosomes.

Transwell migration assay of MFCs in each treatment group. Representative crystal violet‐stained images and quantified migrated cell counts are shown. Statistics: one‐way ANOVA followed by Tukey’s post hoc test.  ∗ p < 0.05,  ∗∗ p < 0.01,  ∗∗∗ p < 0.001.

Scratch wound assay assessing MFC migration under each treatment (CON, BMSC‐Exos, TGF‐β1, and BMSC‐Exos + TGF‐β1). Representative images at 0 and 48 h and quantitative closure rates (ImageJ) are shown. Statistics: one‐way ANOVA followed by Tukey’s post hoc test.  ∗ p < 0.05,  ∗∗ p < 0.01,  ∗∗∗ p < 0.001.

Tables

Table 1. OD450 values of meniscal fibrochondrocytes in the CCK‐8 assay.

Group	CON	BMSC‐Exos	TGF‐β1	BMSC‐Exos + TGF‐β1
OD450	0.988 ± 0.005	1.229 ± 0.008	1.257 ± 0.006	1.324 ± 0.014

Table 2. Quantitative results of DNA in fibrocartilage cells of the meniscus (±s, n = 3).

Group	CON	BMSC‐Exos	TGF‐β1	BMSC‐Exos + TGF‐β1
DNA (ng/μL)	1.97 ± 0.037	2.24 ± 0.016	2.34 ± 0.026	2.59 ± 0.054

Full text

1. Introduction

Meniscal injury is among the most frequent orthopedic conditions and is a major risk factor for the development and progression of knee osteoarthritis [1]. Owing to the complex fibrocartilaginous architecture, low cellularity, and limited vascular supply—particularly in the inner avascular zone—spontaneous healing of the adult meniscus is often inadequate. Current management includes conservative care for symptom control and surgical options, such as partial meniscectomy or meniscal repair; however, these approaches often fail to restore native meniscal structure and long‐term joint biomechanics [2, 3].

Regenerative strategies that enhance meniscal cell proliferation, migration, and matrix synthesis have, therefore, become an active area of research. Bone marrow mesenchymal stem cells (BMSCs) have been explored for cartilage and fibrocartilage repair because of their self‐renewal capacity and multilineage potential [4]. Increasing evidence indicates that many therapeutic benefits of BMSCs are mediated by paracrine signaling rather than direct engraftment [5]. In particular, BMSC‐derived exosomes (BMSC‐Exos), a subset of small extracellular vesicles (~30–150 nm), carry proteins, lipids, and nucleic acids (e.g., miRNAs) and can regulate proliferation, migration, inflammation, and tissue remodeling [6, 7].

Transforming growth factor‐β1 (TGF‐β1) is a canonical chondrogenic and fibrochondrogenic cytokine that promotes proliferation and regulates extracellular matrix production via Smad2/3 signaling, including upregulation of cartilage‐associated genes such as COL2A1, ACAN, and SOX9 [8–10]. Nonetheless, free TGF‐β1 exhibits limited stability and a short half‐life in physiological environments, restricting effective bioavailability and motivating approaches that prolong its activity and local retention [11–13].

Combining growth factors with exosome‐based signaling has emerged as a plausible strategy to create a supportive microenvironment and amplify regenerative responses. Exosomes can protect labile biomolecules, promote cellular uptake, and provide complementary cues via their intrinsic cargo, which together may yield synergistic effects on target cells [14]. In cartilage‐related diseases, small extracellular vesicles have been shown to modulate chondrocyte survival and matrix homeostasis through vesicular miRNAs, highlighting the biological relevance of vesicle‐mediated communication for fibrocartilage repair [15–17].

In this study, we investigated whether BMSC‐Exos combined with TGF‐β1 synergistically enhance meniscal fibrochondrocytes (MFCs) functions relevant to repair. BMSC‐Exos were isolated and characterized by TEM, nanoparticle tracking analysis (NTA), and western blot of exosomal markers. Uptake of PKH26‐labeled exosomes by MFCs was evaluated by fluorescence microscopy. Functional outcomes were assessed by CCK‐8, DNA quantification, Transwell migration, and scratch wound assays.

By clarifying the combinatorial effects of BMSC‐Exos and TGF‐β1 on MFC proliferation and migration in vitro, this work provides an experimental foundation for developing cell‐free, growth‐factor–augmented strategies for meniscus regeneration. Limitations and necessary future work, including analysis of chondrogenic matrix markers, in vivo validation in meniscus–injury models, and mechanistic pathway interrogation, are discussed.

2. Materials and Methods

2.1. Materials

BMSCs (Guangzhou Saiye Biotechnology Co., Ltd.); TGF‐β1 reagent (Beijing Sollebao Technology Co., Ltd.); rabbit meniscus fibrocartilage cells (Shanghai Biyuntian Biotechnology Co., Ltd.); BCA protein quantification kit (Beijing Boaoxin Biotechnology Co., Ltd.); DMEM/F12 medium (Wuhan Bolede Biotechnology Engineering Co., Ltd.); fetal bovine serum (FBS; Shanghai Xiaopeng Biotechnology Co., Ltd.); BMSCs without exosomes complete culture medium (Guangzhou Saiye Biotechnology Co., Ltd.); CCK‐8 kit (Shanghai Biyuntian Biotechnology Co., Ltd.); DNA rapid detection kit (Shanghai Yixing Biotechnology Co., Ltd.); PKH‐26 kit (Shanghai Yixing Biotechnology Co., Ltd.); DAPI kit (Shanghai Biyuntian Biotechnology Co., Ltd.); Stropharia cordiformis peptide kit (Shanghai Biyuntian Biotechnology Co., Ltd.); phosphate buffer solution (Beijing Sollebao Technology Co., Ltd.); RIPA lysis buffer (Shanghai Yixing Biotechnology Co., Ltd.); protein loading buffer (Shanghai Yixing Biotechnology Co., Ltd.); rabbit anti‐CD9 monoclonal antibody (Shanghai Yixing Biotechnology Co., Ltd.); horseradish peroxidase‐labeled goat anti‐rabbit IgG (Shanghai Yixing Biotechnology Co., Ltd.); and ECL chemiluminescence kit (Shanghai Yixing Biotechnology Co., Ltd.).

2.2. Methods

Figure 1 presents a graphical overview of the experimental procedures outlined in Section 2.

Graphical workflow of the experimental procedures described in section 2 (cell culture → exosome isolation/characterization → uptake imaging → treatments → functional assays → statistics).

2.2.1. Extraction of BMSC‐Exos (by Differential Centrifugation Method)

Mix the DMEM/F12 medium with FBS in a ratio of 9:1 to prepare the conventional culture medium. Resuspend the frozen BMSCs in a 37°C water bath and culture them in the conventional medium. Pass them to the 4^th^ generation (P4). Then, replace it with the complete culture medium without exosomes and continue to culture for 24 h. Collect 15 mL of the supernatant, centrifuge at 3000 × g for 10 min to remove cell debris; add the obtained supernatant to the exosome extraction reagent, let it stand at 4°C for 2 h, then centrifuge at 10,000 × g for 60 min to precipitate exosomes. Discard the supernatant, gently resuspend the precipitate with 1 mL of phosphate‐buffered saline (PBS), and finally centrifuge at 12,000 × g for 2 min at 4°C to collect the supernatant, which is the extracted BMSC‐Exos.

2.2.2. Transmission Electron Microscopy (TEM) Observation of BMSC‐Exos

Place the filter paper in a petri dish and place a carbon‐coated copper mesh on it. Add 10 μL of BMSC‐Exos solution onto the copper mesh and incubate it at room temperature for 10 min to facilitate adsorption. Then, gently wash the copper mesh twice with sterile distilled water, and absorb the excess liquid with the filter paper. Add 10 μL of 2% acetic acid diuranate staining solution and let it stain for 10 min. After staining is complete, use the filter paper to absorb the excess staining solution and let the sample dry naturally. Place it under a TEM for observation of the exosome morphology.

2.2.3. Analysis of Particle Size and Concentration of BMSC‐Exos (NTA Method)

The extracted BMSC‐Exos samples were diluted with PBS solution to a concentration range of 1 × 10^7^–1 × 10^9^ particles/mL. The samples were analyzed using a nanoparticle tracking analyzer (ZetaView PMX 110, Particle Metrix, Germany) under a 405 nm laser light source. The particle size distribution, average particle size, and concentration of the exosomes were determined by analyzing the light scattering trajectories caused by Brownian motion, and the particle movement trajectory images were recorded.

2.2.4. Specific Protein Detection of BMSC‐Exos (Western Blot)

Exosomal proteins were lysed in RIPA buffer supplemented with protease inhibitors on ice, followed by centrifugation (12,000 × g, 5 min, and 4°C) to collect supernatants. Protein concentration was determined by BCA assay, and equal total protein amounts (typically 20–30 μg) were loaded per lane. Because exosomes do not consistently contain intracellular housekeeping proteins (e.g., GAPDH/β‐actin), no conventional loading control was used; instead, equal loading was verified by total‐protein normalization (BCA) and membrane staining (e.g., Ponceau S) prior to antibody incubation. Samples were mixed with 5× loading buffer, denatured at 100°C for 5 min, resolved by SDS‐PAGE (10% gel), transferred to PVDF membranes (0.22 μm), blocked with 5% skim milk for 1 h, and incubated overnight at 4°C with primary antibodies against CD9 (1:1000) and TSG101 (1:1000). After TBST washes, membranes were incubated with HRP‐conjugated secondary antibodies (1:5000) for 1 h at room temperature and developed using ECL for imaging.

2.2.5. Determination of Protein Concentration of BMSC‐Exos (BCA Method)

Protein concentration of BMSC‐Exos lysates was quantified using a commercial BCA kit according to the manufacturer’s standard protocol. Briefly, a bovine serum albumin (BSA) standard curve was generated, samples were measured in triplicate using a microplate reader, and concentrations were calculated from the standard curve.

2.2.6. Localization of BMSC‐Exos in Meniscal Fibrocartilage Cells (Immunofluorescence Method)

BMSC‐Exos were labeled with PKH26 according to the manufacturer’s instructions and washed to remove unbound dye. MFCs were seeded in 6‐well plates and incubated with PKH26‐labeled BMSC‐Exos (final protein concentration 100 μg/mL) for 24 h. Cells were then fixed and counterstained with phalloidin for F‐actin (cell boundary) and DAPI for nuclei. Fluorescence images were acquired on an inverted fluorescence microscope equipped with appropriate filter sets (TRITC for PKH26, FITC for phalloidin, and DAPI for nuclei). Exposure time, gain, and illumination intensity were kept constant across groups, and scale bars were added based on the microscope calibration.

Suspend the meniscus fibrocartilage cells in 6‐well plates, add 2 mL of regular culture medium to each well, and culture for 24 h until the cells adhere to the plate. Then, replace with the staining exosome culture medium and continue culturing for another 24 h. After removing the culture medium, rinse once with PBS, add the chrysophycin reagent to stain the cell membrane, incubate at room temperature for 10 min, and then rinse twice with PBS. Next, add the cell fixation solution for 30 min, wash twice with PBS, and finally add the DAPI reagent to stain the cell nuclei. After all the staining steps are completed, observe the localization and distribution of the exosomes within the cells using a fluorescence microscope.

2.2.7. Detection of Fibrocartilage Cell Proliferation Capacity of Meniscus (CCK‐8 Method)

Recover the frozen meniscus fibrocartilage cells and pass them on to the 4^th^ generation. Take 100 μL of the cell suspension and inoculate it into a 96‐well plate. The cell density is 5000 cells per well. Cultivate for 24 h until the cells adhere to the plate. The experiment is divided into four groups, with three replicate wells in each group. The culture medium is replaced with the following four types: (1) regular culture medium; (2) regular culture medium with 100 μg/mL BMSC‐Exos; (3) regular culture medium with 10 ng/mL TGF‐β1; and (4) regular culture medium with 100 μg/mL BMSC‐Exos + 10 ng/mL TGF‐β1. The cells in each group continue to be cultivated for 24 h. After the cultivation, add 10 μL of CCK‐8 reagent to each well and continue to incubate at 37°C for 2 h. Then, use an enzyme reader to measure the OD value of each well at a wavelength of 450 nm and record it.

2.2.8. Detection of DNA Content in Fibrocartilage Cells of Meniscus (Fluorescence Method)

DNA content was quantified using a fluorescence‐based dsDNA assay kit. MFCs were seeded in 96‐well plates (5000 cells/well) and treated for 24 h as described above. After washing with PBS, cells were lysed by papain digestion (125 mg/mL papain, 5 mM L‐cysteine, 100 mM Na2HPO4, 5 mM EDTA, and pH 6.2) at 60°C for 16 h. Aliquots of lysates were incubated with the kit’s dsDNA‐binding fluorescent dye according to the manufacturer’s instructions. Fluorescence was measured using a microplate fluorometer at the recommended excitation/emission settings, and DNA concentration was determined from a standard curve generated with serial dilutions of the provided DNA standard. DNA content was reported as ng/μL (or normalized to well/total protein where appropriate) and used as an additional quantitative readout of cell number/proliferation.

2.2.9. Detection of Meniscus Fibrocartilage Cell Migration Ability (Transwell Method)

The cell migration experiment was conducted using the Transwell chamber. Briefly, 1 × 10^4^ meniscal fibrocartilage cells were suspended in 100 μL of serum‐free medium and inoculated into the upper chamber of a 24‐well Transwell plates. The experiment was divided into four groups, with three replicate wells in each group. In the lower chamber, different amounts of medium were added: (1) regular medium; (2) regular medium with 100 μg/mL BMSC‐Exos; (3) regular medium with 10 ng/mL TGF‐β1; and (4) regular medium with 100 μg/mL BMSC‐Exos and 10 ng/mL TGF‐β1. After the cells were incubated in a 37°C incubator for 24 h, the non‐migrated cells were discarded. The cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet. After staining, the excess dye was washed off with PBS and the cells migrated to the lower chamber were observed and counted using an inverted microscope.

2.2.10. Detection of Meniscus Fibrocartilage Cell Migration Ability (Scratch Assay)

Remove the 6‐well plate and use a marker pen to draw a vertical line on the back of the plate as a reference for scratch positioning. Cultivate the fibrocartilage cells of the semilunar plate in the 6‐well plate, with a cell density of 5 × 10^5^ cells per well. Add 2 mL of regular culture medium to the well plate, then place the 6‐well plate in the constant temperature incubator for 24 h to allow the cells to grow into a monolayer and reach a fusion state of over 90%. Remove the 6‐well plate from the constant temperature incubator, discard the culture medium, and rinse it once with PBS solution. Use a sterile 200 μL pipette to draw a scratch vertically on the cell monolayer along the marker line. Rinse twice with 1 mL of PBS solution to remove the detached cells, take a photo under the microscope, and record the starting width of the scratch. At this time, set it as 0. There are four experimental groups, and each group is repeated three times. Change the culture medium of each group to 2 mL of regular culture medium, 2 mL of regular culture medium containing 100 μg/mL BMSC‐Exos, 2 mL of regular culture medium containing 10 ng/mL TGF‐β1, and 2 mL of regular culture medium containing 100 μg/mL BMSC‐Exos + 10 ng/mL TGF‐β1, respectively. Incubate in the constant temperature incubator. After 48 h, remove the 6‐well plate and take a photo under the microscope to record the healing situation of the scratch. Use the image analysis software ImageJ to measure the scratch width at different time points.

2.3. Statistical Analysis

Statistical analyses were performed using GraphPad Prism (version 8.0.2, GraphPad Software, USA). Data are presented as mean ± standard deviation (SD) from at least three independent experiments (n ≥ 3), unless otherwise specified. For comparisons among multiple groups, one‐way analysis of variance (ANOVA) was applied followed by Tukey’s multiple‐comparisons post hoc test. A p‐value < 0.05 was considered statistically significant and is denoted as  ^∗^ p < 0.05,  ^∗∗^ p < 0.01, and  ^∗∗∗^ p < 0.001.

3. Results

3.1. Characterization of the Physicochemical Properties and Specific Protein Expression of BMSC‐Exos

To verify the physicochemical properties and molecular characteristics of the extracted BMSC‐Exos, TEM, NTA, and western blotting were performed. TEM revealed typical vesicular/cup‐shaped structures with intact membranes (Figure 2A). NTA indicated that particle diameters were mainly distributed within 30–150 nm with an average size of approximately 118 nm (Figure 2B), consistent with exosome characteristics [18]. Western blotting further confirmed the presence of exosomal markers CD9 (~22 kDa) (Figure 1C). As exosomes lack stable intracellular housekeeping proteins, equal loading was ensured by BCA‐based total protein quantification and total‐protein staining of membranes rather than GAPDH/β‐actin controls.

Figure 2(A) TEM image showing typical cup‐shaped morphology of BMSC‐Exos. (B) NTA size distribution of BMSC‐Exos. (C) Western blot analysis of exosomal markers CD9.(A)(B)(C)

3.2. Uptake and Localization of BMSC‐Exos Within Meniscal Fibrocartilage Cells

To determine whether BMSC‐Exos can be internalized by MFCs, exosomes were labeled with PKH26 (red), while F‐actin and nuclei were counterstained with phalloidin (green) and DAPI (blue), respectively. Fluorescence microscopy demonstrated abundant punctate PKH26 signals within the cytoplasmic region delineated by phalloidin, with minimal overlap with the nuclear area, indicating successful uptake and cytoplasmic localization (Figure 3). Notably, cells retained an intact cytoskeletal outline and typical spindle/elongated morphology after incubation, suggesting no overt cytotoxicity or gross morphological disruption under the tested conditions.

Representative fluorescence images showing uptake of PKH26‐labeled BMSC‐Exos (red) by meniscal fibrochondrocytes stained with phalloidin (green, F‐actin) and DAPI (blue, nuclei). Merged images indicate predominant cytoplasmic localization of exosomes.

3.3. BMSC‐Exos Combined With TGF‐β1 Significantly Promotes the Proliferation of Meniscus Fibrocartilage Cells

To evaluate the effects of BMSC‐Exos and TGF‐β1 on the proliferation ability of meniscus fibrocartilage cells, this study used the CCK‐8 method to detect the proliferation status of cells in each experimental group. A total of four groups were set up in the experiment: the blank control group (CON group), the BMSC‐Exos group, the TGF‐β1 group, and the combined treatment group of BMSC‐Exos + TGF‐β1. The results showed that compared with the CON group, the OD_450_ values of the BMSC‐Exos group and the TGF‐β1 group were significantly increased (p < 0.01), indicating that both could effectively promote cell proliferation. The combined treatment group (BMSC‐Exos + TGF‐β1) had the highest OD_450_ value and the most significant proliferation‐promoting effect, with a statistically significant difference compared to the single treatment groups (p < 0.001) (Figure 4A, Table 1).

Figure 4(A) CCK‐8 assay showing cell viability/proliferation under different treatments (CON, BMSC‐Exos, TGF‐β1, and BMSC‐Exos + TGF‐β1). (B) Fluorescence‐based dsDNA quantification of cells in each group. Statistics: one‐way ANOVA followed by Tukey’s post hoc test.  ^∗^ p < 0.05,  ^∗∗^ p < 0.01,  ^∗∗∗^ p < 0.001.(A)(B)

3.4. Quantitative Analysis of DNA in Fibrocartilage Cells of the Meniscus

To further corroborate proliferation outcomes, cellular DNA content was quantified using a fluorescence‐based dsDNA assay. Fluorescence signals were converted to DNA concentration using a DNA standard curve prepared in parallel, thereby providing a quantitative surrogate for cell number.

Compared with the CON group, DNA content increased significantly in both the BMSC‐Exos and TGF‐β1 groups (p < 0.01). The combined BMSC‐Exos + TGF‐β1 group showed the highest DNA content and was significantly higher than either single‐treatment group (p < 0.001) (Figure 4B, Table 2).

Together with the CCK‐8 results, these data indicate that BMSC‐Exos and TGF‐β1 each promote MFC proliferation, and that their combination produces a synergistic enhancement.

3.5. BMSC‐Exos Combined With TGF‐β1 Enhances the Migration Ability of Meniscal Fibrocartilage Cells (Transwell Assay)

To evaluate the effects of BMSC‐Exos and TGF‐β1 on the migration ability of meniscus fibrocartilage cells, a Transwell chamber experiment was performed to detect the cell migration under different treatment conditions. The experiment was set up with four groups: CON group, BMSC‐Exos group, TGF‐β1 group, and BMSC‐Exos + TGF‐β1 group.

Both the BMSC‐Exos and TGF‐β1 groups exhibited significantly greater numbers of cells migrating to the lower chamber compared with the control group (p < 0.01), indicating that each treatment effectively enhanced MFC migratory capacity. The combined treatment group of BMSC‐Exos + TGF‐β1 had the most migrating cells and significantly enhanced the migration ability, with a significant difference compared to the single treatment groups (p < 0.001) (Figure 4).

These findings demonstrate that both BMSC‐Exos and TGF‐β1 independently enhance the migratory capacity of MFCs, while their combined application exerts a markedly synergistic effect. Such synergy may substantially improve the cellular responses required for effective meniscal repair.

3.6. BMSC‐Exos Combined With TGF‐β1 Further Enhanced the Migration Ability of Meniscal Fibrocartilage Cells (Scratch Assay)

To further verify the effects of BMSC‐Exos and TGF‐β1 on the migration ability of meniscus fibrocartilage cells, the scratch assay was used to evaluate the healing status of cells in each group within 48 h. The experimental design was consistent with the Transwell experiment, including the CON group, BMSC‐Exos group, TGF‐β1 group, and the combined BMSC‐Exos + TGF‐β1 group.

Microscopic observation showed that the scratch healing in the CON group was the least, while the scratch healing in the BMSC‐Exos group and the TGF‐β1 group was to some extent. The combined treatment group showed a significantly reduced scratch width and the most significant healing effect (Figure 5). Quantitative analysis of scratch area changes using ImageJ software also further confirmed the above observations. Compared with the CON group, the scratch healing rates of all experimental groups were significantly increased (p < 0.01), and the migration ability of the BMSC‐Exos + TGF‐β1 group was the strongest, with a highly statistically significant difference (p < 0.001).

Transwell migration assay of MFCs in each treatment group. Representative crystal violet‐stained images and quantified migrated cell counts are shown. Statistics: one‐way ANOVA followed by Tukey’s post hoc test.  ∗ p < 0.05,  ∗∗ p < 0.01,  ∗∗∗ p < 0.001.

The results indicate that both BMSC‐Exos and TGF‐β1 can promote the migration of meniscus fibrocartilage cells, and the combined application has a better effect in promoting scratch healing. (Figure 6)

Scratch wound assay assessing MFC migration under each treatment (CON, BMSC‐Exos, TGF‐β1, and BMSC‐Exos + TGF‐β1). Representative images at 0 and 48 h and quantitative closure rates (ImageJ) are shown. Statistics: one‐way ANOVA followed by Tukey’s post hoc test.  ∗ p < 0.05,  ∗∗ p < 0.01,  ∗∗∗ p < 0.001.

4. Discussion

Meniscus injury is a common joint cartilage disorder. The treatment methods for it mainly include conservative treatment and surgical intervention [19]. Although conservative treatment can alleviate pain and control symptoms, it is usually difficult to reverse the degeneration of cartilage [20, 21]. Surgical treatments, such as meniscus resection or joint replacement, do have certain efficacy in improving the force line and alleviating symptoms. However, they still have problems such as significant trauma, limited repair capabilities, and higher risks of postoperative complications [22]. Therefore, it is particularly important to explore new treatment strategies that have better biocompatibility and stronger repair capabilities.

In recent years, with the development of tissue engineering and regenerative medicine, the potential of MSCs and their derived exosomes in cartilage repair has received widespread attention [23]. Exosomes are a type of extracellular vesicles with a diameter of 30–150 nm. They have a typical double‐layered membrane structure and are rich in proteins, lipids, and nucleic acids. They can mediate intercellular signal transmission and regulate cell proliferation, migration, differentiation, and apoptosis [24–26]. Among them, BMSC‐Exos has significant advantages in the field of cartilage injury repair due to its low immunogenicity and good plasticity [27].

TGF‐β1 is a key member of the TGF‐β family and is widely involved in the development and regeneration process of fibrocartilage [28]. Studies have shown that TGF‐β1 not only promotes the proliferation of fibrocartilage cells, but also enhances the expression of type II collagen and glycosaminoglycans [29]; and by activating the Smad2/3 signaling pathway, it upregulates the expression of cartilage marker genes such as Col2a1, Sox9, and Acan [30]. Furthermore, the absence of the TGF‐β1 signal has been proven to be closely related to the degeneration of articular cartilage [31]. Therefore, TGF‐β1 is of great significance in maintaining the integrity of cartilage and slowing down the degeneration of cartilage [32, 33]. However, its short half‐life and unstable nature have limited the wide application of TGF‐β1 in clinical practice [34].

This study verified the combined effect of BMSC‐Exos and TGF‐β1 on meniscal fibrocartilage cells. The BMSC‐Exos were characterized and identified by methods such as TEM, NTA, and western blot. The results showed that they had the typical morphology of exosomes and the expression of characteristic proteins. The PKH26 staining experiment confirmed that BMSC‐Exos could be phagocytosed by meniscal fibrocartilage cells and localized in the cytoplasm, laying the foundation for their subsequent functional expression.

In the cell function experiments, both BMSC‐Exos and TGF‐β1 significantly enhanced MFC proliferation (CCK‐8 and dsDNA quantification) and migration (Transwell and scratch assays). Importantly, the combined BMSC‐Exos + TGF‐β1 group consistently produced the strongest responses, suggesting a possible synergistic interaction. In addition to quantitative endpoints, fluorescence and phase‐contrast observations indicated preserved cell morphology (intact F‐actin outlines and typical spindle/elongated fibrochondrocyte shapes) after exosome exposure, supporting biocompatibility under the tested conditions and consistent with the enhanced migratory phenotype.

From a morphological perspective, migratory MFCs typically exhibit increased spreading, polarized protrusions, and aligned actin stress fibers. Future work will include systematic morphometric analysis (cell area, aspect ratio, and circularity) and cytoskeletal quantification (F‐actin intensity and focal adhesion markers) to objectively link the observed functional improvements to changes in cell shape and motility programs.

It is worth emphasizing that the combined use of BMSC‐Exos and TGF‐β1 may provide a more stable extracellular microenvironment, which helps to prolong the biological activity time of TGF‐β1, and at the same time enhances the activation effect of its downstream signaling pathways, thereby further improving the repair efficiency of damaged tissues [35]. This combined application strategy is expected to become a new direction in the treatment of meniscus injuries and has promising application prospects.

Although these findings provide strong evidence for the possible synergistic effect between BMSC‐Exos and TGF‐β1, several limitations remain. (1) Cartilage/fibrocartilage formation was not directly assessed; future studies will quantify chondrogenic and fibrochondrogenic markers (e.g., COL2A1, ACAN, SOX9, and COL1A1) by qPCR/immunoblotting and evaluate matrix deposition by alcian blue/safranin O staining and immunofluorescence. (2) This study focused on in vitro assays and did not include in vivo meniscus‐injury repair; subsequent work will test therapeutic efficacy in established animal models (e.g., rat or rabbit meniscal defect/tear models) using histological scoring, biomechanical testing, and imaging‐based evaluation. (3) The underlying mechanism was not interrogated. Candidate pathways include TGF‐β/Smad2/3 signaling and exosome cargo–mediated regulation (miRNAs/proteins) that influence proliferation and migration. Future experiments will combine pathway inhibitors/siRNA (e.g., TGF‐β receptor blockade) with exosome cargo profiling to delineate causal mechanisms. Addressing these points will strengthen the translational relevance and provide a more complete mechanistic story.

Author Contributions

Puzhen Song: data curation, formal analysis, investigation, methodology, visualization, writing – original draft, writing – review and editing. Hebin Ma: data curation, formal analysis, investigation, methodology. Hongguang Chen: formal analysis, investigation. Yuanbo Zhou: data curation, editing.

Funding

This work was supported by the National Natural Science Foundation of China (Grant 82072451) and the Beijing Municipal Science and Technology Commission, Administrative Commission of Zhongguancun Science Park (Grant Z221100007422014).

Disclosure

All claims expressed in this article are solely those of the authors and do not necessarily reflect the views of their affiliated institutions, the publisher, the editors, or the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Ethics Statement

All animal experiments were approved by the Animal Care and Use Committee of Beijing Keyu Animal Center (Approval Number: KY20240328011) and were conducted in accordance with institutional guidelines and national regulations on the ethical treatment of laboratory animals. All procedures complied with the ARRIVE guidelines and were designed to minimize animal suffering.

Conflicts of Interest

The authors declare no conflicts of Interest.