Exosomes derived from Schwann cells (SCDE) promote tendon repair through the PTEN/PI3K/Akt signaling pathway

Abstract

Tendon injury is one of the most frequently encountered soft tissue injuries with slow repair and poor functional recovery. Over the past few years, there’s been a buzz about how exosomes might boost tissue repair and regeneration. This research dive delves into the question of whether Schwann cell-derived exosomes (SCDEs) can jumpstart tendon healing by tweaking the PTEN/PI3K/Akt signaling chain.been concentrated on the effects of exosomes in promoting tissue regeneration and repair. In the present study, we would like to explore whether exosomes derived from Schwann cells (SCDE) could promote tendon repair via modulation of PTEN/PI3K/Akt signaling cascade. This study integrates experiments performed both in vitro and in vivo. Tendon cells were categorized into the NC and SCDE groups, with a scratch assay employed.vitro and in vivo conditions tendo cells were divided into the NC group and SCDE group, and scratch assay, Transwell migration assay, flow cytometry for cell cycle detection, and Western blot and qRT-PCR analyses were performed to measure SCX, DCN, COL1A1, p-AKT, PTEN, and other markers.In the in vivo experiments, a rat model of 1/3 patellar tendon defect was established, and hydrogel or hydrogel + SCDE was injected. Tissues were gathered at 2, 4, and 8 weeks following the surgical procedure, and HE, Masson, and Sirius Red stains were applied to gauge the healing process. Important protein levels were assessed at the 2 week mark after the operation.Masson, and Sirius Red staining were performed to evaluate tissue repair. Key protein expression was measured at 2 weeks post-surgery.

Background

Tendons, as important tissue structures connecting bones and muscles, play a key role in movement and force transmission [1]. Tendons are specialized dense connective tissues characterized by a hierarchically organized and polarized architecture, which enables them to sense and adapt to the mechanical forces generated by muscle contractions and conveyed to the bones, thereby facilitating movement and contributing to postural stability [2]. However, due to their poor blood supply and low metabolic activity, tendon injuries often heal slowly and incompletely, easily forming fibrotic scars, which lead to a decline in mechanical performance and an increased risk of recurrence [3]. Effective treatment options are limited. Therefore, new treatment methods, such as stem cell–based interventions and exosome-derived therapies, have been suggested and progressively developed. become more popular [4]. Stem cells employed in regenerative medicine exhibit notable therapeutic potential; however, their limitations and associated risks remain insufficiently defined [5]. With the aging population and the increasing incidence of sports injuries, tendon injury diagnosis and treatment have become a major challenge in orthopedics and sports medicine. Although clinical practices widely employ techniques such as suture repair, tendon transplantation, platelet-rich plasma (PRP) injection, and physical rehabilitation, these strategies are generally ineffective in achieving functional healing, especially in the tendon-bone interface region, where reconstructing native tissue structures and collagen fiber arrangement is difficult, thus affecting long-term mechanical stability and functional recovery [6]. Therefore, developing new, safe, and effective tendon repair strategies, particularly those capable of precisely regulating cell behavior and tissue microenvironments, holds significant clinical importance and application potential [7]. Exosomes represent extracellular vesicles with a diameter of roughly 30–150 nm.diameter [8], formed and secreted through the multivesicular body pathway [9]. They are enriched with bioactive molecules, proteins, and lipids, which can deliver information between cells, enabling intercellular communication and targeted regulation [10]. Earlier research has demonstrated that exosomes are critically involved in processes such as tissue regeneration, modulation of immune responses, and the advancement of tumor development [11]. Mesenchymal stem cell-derived extracellular vesicles are emerging as a promising alternative treatment for inflammation. Previous research has shown that exosomes are crucial in the process of tissue repair, the modulation of immune responses, and the development of cancer.become an alternative therapy for treating inflammation Earlier investigations have indicated that exosomes play a pivotal role in tissue regeneration, regulation of immune function, and the progression of tumors [12]. Compared to cell therapy, exosome therapy offers advantages such as higher safety, lower immunogenicity, stronger biocompatibility, and more convenient storage and transportation, making it a hot topic in regenerative medicine research [13]. Mass spectrometry (MS) has been used to investigate variations in exosomal protein expression and to discover potential biomarkers [14]. Regarding exosome sources, Schwann cells, as important supporting cells of the peripheral nervous system, have strong secretion and paracrine regulatory capabilities [15]. Recent evidence indicates that Schwann cell–derived exosomes (SCDEs) contribute to tendon healing by influencing cellular proliferation, migration, and extracellular matrix synthesis through the transfer of bioactive molecules, thereby exerting a potentially beneficial effect on tendon repair [16]. Nevertheless, the precise mechanisms remain insufficiently understood. Among the critical regulatory networks, The PI3K/Akt pathway is crucial for modulating cell viability, growth, motility, and metabolic processes.proliferation, migration, and metabolism, and has been demonstrated to serve a pivotal function in the regenerative processes of diverse tissue types [17]. PTEN, known as a traditional inhibitor of the PI3K/Akt signaling pathway,suppresses Akt activation by catalyzing the dephosphorylation of PIP₃, thereby suppressing cellular biological functions [18]. Research has shown that stem cells and their exosomal secretions enhance cell proliferation and motility by inhibiting PTEN and subsequently triggering Akt activation.the exosomes secreted by them can promote cell growth and migration through suppression of PTEN and subsequent activation of Akt. However, it is not clear Whether Schwann cell-originated exosomes (SCOs) influence tendon repair via alteration of the PTEN/PI3K/Akt signaling axismodulating the PTEN/PI3K/Akt signaling pathway [19].

Research purpose

Thus, the objective of this research was to explore the contributory role of Schwann cell-originated exosomes in the healing process of tendons. Our focus was to ascertain whether these Schwann cell-derived exosomes, or SCDEs, possess the potential to boost the proliferation, migration, and collagen synthesis of tendon cells. This was achieved by modulating PTEN expression levels and triggering the PI3K/Akt signaling cascade. To delve into these mechanisms, a combination of in vitro and in vivo studies were meticulously carried out.derived exosomes in tendon repair. Specifically, we aimed to determine whether SCDEs can enhance tendon cell proliferation, migration, and collagen production by downregulating PTEN expression and by activating the PI3K/Akt signaling pathway. To this end, both in vitro and in vivo and in vivo experiments were performed. Our findings may provide new mechanistic insights and potential novel therapies for tendon regeneration. This may also facilitate the translational application of exosomes in orthopedic tissue engineering.

Conclusion: Exosomes derived from Schwann cells, often referred to as SCDEs, could potentially stimulate the growth, movement, and collagen production in tendon cells by turning down PTEN and kicking off the PI3K/Akt signaling process. This research hints that SCDEs could be the key to healing tendon damage and opens up some exciting avenues for novel biological therapies for treating injured tendons.

Ethical statement

Healthy male Sprague–Dawley (SD) rats were used as the animal model in this study, 4–8 weeks of age, with body weights of 100–200 g. Each creature was kept solo and nourished in a controlled environment, with unfettered access to sustenance and hydration, not to mention the usual commercial feed. The critters were kept cozy at a balmy 24 to 26 °C, with a humidity level between 60 and 80 percent, fresh air, and a 12 h light/dark schedule to keep their internal clocks ticking. The hospital’s Animal Care and Use Committee gave the green light to all the experimental procedures.

Isolation, purification, and characterization of schwann cell–derived exosomes (SCDEs)

Upon reaching passages P3–P5, Schwann cells were cultivated in serum medium free of exosomes, refreshed every three days, and the supernatant was then harvested for subsequent procedures.with exosome-depleted serum medium and refreshed every three days and the supernatant was collected for the following steps. The medium samples were spun at 300 × g for 5 min at 4 °C to isolate viable cells, with the supernatant then frozen at − 20 °C [20]. Sequential ultracentrifugation was employed to isolate the exosomes. To begin with, the conditioned medium was spun at 2000 × g at 4 °C for 20 grueling minutes to sift out cellular debris and big guys. That was then topped off with a second round of spinning at a higher g-force 10,000 × g for an hour at the chilly 4 °C setting, using the trusty Eppendorf 5810R centrifuge from Germany.centrifugation at 10,000 × g for 60 min at 4 °C using an Eppendorf 5810R centrifuge (Eppendorf, Germany). The supernatant was then strained through a 0.22 µm filter to eliminate any lingering contaminants. To top it off, we subjected the mixture to high-speed centrifugation at 130,000 g for a solid 70 min, all while keeping it nice and chilly at 4 °C. We did this magic with a trusty Beckman Optimal − 100 ×  P ultracentrifuge, a gem from Beckman Coulter in Germany. Post centrifugation, the cell sediment was gently reconstituted in sterile, distilled H₂O. These pristine Schwann cell-derived exosomes were then employed for the subsequent experiments [21].

Exosome protein concentration and characterization

Protein concentration in the complete culture medium was assessed using the BCA method. Exosome dimensions and structure were analyzed via transmission electron microscopy [22]. Nanoparticle tracking analysis was employed to assess the size distribution and concentration of the exosomes, whereas western blotting was used to detect exosome-specific surface markers (Alix, CD9, and CD63) to verify their exosomal identity [23].

SCDE effect on tendon cell migration ability in vitro

To examine how Schwann cell–derived exosomes (SCDEs) influence tendon cell migration, transwell migration and scratch wound healing assays were conducted.

Transwell assay

To investigate the effects of second-passage rat tendinous cells (with a concentration of 5 × 10⁴ cells), these were seeded in the upper compartment of a 24-well Transwell insert featuring an 8 μm pore. Once the cells had taken hold, they were split into two groups. In the NC group, an equal volume of phosphate-buffered saline (PBS) was introduced to the lower chamber. For the SCDE group, 100 μL of serum-free medium laden with SCDE was poured into the lower compartment. The cells were then placed into a 37 °C incubator with 5% CO₂ for incubation. In a span of 48 h, the apparatus was dismantled, and the specimens were subjected to a 30 min fixation using 4% paraformaldehyde. Following this, a 0.5% crystal violet solution was applied to the cells for a duration of 5 min. Once the PBS wash was complete, any cells that hadn’t moved to the bottom chamber were delicately scraped away with a cotton bud. Then, five randomly selected fields from each well were photographed at 100 × magnification, and the count of migrating cells was tallied by two researchers who were unaware of the sample identities.

Scratch assay

Second-passage tendon cells were cultured in 24-well plates, and upon reaching confluence to form a uniform monolayer, a linear scratch was created at the center of each well using a sterile 200 μL pipette tip. After PBS washing to remove suspended cells, either SCDE-containing medium (SCDE group) or medium without exosomes (NC group) was added, 200 μL per well. Images of the scratch wound were obtained at 0, 24, and 48 h. The scratch area was quantified using ImageJ software, and cell migration rates were calculated to assess the impact of Schwann cell–derived exosomes (SCDEs) on tendon cell motility.

Flow cytometry evaluation of tendon cell cycle distribution

In order to investigate the effect of Schwann cell–derived exosomes (SCDEs) on the proliferative ability of tendon cells, cell cycle analysis was performed via flow cytometry analysis.Second-generation tendon cells were used in this experiment. When the confluence reached about 60%, tendon cells were digested, and then resuspended in PBS. NC group: PBS with an equal volume was added. SCDE group: 150 μL of above-mentioned isolated and identified SCDE was added. After 24 h of treatment, cells were resuspended according to the instructions of cell cycle detection kit. In brief, cells were firstly washed with PBS, and then collected. After that, 70% ice-cold ethanol was used to fix cells, and then the cell samples were stored at 4 °C for over night. After that, RNase A and PI (propidium iodide) mixture was added, and then the cell samples were incubated at room temperature for 30 min in the dark. Finally, flow cytometry (e.g., BD FACSCanto II) was used to collect  ≥  10,000 cell events. FlowJo software was used to quantify the cell number in G0/G1, S and G2/M phase. The cell cycle profile was compared between these two groups. Were these two cell groups in the same phase? If not, the cell cycle distribution was changed. Then, we could further explore whether the cell cycle process was affected by Schwann cell–derived exosomes (SCDEs) on tendon cells.

qRT-PCR detection of scde regulation on tendon cell gene expression

To assess how SCDE affects key tendon-cell genes, we performed qRT-PCR to quantify mRNA levels of TGF-β, COL1A1, DCN, SCX, PTEN, and other related genes, comparing the NC control group with the SCDE-treated group. To initiate the experiment, we extracted total RNA with the help of TRIzol reagent and then synthesized cDNA with a reverse transcription kit. GAPDH served as our go-to internal control gene, while we employed SYBR Green dye for the amplification process. We adhered to the protocol outlined in the kit’s instructions for the amplification parameters.

Western blot detection of scde impact on key protein expression

To further verify the regulatory effect of Schwann cell-derived exosomes (SCDE) on tendon cell function, Western blotting was performed to assess the expression of tendon-related proteins (DCN, COL1A1, SCX) and signaling pathway proteins (p-AKT, PTEN) in the NC group and SCDE treatment group. The steps are as follows: The membrane was incubated overnight at 4 °C with the primary antibody, then probed with an HRP-conjugated secondary antibody and protein bands detected by chemiluminescence. with GAPDH or β-actin serving as internal loading controls. ImageJ software was used for semi-quantitative analysis of band intensity. By comparing the protein expression levels between the two groups, the regulatory effect of SCDE on the PI3K/Akt pathway and its potential mechanisms in promoting tendon cell proliferation and differentiation were evaluated.

SCDE impact on in vivo tendon repair

Thirty male Sprague–Dawley (SD) rats, 7–8 weeks old and weighing 250–320 g, were used, were utilized to establish the patellar tendon injury model in this study. The central 1/3 of the patellar tendon was excised under sterile conditions to create a defect. Post-surgery, the animals were randomly divided into two groups:NC group: Hydrogel treatmentSCDE group: Hydrogel + SCDE treatment.

Observation points (n = 5) were set at the 2nd, 4th, and 8th weeks post-surgery. After surgery, the animals were allowed to move freely with no additional intervention. After completing the injection, the hydrogel containing SCDE was exposed to ultraviolet light for 1 min, causing it to gel in situ at the defect site, forming a scaffold structure.Patellar tendon tissue specimens were harvested at 2, 4, and 8 weeks after surgery for subsequent histological study. All specimens were fixed, dehydrated, embedded, Segmented and subsequently dyed using hematoxylin - eosin (HE), Masson’s trichrome, and Sirius Red stains. The study aimed to delve into how Schwann cell-derived exosomes, or SCDEs, influence the architecture of tendon tissue, the arrangement of collagen, and the reorganization of the extracellular matrix, or ECM. To differentiate between type I and type III collagen, the researchers employed Sirius Red staining. Under polarized light, type I collagen exhibited a range of red to orange-red hues, whereas type III collagen presented with a shift from green to yellow-green shades. The I/III collagen ratio was semi-quantitatively analyzed by ImageJ software to investigate the effect of SCDE on fiber maturity.The design of this part of the experiment was to verify the promoting effect of SCDE on tendon repair and provide a morphological basis for the research on molecular mechanism.

Mechanical properties testing

At the 8th week post-surgery, the biomechanical properties of the regenerated tendon were evaluated using an electronic universal testing machine (Instron, USA). During the procedure, patellar tendon samples containing the repaired tissue were collected from the bone-tendon junction area, trimmed, and fixed in the testing machine clamps. A preliminary load of 0.1 Newtons was used to take up any initial slackness, and the specimens were then stretched at a rate of 40 mm per minute until they broke. The ultimate tensile strength and Young’s modulus were recorded to gauge the mechanical characteristics of the tendon tissue.samples were stretched at a displacement rate of 40 mm/min until failure. The maximum tensile strength (ultimate load) and Young’s modulus were measured to evaluate the mechanical properties of the tendon tissue.

Time-dependent changes in key proteins

To explore the underlying mechanisms in greater depth, repair tissue from each group was collected at 2 and 8 weeks post-surgery for Western blot analysis. Target proteins were extracellular matrix related markers (DCN, COL1A1), tendon specific transcription factor scleraxis (SCX) and key signaling proteins (p-AKT, PTEN). Total protein was extracted and conventional Western experiments were performed. GAPDH or β-actin were used as an internal control of loading. The protein band intensity was analyzed by ImageJ software to calculate the relative expression of each target protein. Based on the changes in protein expression at different time points, we systematically studied the regulatory effects of Schwann cell–derived exosomes (SCDEs) on tendon repair and provided evidence that SCDEs were involved in modulating PTEN/PI3K/Akt signaling pathway.

Results

Tendon cell identification

Based on the microscopic analysis, the primary cultured tendon cells were found to be spindle-shaped or irregularly long in morphology. The cells were successfully attached to the culture layer and were spiral or bundle-like in arrangement, which are the typical morphology of tendon cells. With the culture time, the cells kept proliferating and fused gradually. The primary cultured tendon cells were still in typical morphology even after passaging.To further verify the identity of the tendon cells, the COL1A1 (type I collagen) was further stained by the immunofluorescence method. Positive red fluorescence signal was observed in the cytoplasm of cells, which indicated the high expression of type I collagen and the cultured cells were in tendon-specific culture.In conclusion, combined with the morphological analysis and COL1A1 immunofluorescence staining, the successful isolation and culture of tendon cells is verified. Tendon cells are well-established in culture, which provides cellular basis for the latter experiments (Fig. 1).

Fig. 1.

Morphological observation of tendon cells and type I collagen immunofluorescence staining results

SCDE identification results

Schwann cell–derived exosomes (SCDEs) were isolated by ultracentrifugation. TEM analysis showed that vesicles with cup-shaped or sphere morphology were typical of exosomes. The majority of particles were sized between 30 and 150 nm according to NTA, with a peak at 139 nm. Western analysis demonstrated the expression of previously identified exosomal surface proteins, such as CD9, CD63, CD81, and TSG101, whereas the endoplasmic reticulum protein Calnexin was not detected. This profile of markers is consistent with known exosomal signatures and confirms that the vesicles obtained from Schwann cell–conditioned medium were genuine exosomes (Fig. 2).

Fig. 2.

Identification of schwann cell–derived exosomes (SCDEs): characterization by TEM, NTA, and western blot analysis of exosomal markers

SCDE promotes tendon repair by regulating the PTEN/PI3K/Akt pathway

In exploring the molecular mechanisms through which Schwann cell-derived exosomes (SCDEs) influence tendon regeneration, this study zeroed in on the PTEN/PI3K/Akt signaling cascade. Western blot data revealed a notable drop in PTEN expression within tendon cells exposed to SCDEs when stacked up against the negative control group. At the same time, phosphorylated AKT levels shot up significantly (p < 0.05). These findings point to SCDEs putting a damper on PTEN while giving a green light to the PI3K/Akt pathway. Furthermore, tendon phenotype–related proteins SCX, DCN, and COL1A1 were significantly upregulated in the SCDE group. The expression of these proteins was significantly upregulated, and tendon cells exhibited increased differentiation and collagen synthesis.The above in vitro results were further verified in vivo. At 2 and 8 weeks after surgery, the expression of p-AKT was upregulated, and the expression of PTEN was downregulated in the repaired patellar tendon tissue in the SCDE group. The expression of SCX, COL1A1, and DCN were significantly upregulated. Masson’s trichrome and Sirius Red staining revealed that thethe collagen fibers were denser and regularly arranged in the SCDE group, and the type I/III collagen ratio was significantly increased.Moreover, mechanical testing revealed significantly greater ultimate tensile strength and Young’s modulus in the SCDE group compared to controls (p < 0.05), indicating a substantial improvement in tendon repair quality.

In summary, Schwann cell–derived exosomes (SCDEs) facilitate tendon repair by suppressing PTEN expression and activating the PI3K/Akt signaling pathway,enhancing tendon cell function, and promoting ECM remodeling, thereby improving tendon repair outcomes (Fig. 3).

Fig. 3.

Schematic diagram of SCDE-regulated PTEN/PI3K/Akt signaling pathway promoting tendon repair

SCDE promotes tendon cell migration and proliferation

The scratch assay results showed that the wound healing area in the SCDE-treated group was significantly larger than in the NC group at both 24 h and 48 h. Quantitative analysis indicated that the migration distance in the SCDE group was significantly increased at 24 h and 48 h, suggesting that SCDE effectively promotes tendon cell horizontal migration.

In the Transwell migration assay, the number of tendon cells migrating to the lower membrane in the SCDE group was significantly higher than that in the NC group. Crystal violet staining revealed that the cell migration ability in the The SCDE-treated group showed markedly more migrated cells than those in the NC group, indicating that SCDEs can significantly increase the chemotactic capacity of tendon cells.Flow cytometric cell cycle analysis revealed that the SCDE-treated group had a significantly increased proportion of cells in the S and G2/M phases, and a decreased proportion of cells in the G0/G1 phase. These results demonstrated that SCDEs can accelerate cell cycle progression, and promote the migration of tendon cells to a proliferative state.CCK-8 assay results also demonstrated that the absorbance (OD450) values of tendon cells in the SCDE-treated group at 24, 48 and 72 h were significantly higher than those in the NC group. These results demonstrated that SCDEs can significantly increase the metabolic activity of tendon cells, and promote the proliferative ability of tendon cells.In summary, SCDE can significantly promote tendon cell migration, and enhance tendon cell proliferation, and cell cycle progression in vitro. These results provided cellular evidence for the tendon repair effects of SCDEs (Fig. 4).

Fig. 4.

Effect of schwann cell-derived exosomes (SCDE) on tendon cell migration and proliferation ability. A Representative images of scratch healing assay (0 h, 24 h, 48 h); B Transwell cell migration assay images; C Flow cytometry cell cycle analysis; D Scratch healing area statistical results showing that the migration distance in the SCDE group was significantly increased at 24 h and 48 h (p < 0.01 vs. NC); E CCK-8 assay results showing that cell viability in the SCDE group at 24 h, 48 h, and 72 h was significantly higher than the NC group (p < 0.05); F Flow cytometry cell cycle statistical results showing that the proportions of cells in the S phase and G2/M phase in the SCDE group were significantly increased, while the G0/G1 phase proportion decreased (p < 0.05 versus. NC)

Exosome regulation of tendon repair-related gene and protein expression

SCDE regulation of key gene and protein expression in tendon cells

qRT-PCR analysis revealed that compared with the NC group, tendon cells treated with Schwann cell–derived exosomes (SCDEs) exhibited significantly upregulated mRNA expression of tendon-specific genes, such as SCX, COL1A1, and DCN. Expression of PTEN, a negative regulator of PI3K/Akt signaling pathway, was significantly downregulated (p < 0.01). These results indicate that the tendon-specific phenotypes of tendon cells may be upregulated by SCDEs through downregulating PTEN and further activating downstream signaling pathways. Western blot analysis also consistent with the transcription results. Expression of SCX, COL1A1, and DCN was significantly upregulated in the SCDE-treated group compared with the NC group (p < 0.05), while expression of PTEN was significantly downregulated (p < 0.01). Meanwhile, the p-AKT/AKT ratio was significantly upregulated (p < 0.01), indicating the strong activation of PI3K/Akt signaling pathway.In summary, our results demonstrated that SCDEs could upregulate tendon cell phenotype stability and function enhancement by downregulating PTEN and further activating downstream signaling pathways (PI3K/Akt), and upregulating expression of related genes and proteins involved in tendon repair. These findings provided strong mechanistic evidence for the potential use of SCDEs in tendon tissue regeneration (Fig. 5).

Fig. 5.

Effect of schwann cell-derived exosomes (SCDE) on the expression of key factors in tendon cells. A, B qRT-PCR results showed that, compared to the NC group, the mRNA expression of SCX, COL1A1, and DCN in the SCDE group was significantly increased (*p < 0.05, **p < 0.01, ***p < 0.001), while the mRNA expression of PTEN was significantly decreased (***p < 0.001); C Western blot band images showed that the proteins COL1A1, DCN, and SCX were upregulated in the SCDE group, PTEN was downregulated, and p-AKT expression was enhanced; D Quantitative protein analysis revealed that the ratios of COL1A1, DCN, SCX, and p-AKT/AKT were significantly increased in the SCDE group (**p < 0.01, ***p < 0.001), while PTEN protein levels were significantly decreased (**p < 0.01)

In vivo experimental results

A rat tendon defect model was established by surgically excising the central one-third of the patellar tendon. Following the procedure, the defect site was treated with either hydrogel alone (negative control, NC group) or hydrogel combined with Schwann cell–derived exosomes (SCDE group). Tissue repair and regeneration were evaluated at 2, 4, and 8 weeks post-surgery. The results demonstrated that in the NC group, the defect area remained evident throughout the observation period, whereas in the SCDE-treated group, the defect progressively decreased over time, accompanied by gradual restoration of tissue continuity. 8 weeks post-surgery, regenerated tendon tissues were harvested for biomechanical evaluation. Compared with the NC group, the SCDE-treated group exhibited significantly higher ultimate load and Young’s modulus values, indicating that SCDEs enhance the mechanical strength and functional properties of repaired tendons (Fig. 6).

Fig. 6.

Animal experimental design and tissue repair status A Electronic universal testing machine used for ultimate load and Young’s modulus testing; B Photographic image of the tissue samples collected from the patellar tendon post-surgery; C, D H&E staining to observe the morphology of the repaired tissue, with C representing the NC group and D representing the SCDE group

At 2 weeks post-surgery, Western blot analysis revealed that protein expression levels of TGF-β, COL1A1, DCN, and SCX were significantly higher in the SCDE-treated group compared with the NC group, whereas PTEN expression was markedly reduced. In parallel, p-Akt expression was significantly upregulated in the SCDE group (p < 0.05). These findings were consistent with the in vitro results and further confirmed that SCDEs promote tendon repair by downregulating PTEN and activating the PI3K/Akt signaling pathway (Fig. 7).

Fig. 7.

Molecular mechanism and mechanical properties of SCDE-promoted in vivo tendon repair A, B At 2 and 8 weeks post-surgery, expression levels of COL1A1 and DCN were significantly higher in the SCDE-treated group compared with the NC group (p < 0.01, *p < 0.001 versus. NC). C PTEN expression was markedly reduced in the SCDE group (p < 0.01 versus. NC). D The p-Akt/Akt ratio was significantly elevated in the SCDE group (p < 0.01 versus. NC).E SCX expression was significantly upregulated in the SCDE group compared with the NC group (p < 0.01 versus. NC). F Representative Western blot images showing upregulation of COL1A1, DCN, and SCX, downregulation of PTEN, and increased p-Akt levels in the SCDE group. G Biomechanical testing of ultimate load demonstrated significantly higher values in the SCDE group compared with the NC group (p < 0.05). H Young’s modulus was significantly greater in the SCDE group compared with the NC group (p < 0.05)

At the 2nd, 4th, and 8th weeks post-surgery, the NC group and SCDE group were subjected to HE (upper row), Masson trichrome (middle row), and Sirius Red (polarized light, lower row) staining. Representative images showed that the collagen bundles in the SCDE group were denser and more orderly arranged, and the inflammatory cell infiltration was significantly decreased. The type I/III collagen ratio was enhanced in the tendon of SCDE group as observed by Sirius Red staining (Fig. 8). It has been reported that higher type I/III collagen ratio reflected better collagen maturation and tissue organization.

Fig. 8.

Histological evaluation of tendon tissue repair in rats by SCDE (HE, Masson, Sirius red)

Ultimate load and Young’s modulus evaluation of tendon biomechanical properties

The ultimate load and Young’s modulus of tendons were measured as indicators of tendon biomechanics at 8 weeks after surgery in NC and SCDE groups. The results showed that the ultimate load of tendons in SCDE group was significantly higher than that in NC group (52.67 ± 17.15 N versus. 30.67 ± 3.06 N, p < 0.05), indicating that the tensile strength of tendons in SCDE group was significantly enhanced (Fig. 5).Furthermore, the Young’s modulus of tendons in SCDE group was significantly higher than that in NC group (28.57 ± 1.72 MPa versus. 22.06 ± 2.66 MPa, p < 0.05) (Fig. 7). The mechanical strength of tendons was significantly enhanced and the tissue elasticity was also improved in the tendon of SCDE group.

Pathway inhibitor experiment results

To further validate the involvement of the PI3K/Akt signaling pathway in SCDE-induced tendon cell proliferation, three experimental groups were established: a negative control (NC) group, an SCDE-treated group, and an SCDE + PI3K/Akt inhibitor group (X).After the addition of PI3K/Akt inhibitor (X group), the above changes were significantly reversed, with a decrease in the S phase and an increase in G0/G1, approaching NC levels. This suggests that SCDE-induced cell cycle progression is dependent on the PI3K/Akt pathway.EdU Probe Detection (Figure B): The SCDE group showed a significant increase in EdU-positive cells, indicating enhanced DNA replication activity. After the addition of the inhibitor (X group), the EdU positivity rate significantly decreased, approaching the NC group level, further supporting that SCDE’s proliferative effect relies on PI3K/Akt activation.Protein Expression (Figure C): In the SCDE-treated group, PTEN was downregulated, and p-AKT was upregulated (total AKT remained unchanged). Simultaneously, the expression of tendon phenotype/matrix-related proteins, such as SCX, DCN, and COL1A1, increased. After the addition of the inhibitor (X group), p-AKT levels were suppressed, and the expression of SCX, DCN, and COL1A1 decreased (compared to the SCDE group), while PTEN levels did not fully return to NC levels.In summary, SCDE promotes tendon cell proliferation and tendon-like matrix synthesis by downregulating PTEN and activating the PI3K/Akt pathway, and its effect is dependent on the activation of the PI3K/Akt pathway (Fig. 9).

Fig. 9.

Pathway dependence of SCDE on proliferation and matrix synthesis. A Cell cycle flow cytometry histograms (from left to right: NC, SCDE, SCDE + inhibitor (X)); the SCDE group showed an increased proportion of cells in the S phase, and the X group reversed this, approaching NC levels. B EdU staining (green: EdU positive; blue: DAPI). The SCDE group showed an increase in EdU-positive cells, while the X group showed a decrease. C Western blot detection of PTEN, DCN, COL1A1, SCX, p-AKT, and AKT (GAPDH as the internal reference); in the SCDE group, PTEN was downregulated (PTEN↓), p-AKT was upregulated (p-AKT↑), and SCX, DCN, and COL1A1 were upregulated (SCX/DCN/COL1A1↑); in the X group, p-AKT and downstream markers were downregulated

Discussion and conclusion

The results of this study indicate that Schwann cell-derived exosomes (SCDE) can enhance the structure and mechanical properties of tendon repair tissues, and exosomes are emerging as a promising regenerative therapy [24]. Stem cell-derived exosomes have shown positive effects in enhancing the regeneration of injured tendons/ligaments [25]. The mechanism of action has been validated at both the cellular and animal model levels, suggesting their potential application value in tendon regeneration [26].

In vitro, SCDE significantly promoted tendon cell migration and proliferation. Scratch and Transwell assays confirmed their promotive effect on cell motility, flow cytometry analysis showed that SCDE could drive tendon cells from the G0/G1 phase into the S and G2/M phases, and CCK-8 assays indicated enhanced cell metabolic activity [27]. Molecular Level Detection and Study Limitations Molecular level detection further revealed that SCDE can downregulate PTEN, activate the PI3K/Akt pathway, and upregulate key tendon-related genes and proteins such as SCX, COL1A1, and DCN. In vivo, SCDE improved the histological repair in the rat patellar tendon defect model, promoting the ordered arrangement of collagen fibers and increasing the I/III collagen ratio, while significantly enhancing the mechanical properties of the repaired tissue. Western blot analysis further confirmed that SCDE can downregulate PTEN, activate the PI3K/Akt pathway at the tissue level, and thereby promote ECM remodeling and tendon repair.However, this study also has limitations. First, the animal model was limited to small animals (rats), and the observation period was relatively short (8 weeks), which is insufficient to address key questions regarding the long-term stability, tissue integration, and durability of SCDE under prolonged physiological stress [28]. Study Limitations, Clinical Application Challenges, and Future Directions Moreover, despite the potential translational value of the results, clinical application still faces significant challenges. The immunogenicity of SCDE, potential off-target effects, and issues related to large-scale production and quality control have not been systematically evaluated [29]. Therefore, the current findings are more of a proof-of-concept, and the applicability for clinical translation should be interpreted with caution. Future research should extend the observation period, use large animal models, and combine mechanical load and stress conditions to comprehensively evaluate the effects and safety of SCDE in long-term repair [30]. Further investigations are required to elucidate the molecular mechanisms underlying tendon healing and homeostasis. Although the present study primarily focused on tendon cells, it is important to recognize that tendons function as complex “organs” with intricate anatomical structures and highly dynamic physiological properties. While the use of a single cell type in tissue engineering may yield partial success, future strategies are likely to involve co-culture systems incorporating multiple tendon-resident cell populations, together with appropriate biochemical and mechanical cues. Such approaches should also take into account the contribution of vascular and neural elements, which form integral components of the tendon unit [31]. In addition, there is a need for in-depth analysis of the internal active components (such as miRNA, proteins, etc.) and the establishment of standardized preparation, delivery, and quality control systems to promote the sustainable development of SCDE in regenerative medicine [32]. In conclusion, Schwann cell–derived exosomes (SCDEs) enhance tendon cell function and promote extracellular matrix (ECM) remodeling by downregulating PTEN and activating the PI3K/Akt signaling pathway, thereby facilitating tendon repair. Although the findings of this study provide valuable experimental evidence supporting the therapeutic potential of SCDEs in tendon regeneration, further comprehensive investigations are required to confirm their long-term efficacy and safety. Such studies will be essential to establish a solid foundation for the future clinical translation of SCDE-based therapies.

Author contributions

Chunbo Liu: Conceptualization, Methodology, Investigation, Writing—Original Draft. Jia Li: Investigation, Data Curation, Visualization. Jian Zhang: Investigation, Data Curation, Visualization. Yaxian Gao: Formal Analysis, Validation. Chenwei Guan: Methodology, Resources, Writing—Review & Editing. Cong Xu: Supervision, Funding Acquisition, Project Administration, Writing—Review & Editing, Corresponding Author.

Funding

This work was supported by the Natural Science Foundation of Hebei Province (Grant No. H2021406023).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Chengde Medical University Affiliated Hospital and were formally approved. Animal welfare and ethical principles were strictly followed throughout the study.

Consent for publication

All authors have read and approved the final version of this manuscript and consent to its submission for publication.

Competing interests

The authors declare that they have no competing interests.