The method for obtaining antler stem cells (ASCs) followed the procedures outlined by Wang, and previous studies have identified the cells [17]. Specifically, antler tissue was collected from three 8-month-old sika deer immediately post-slaughter, minced into small pieces, and digested in dulbecco’s modified eagle medium (DMEM) containing 150 U/mL collagenase (Gibco, 11965092, Grand Island, NE, USA). ASCs were successfully isolated through this process and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, 10100147C, USA) and 1% penicillin-streptomycin solution (Gibco, 15140163, USA) at 37 °C, under saturated humidity with 5% CO₂ concentration. The study also utilized other cell lines obtained from Procell (Wuhan, China), including rat bone marrow mesenchymal stem cells (rat-BMSC, product number: CP-R131), human adipose-derived stem cells (hADSC, product number: CP-H202), and rat chondrocytes (product number: CP-R087). All cells were tested for mycoplasma to ensure that they were not contaminated with mycoplasma, and human cells were identified by short tandem repeats (STR).

Cells were seeded in T75 cell culture flasks and expanded in DMEM medium supplemented with 10% FBS (Exos removed) at 37 °C and 5% CO₂. Once the cell density reached 80%, the medium was collected and centrifuged at 4 °C, 300 g for 10 minutes to remove cells. The supernatant was collected, transferred to a new centrifuge tube, and centrifuged at 4 °C, 2000 g for 20 minutes to remove cell debris. The supernatant was then filtered through a 0.22 µm filter (Merck, SVGP010, South San Francisco, CA, USA) and centrifuged at 4 °C, 10,000 g for 30 minutes. The resulting supernatant was transferred to an ultracentrifuge tube and ultra-centrifuged at 4 °C, 100,000 g for 60 minutes. After centrifugation, the supernatant was discarded, and the exosomes were resuspended in PBS. The exosomes were centrifuged again at 100,000 g for 60 minutes at 4 °C. After resuspension in Phosphate Buffered Saline (PBS), the exosomes were transferred into sterile eppendorf (EP) tubes and stored in a refrigerator at –80 °C for subsequent experiments

Eight-week-old male Wistar rats (250 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Shenyang, China). All experiments followed the guidelines of the Ethics Committee for Animal Experiments of the Institute of Special Animal and Plant Sciences. To create cartilage defects, the knee joints were exposed by incising the skin and muscle, and full-thickness cylindrical defects (2 mm in diameter, 1 mm in depth) were drilled. Thirty rats were randomly assigned to five groups: (1) Control (Untreated), (2) DMEM (negative control), (3) ASC-Exos (200 µg/mL), (4) hADSC-Exos (200 µg/mL, positive control), and (5) Sham -operated. In groups 1, 2, 3, and 4, cartilage defects were created. After anesthesia with 1.5% sodium pentobarbital (30 mg/kg), exosomes mixed with hydrogel (SunP Gel G1, Shangpu Bio, Shanghai, China) were implanted into the defects of groups 3 and 4. DMEM mixed with hydrogel was implanted into the defects of group 2. Postoperative antibiotics (gentamicin 80,000 U, intramuscularly, once daily for three days) were administered to prevent infection. Samples were collected after 6 weeks.

At the end of the experiment, the rats were euthanized by CO₂, and the muscle tissues around the rat knee joints were removed. The right and left knee joints of six rats from each group were selected. Photos of the rat knee joint cartilage were taken with a high-resolution camera (Canon, Tokyo, Japan) and the overall morphology repair effect was evaluated according to the International Cartilage Repair Society (ICRS) morphological assessment criteria [19].

After embedding the tissue, sections were prepared and stained with hematoxylin (Beyotime, C0107, China) for 10 minutes. The excess stain was subsequently rinsed off for 10 minutes, followed by a 2-minute rinse with distilled water. The sections were then stained with eosin (Beyotime, C0109, China) for 15 seconds. For mounting, the sections were sequentially placed in 70%, 80%, 90% ethanol, and then in anhydrous ethanol for 15 seconds each. Subsequently, they were placed in xylene I and xylene II for 10 minutes each, and finally mounted with neutral gum.

For Safranin-O/fast green staining (Solarbio, G1371, Beijing, China), the sections were rehydrated and stained with Weigert’s staining solution for 3–5 minutes, differentiated with a neutral solution for 15 seconds, and washed with distilled water for 1 minute. Subsequently, the sections were immersed in fast green staining solution for 5 minutes, followed by a 2-minute wash with distilled water. Next, the sections were immersed in Safranin O staining solution for 2 minutes, followed by another 1-minute wash with distilled water. To remove excess fast green, the sections were rinsed with acetic acid solution for 2 minutes and washed with distilled water for 2 minutes. Finally, the sections were briefly treated with 95% ethanol for 2 seconds, dehydrated with anhydrous ethanol for 2 seconds, clarified with xylene for 10 minutes, and sealed with neutral resin.

Histological observations were evaluated based on the International Society for Cartilage Repair’s scoring criteria for cartilage defect repair [19].

Washed the tissue sections in PBS for 5 minutes, followed by replacing PBS with 1% antigen retrieval solution. Heated in a water bath for 8 minutes. Rinsed the cooled sections with PBS three times for 3 minutes each. Encircled the tissue with an immunohistochemical pen. Follow-up experiments with ready-to-use rapid immunohistochemistry MaxVision TM kits (MXB, KIT-5001, Shanghai, China). Applied Reagent A and Reagent B, then added 1 drop of Collagen II (COLII) primary antibody (1:200, GB11021, Servicebio, Wuhan, China) to each section and incubated overnight at 4 °C. Subsequently, applied Reagent C and Reagent D, followed by incubating with 3,3${}^{\prime }$-Diaminobenzidine Tetrahydrochloride Solution (DAB) solution on each section for 10 minutes within a 15-minute period. Rinsed with tap water and counterstain with hematoxylin for 15 minutes, then rinsed with tap water until the sections returned to blue color. Dehydrated the sections using a series of ethanol gradients, cleared with xylene, and sealed with neutral resin.

Added 5 µL of PKH26 fluorescent (Merck, PKH26PCL, USA) dye to the exosome suspension, mixed thoroughly, and incubated at 37 °C for 10 minutes. Centrifuged the cells at 300 g for 5 minutes to collected the cell pellet. Rinsed the cells several times with PBS, then resuspended them in serum-free medium and seeded them into a culture flask. Once the cell density in 6-well plates reached 60%, added 100 µL of fluorescent-stained exosomes to each well. Gently agitated the plates to ensure even distribution of exosomes, then incubated for 12 hours. Stained the cell nuclei with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) for 10 minutes, washed with PBS three times, and examined the uptake of exosomes using an inverted fluorescence microscope. Captured images of the findings.

Cell counting kit-8 (CCK-8) assay: Rat chondrocytes and MSCs were thawed and cultured in petri dishes until they reached 80% confluence. The cells were then harvested, counted, and seeded into 96-well plates at a density of 3000 cells per well. Cultures were maintained at 37 °C with 5% CO₂ and saturated humidity for 5 days. Cell proliferation was measured daily using the CCK-8 assay. For the assay, 10 µL of CCK-8 (Beyotime, C0038, China) was added to each well and incubated with the cells for 2 hours. Proliferation was quantified by measuring the absorbance at 450 nm using an Enzyme-Linked Immunosorbnent Assay (ELISA) plate reader. Data are presented as mean $\pm$ standard deviation from three independent replicates.

5-Ethynyl-2${}^{\prime }$-Deoxyuridine (EdU) method: Rat chondrocytes and MSCs were inoculated in 24-well plates. Equal volumes of preheated EdU working solution (Beyotime, C0071S, China) were added to each well and incubated for 2 hours. After incubation, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Following fixation, cells were washed three times with 3% Bovine Serum Albumin (BSA) in PBS, with each wash lasting 5 minutes. Subsequently, 1 mL of 0.3% Triton X-100 was added to each well for 15 minutes, followed by three additional washes with the washing solution, each for 5 minutes. Hoechst 33342 (1000$\times$ dilution in PBS, 1:1000) was then added to each well and incubated in the dark for 10 minutes, followed by three washes with the washing solution, each for 5 minutes.

Culture inserts (Ibidi, 80209, Munich, Germany) were placed in each well, and 70 µL of 3 $\times$ 10⁵ rat MSCs were seeded. The cells were cultured until they reached 80% confluence, then detached, suspended, and incubated at 37 °C. Upon reaching 100% confluence, the culture inserts were removed. After a 12-hour incubation period, ASC-Exos, hADSC-Exos, and DMEM were introduced into the wells. Cell migration was subsequently monitored using a microscope (Olympus, Tokyo, Japan).

We co-cultured exosomes with chondrocytes for 7 generations to observe the exosomes’ impact on maintaining chondrocyte markers. RNA and protein were extracted from passages 1, 4, and 7 for subsequent experiments.

After exosome was stored in liquid nitrogen, it was sent to Guangzhou Kidio Biotechnology Co. for Data-Independent Acquisition (DIA) proteomic quantitative analysis.

Statistical analyses were performed using Prism 8 (GraphPad Software, GraphPad Software, Inc., San Diego, CA, USA). All data were assessed for normal distribution. One-way ANOVA with Dunnett’s multiple comparison test was employed for comparisons involving more than more than two groups. Repeated measures ANOVA was used for data collected at various time points in the CCK-8 assay. All quantitative data are presented as mean $\pm$ SD from at least three independent experiments. Statistical significance was set at p 0.05.

Electron microscopy results showed that the particle size range of both types of exosomes predominantly ranged between 50–100 nm range (Fig. 1A). Western blotting detected positive expression of Alix, CD63, and TSG101, and negative expression of GM130 (Fig. 1B), which are characteristic surface markers of hADSC-Exos and ASC-Exos. The presence of these specific markers, coupled with the observed size distribution, confirms the identity of the isolated exosomes, thereby ensuring the reliability and purity of the sample isolation process. Furthermore, NTA analysis revealed that both types of exosomes primarily exhibited particle sizes in the range of 50–100 nm, further validating their purity (Fig. 1C).

Fig. 1.

Identification of exosomes derived from antler stem cells (ASC-Exos) and exosomes derived from human adipose-derived stem cells (hADSC-Exos). (A) Electron microscopy results depicting the particle size distribution of ASC-Exos and hADSC-Exos, predominantly within the 50–100 nm range. Scale bar = 100 nm. (B) Western blot assay results demonstrating positive expression of the specific surface markers Alix, CD63, TSG101 and GM130. (C) The results of Nanoparticle Tracking Analysis (NTA) detection for Exosomes’ particle size. (D) Assessment of ASC-Exos uptake by Chondrocytes and Rat Bone Marrow Stromal Cells, conducted in triplicate (n = 3). Scale bar = 200 µm or 50 µm. TSG101, Tumor Susceptibility Gene 101; GM130, Golgi Matrix Protein 130.

To investigate the uptake of ASC-Exos by chondrocytes and rat BMSCs, ASC-Exos were labeled with PKH26 and co-cultured with chondrocytes and rat BMSCs for 24 hours. The uptake of ASC-Exos was subsequently visualized using a fluorescence microscope. A significant amount of red granular material was observed aggregated around the nuclei of both chondrocytes and rat BMSCs, indicating successful uptake of ASC-Exos after 24 hours of co-culture (Fig. 1D).

To achieve sustained in vivo release of ASC-Exos, hydrogels containing ASC-Exos (50 µg/mL), hADSC-Exos, and DMEM were prepared for topical administration to cartilage defects. After 6 weeks, samples were macroscopically assessed to evaluate the impact of ASC-Exos on cartilage repair (Fig. 2A). In the blank control group, the drilled defects exhibited less regenerated tissue with a rough surface and a clear boundary between the new and original cartilage. In the DMEM group, new tissue filled the defect, but the surface remained rough, and there was an obvious interface between the new and original cartilage. The hADSC-Exos group showed new tissue with a relatively smooth surface, but with a distinct boundary between the new and original cartilage. In contrast, the ASC-Exos group displayed new tissue similar to the sham-operated group, with a complete and smooth surface that integrated well with the surrounding cartilage. According to the International Cartilage Repair Score (ICRS) criteria, the ASC-Exos group achieved a higher macroscopic score compared to the hADSC-Exos, DMEM, and blank control groups, and approached that of the sham-operated group (Fig. 2B).

Fig. 2.

Histologic evaluation of cartilage defect. (A) Macroscopic observation of cartilage defects 6 weeks after surgery. (B) Macroscopic ICRS scores of different groups. Data are expressed as mean $\pm$ SD of three replicates. Statistical analysis was performed using a t-test for each group to compare the mean scores. n = 3, ****p 0.0001. (C) Safranin O/fast green staining of repaired cartilage. Scale bar = 250 µm. (D) ICRS visual histology scores of different groups. Data are expressed as mean $\pm$ SD of three replicates. Statistical analysis was conducted using a t-test for each group to compare the mean scores. n = 3, ****p 0.0001. (E) Immunohistochemical staining of COL II. Scale bar = 250 µm. (F) Quantitative analysis (Positive area and Staining intensity) of immunohistochemical staining of collagen II. n = 3, ***p 0.001, ****p 0.0001. ns: there was no significant effect observed between the groups (p $>$ 0.05). ICRS, International Cartilage Repair Society; DMEM, Dulbecco’s Modified Eagle Medium.

For histological evaluation, Safranin O/fast green staining was employed to assess cartilage regeneration (Fig. 2C). The blank control and DMEM groups exhibited the most severe cartilage defects, characterized by irregular and rough surfaces. In the hADSC-Exos group, the new tissue almost completely filled the defects but showed little red staining, indicating a non-smooth surface. Conversely, the regenerated cartilage in the ASC-Exos group displayed intense red staining and a smooth surface, resembling natural cartilage in the sham-operated group. According to the ICRS for histology, the ASC-Exos group achieved higher scores compared to the hADSC-Exos, DMEM, and blank control groups, and approached scores observed in the sham-operated group (Fig. 2D). These findings suggest that ASC-Exos effectively promoted cartilage regeneration.

Immunohistochemical staining was employed to assess the expression of COL II in the newly formed tissues. The ASC-Exos group exhibited strong positive staining for type II collagen, similar to the sham-operated group. In contrast, the DMEM and hADSC-Exos groups showed comparatively weaker staining for type II collagen. Surprisingly, the intensity of type II collagen staining was even higher in the ASC-Exos group than in the sham group. On the other hand, the area of type II collagen positivity in the ASC-Exos group was not significantly different from that in the hADSC-Exos group, but was significantly higher than that in the DMEM group. This enhanced expression of type II collagen in the ASC-Exos group suggests that ASC-Exos promoted the formation of the extracellular matrix (Fig. 2E,F).

The CCK-8 assay results demonstrated that the ASC-Exos group exhibited faster proliferation compared to the hADSC-Exos and the DMEM groups in both cell types (Fig. 3A,B). The findings indicate that ASC-Exos significantly enhance the proliferation of chondrocytes and rat BMSCs. Using the EdU detection kit and inverted fluorescence microscopy, we observed that ASC-Exos treated both chondrocytes and rat BMSCs had the highest percentage of EdU-positive cells compared to the two control groups, respectively. Conversely, the DMEM group displayed the lowest percentage of EdU-positive cells. These results indicate that ASC-Exos significantly increase the proliferation potential of chondrocytes and rat BMSCs, surpassing the effect of hADSC-Exos (Fig. 3C).

Fig. 3.

ASC-exosomes promote the proliferation of rat chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs). (A) Effect of ASC-Exos on the proliferation of chondrocytes, n = 3, *p 0.05, **p 0.01, ***p 0.001 vs. Dulbecco’s Modified Eagle Medium (DMEM) group at the same time point. ns: there was no significant effect observed between the groups (p $>$ 0.05). (B) Effect of ASC-Exos on the proliferation of rat BMSCs, n = 3, ***p 0.001, ****p 0.0001. (C) Effect of ASC-Exos on the proliferation of chondrocytes and rat BMSCs using EdU staining. Scale bar = 400 um. Percentage of EdU-positive cells in chondrocytes and rat BMSCs, n = 3, **p 0.01, ***p 0.001, ****p 0.0001.

In a cell migration assay, rat BMSCs treated with ASC-Exos exhibited the highest migration rate compared to those treated with hADSC-Exos and DMEM. This indicates that ASC-Exos significantly enhance the migration of rat BMSCs (Fig. 4A,B).

Fig. 4.

ASC-exosomes promote the migration of rat BMSCs in vitro. (A) Migration assay of rat BMSCs. Scale bar = 1000 µm. (B) Quantitative analysis of migrated rat BMSCs, n = 3, *p 0.05.

Chondrocytes undergo differentiation and gradually lose their characteristic phenotype in vitro. Maintaining the chondrocyte phenotype is crucial for cartilage repair. In this study, we investigated the impact of ASC-Exos on the expression of chondrogenic genes and proteinsin the first, fourth, and seventh generations of chondrocytes. ASC-Exos treatment significantly up-regulated or maintained the mRNA expression levels of Aggrecan, COL II, and Sox-9, demonstrating more pronounced effects compared to the hADSC-Exos and DMEM groups (Fig. 5A,B).

Fig. 5.

ASC-exosomes regulate the expression of cartilage-related genes and proteins. (A) Relative gene expression levels of Aggrecan, Collagen II, and Sox9 in chondrocytes detected by qPCR. n = 3. #p 0.05, ##p 0.01, ###p 0.001, ####p 0.0001 vs. DMEM group in the same cell passage. ^p 0.05, ^^p 0.01, ^^^p 0.001, ^^^^p 0.0001 vs. hADSC-Exos group in the same cell passage. (B) Relative protein expression levels of Aggrecan, Collagen II, and Sox9 in chondrocytes detected by Western blot analysis. n = 3. #p 0.05, ##p 0.01, ###p 0.001, ####p 0.0001 vs. DMEM group in the same cell passage. ^p 0.05, ^^p 0.01, ^^^^p 0.0001 vs. hADSC-Exos group in the same cell passage. ns: there was no significant effect observed between the groups (p $>$ 0.05).

We analyzed the protein composition of ASC-Exos using a DIA assay and proteomics data has been deposited in ProteomeXchange (PXD053936). We identified several potential proteins with high relative expression levels, protein function obtained from uniprot (https://www.uniprot.org/) (Table 2). These proteins were found to play roles in promoting cell proliferation and migration, as well as protecting cartilage.