Small extracellular vesicles originating from TNFAIP6-ADSCs subpopoulation identified by single-cell RNA sequencing promote tendon healing

Small extracellular vesicles originating from TNFAIP6-ADSCs subpopoulation identified by single-cell RNA sequencing promote tendon healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Small extracellular vesicles originating from TNFAIP6-ADSCs subpopoulation identified by single-cell RNA sequencing promote tendon healing Hengchen Liu, Aodan Zhang, Manyu Shi, Jingyao Zhang, Tingting Zhang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7279057/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 16 You are reading this latest preprint version Abstract Background Small extracellular vesicles originating from adipose-derived mesenchymal stromal cells (ADSC-sEVs) have excellent therapeutic value in tendon injury healing, but its mechanism and effect have not been fully elucidated. This study aimed to identify the key subsets and mechanisms involved in ADSC-sEVs contributing to tendon healing. Methods Based on our previous research of ADSC-sEVs improving the quality of tendon healing, we utilizing second-generation sequencing and bioinformatics methods to predict the key role of the TNFAIP6 − ADSCs subgroup in the treatment of tendon injury. We constructed different ADSC-sEVs through transfection ADSCs and treated to tendon stem cells (TSCs) for further exploration. The EdU, cell scratch, and transwell assays were used for proliferation proliferation and migration assays. Western blot and qRT-PCR analyses were used for qualitative analysis. Histopathological, immunohistochemical and, biomechanical testing were used for in vitro validation. Results TNFAIP6 − ADSC-sEVs significantly improves the therapeutic effect of ADSC-sEVs on tendon injury, which is related to the high expression of let-7c-5p. Based on application of different ADSC-sEVs in vitro and vivo, we identified CRCT1/JAK2/STAT3 as a key downstream signaling pathway regulated by let-7c-5p. Conclusions Our findings contributed to deeper understanding of how TNFAIP6 − ADSC-sEVs promote tendon healing through the let-7c-5p/CRCT1/JAK2/STAT3 signaling pathway. Furthermore, this study proposes a concept for constructing conditional ADSC-sEVs to enhance its inherent therapeutic effect. TNFAIP6 Adipose-derived mesenchymal stromal cells Tendon stem cells Small extracellular vesicles Tendon healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Tendons are integral structures within the musculoskeletal system, playing a crucial role in maintaining and regulating the biomechanics of the body [ 1 , 2 ]. The unique fiber structure of tendons contributes to their robust mechanical load performance [ 3 ]. However, certain sports activities or external forces can lead to tendon injuries, affecting approximately 30 million patients annually and resulting in a substantial medical expenditure burden of up to $ 180 billion [ 4 ]. The management of tendon injuries has become a global health problem. Moreover, the sparse distribution of blood vessels and the low metabolic rate of cells in tendon tissues make restoring their original biological function difficult, rendering them susceptible to re-rupture even after healing [ 5 ]. Despite advancements in surgical techniques and rehabilitation methods for tendon repair, mitigating long-term complications and re-rupture remains challenging, leading to premature career terminations for many renowned athletes [ 6 ]. Consequently, there is a pressing need to develop high-quality tendon repair methods. Tendon stem cells (TSCs) are unique cells with a remarkable capacity for self-renewal and differentiation and play a crucial role in tendon repair [ 7 ]. TSCs undergo extensive proliferation within a short period following tendon injury and are highly prone to tenogenic differentiation during tendon healing [ 8 ]. Therefore, adjusting the activity of TSCs is anticipated to enhance tendon healing and minimize the need for surgery. Mesenchymal stromal cell (MSC) transplantation shows promise as a cutting-edge biological therapy method, given MSC’s ability to regulate immune responses, improve the extracellular microenvironment, and activate endogenous stem cells [ 9 – 11 ]. For instance, Uysal et al. demonstrated that transplanting adipose-derived mesenchymal stromal cells (ADSCs) into injured tendons effectively promotes primary tendon healing [ 12 ]. However, limitations such as heterogeneity between cell populations, low cell retention, and ectopic osteogenesis persist in MSC transplantation [ 13 , 14 ]. Consequently, small extracellular vesicles, as the primary carriers of MSCs, have become a focal point in cell-free biotherapy research. Small extracellular vesicles (sEVs) are membrane-bound extracellular vesicles facilitating cellular communication through processes such as endocytosis, membrane fusion, or receptor-ligand interactions [ 15 ]. MSC-sEVs exhibit therapeutic potential across several diseases [ 16 – 18 ]. In a previous study, we utilized gelatin methacryloyl (GelMA) loaded ADSC-sEVs for the first time to treat tendon injuries [ 19 ]. Our findings revealed that treatment with ADSC-sEVs loaded with GelMA effectively regulated the biological characteristics of TSCs (proliferation, migration, and tenogenic differentiation), significantly improving tendon healing quality. Small extracellular vesicles, being lipid-rich structures, harbor diverse bioactive molecules, such as proteins, mRNAs, miRNAs, and DNAs. While the specific mechanisms through which small extracellular vesicles endogenously regulate the biological characteristics of TSCs are not fully understood, miRNAs have been identified as key players. As integral components of gene regulatory networks, miRNAs mediate the effects of small extracellular vesicles [ 20 ]. Additionally, as MSCs consist of a heterogeneous population of cells, there is limited research exploring the functions and therapeutic effects of ADSC-sEVs released by different ADSC subsets. This study aimed to identify critical subsets and miRNAs in ADSC-sEVs that contribute to tendon healing. Through second-generation sequencing technology and bioinformatics analysis, we predicted and verified that TNFAIP6 − ADSC-sEVs regulate the biological characteristics of TSCs through the let-7c-5p-mediated CRCT1/JAK2/STAT3 signaling pathway. Overall, based on cellular heterogeneity, our study presents a concept of constructing GelMA-loaded conditioned ADSC-sEVs to enhance the treatment of tendon injury, which also provides new ideas for cell-free regenerative medicine. Materials and methods Animals A total of 108 male SD rats (8–10 weeks, 180–230 g) were sourced from the Animal Experimental Center of Harbin Medical University. Experimental SD were given an adequate diet and maintained on a regular 12-h light/dark cycle. This study followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the ethics committee (No.ky2018-135). Isolation and culture of ADSCs and TSCs Following methods employed in previous studies, TSCs and ADSCs were isolated from the patellar tendon and inguinal adipose tissues of SD rats using collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) digestion [19]. TSCs were cultured in DMEM (Invitrogen, Carlsbad, CA, USA), and ADSCs were cultured in DMEM/F12 (Invitrogen). Each culture medium was supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin (Beyotime, Shanghai, China). The cells were cultured in a cell incubator at 37℃ and 5% CO 2 , with cells from the 3rd to 5th passages selected for this study. Isolation and identification of ADSC-sEVs The isolation method for ADSC-sEVs mirrored that of a previous study [19]. Briefly, ADSCs were incubated with an small extracellular vesicle-free medium (Biological Industries) for 24 h, and ADSC-sEVs in the supernatant were isolated through differential centrifugation (300 g, 10 min; 3000 g, 10 min; 10000 g, 30 min; 100000 g, 2 h). ADSC-sEVs were identified using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting. ADSC-sEVs were stained with the lipophilic membrane dye PKH26 (Sigma-Aldrich) for tracking experiments. GelMA loaded with 100 μg ADSC-sEVs was immersed in PBS for the ADSC-sEVs release analysis. The supernatant was collected every 24 h and the extent of release was performed using the BCA protein assay kit (Beyotime, China). Transfection of cells Gene expression in ADSCs was regulated through transfection using Lipofectamine 3000 (Invitrogen). Let-7c-5p mimic, let-7c-5p inhibitor, and their corresponding negative controls were designed by GenePharma. For transfection, 7.5 μL of Lipofectamine 3000 was incubated with 75 μL of RNAs (let-7c-5p mimics, let-7c-5p inhibitors, and their negative controls) in 250 μL of MEM (Invitrogen) for 15 min and then added to ADSCs for 48 h incubation period. Similarly, third-generation TSCs were transfected using Lipofectamine 3000 (small interfering RNAs against CRCT1 and negative control were purchased from GenePharma). The RNA sequences are presented in Table S1. Dual-luciferase reporter assay The potential downstream target CRCT1 of let-7c-5p was predicted through TargetScan, DIANA-microT, and miRanda databases. We constructed the wild and mutant-type (WT/MUT-CRCT1) pmirGLO luciferase reporter plasmids for the CRCT1-3’ UTR. Using Lipofectamine 3000, we transfected these plasmids along with let-7c-5p mimics and their respective negative controls into TSCs. After 24 h of transfection, fluorescence was detected using the Dual-Luciferase Reporter Assay Kit (Beyotime). Treatment of TSCs with different ADSC-sEVs TSCs were seeded in 6-well plates at a cell density of 5×10 6 /well to investigate the impact of various ADSC-sEVs. Each group of TSCs was treated with 50 μg/mL of different ADSC-sEVs. The TSCs were randomly categorized into six groups as follows: (1) Control: TSC medium was replaced with an small extracellular vesicle-free medium. (2) ADSC-sEVs: normal ADSC-sEVs were added to the small extracellular vesicle-free medium. (3) NC-mimic ADSC-sEVs: mimic-negative controls transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (4) let-7c-5p-mimic ADSC-sEVs: let-7c-5p-mimic transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (5) NC-inhibitor ADSC-sEVs: inhibitor-negative controls transfected ADSCs derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (6) let-7c-5p-inhibitor ADSC-sEVs: let-7c-5p-inhibitor transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. Subsequently, we explored the regulation of let-7c-5p on downstream targets and introduced inhibitor-let-7c-5p ADSC-sEVs and AG490 (a JAK2/STAT3 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA) to TSCs transfected with si-CRCT1 or siNC. Additionally, we treated TSCs with TNFAIP6 + and TNFAIP6 - ADSC-sEVs to further explore the signaling pathways. Cell proliferation assay The proliferation capacity of TSCs was assessed by Edu Assay Kit (UE, China). TSCs in each group were pretreated with 50 μmol/L EdU for 4 h. Following fixation with 4% paraformaldehyde, the Click-iT EdU working solution was prepared according to the manufacturer’s instructions. Subsequently, 5 μg/mL Hoechst 33342 (UE) was used to label nuclei for 20 min, and the cell proliferation was counted using a fluorescent fiber microscope (Leica, Wetzlar, Germany). Cell migration assay In the upper chamber of the Transwell plate, TSCs (1x10 4 ) were seeded, and an small extracellular vesicle-free medium containing different ADSC-sEVs was added to the lower chamber based on the experimental groups. After incubation at 37℃ and 5% CO 2 for 24 h, the TSCs in the upper chamber were stained with 0.1% crystal violet, and the extent of TSCs migration was counted under a light microscope (Leica). Additionally, a cell scratch assay was performed to evaluate TSCs migration. TSCs were seeded in 6-well plates with a cell density of 2×10 5 /well and cultured overnight at 37℃ and 5% CO 2 . The assay involved scratching a straight line in the cultured cells and adding different ADSC-sEVs to an small extracellular vesicle-free medium. Changes in wound healing at 0 h and 24 h were recorded to evaluate the migration of TSCs. Western blot analyses Proteins were extracted from TSCs using RIPA buffer (Beyotime), and 20 μg of protein from each group was selected for analyses. Immunoblotting was conducted with primary antibodies: anti-CD9 (ab92726; Abcam, Cambridge, UK), abti-TSG101 (ab125011; Abcam), anti-Hsp70 (ab2787; Abcam), anti-scleraxis (DF13293; Affinity Biologicals, Ancaster, ON, Canada), anti-tenomodulin (DF13715; Affinity), anti-collagen I (AF7001; Affinity), anti-phospho(p)-JAK2 (AF3024; Affinity), anti-JAK2 (AF6022; Affinity), anti-phospho(p)-STAT3 (AF3293; Affinity), anti-STAT3 (AF6294; Affinity), anti-TNFAIP6 (PA5-75332; Thermo Fisher, Massachusetts, USA), and a horseradish peroxidase-conjugated goat anti-rabbit IgG (BA1055; Boster, Wuhan, China) served as a secondary antibody. A chemiluminescence imaging system (ChemiScope 6200T, Clinx Science Instruments, Shanghai, China) was used for obtaining images. Protein bands were quantified using ImageJ software. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses Total RNA in TSCs was extracted using Trizol reagent (Beyotime), and total RNAs from small extracellular vesicles were extracted using a Total RNA Isolation Kit (Thermo). RNAs were reverse transcribed into cDNA using Premix Ex Taq II. The qRT-PCR primers were obtained from GenePharma (Table S2). RNA expression in the samples was assessed using real-time PCR with SYBR Green (Takara, Japan) on an ABI StepOnePlus system. Experimental protocols and surgical procedures A total of 108 Sprague-Dawley rats were randomly divided into 5 groups: (1) Control ( n = 36): Animals underwent partial patellar tendon resection surgery. (2) ADSC-sEVs ( n = 24): Animals were treated with 200 μg ADSC-sEVs after partial patellar tendon resection surgery. (3) mimic-let-7c-5p ADSC-sEVs ( n = 24): Animals were treated with 200 μg mimic-let-7c-5p ADSC-sEVs after partial patellar tendon resection surgery. (4) TNFAIP6 + ADSC-sEVs ( n = 12): Animals were treated with 200 μg TNFAIP6 + ADSC-sEVs after partial patellar tendon resection surgery. (5) TNFAIP6 - ADSC-sEVs ( n = 12): Animals were treated with 200 μg TNFAIP6 - ADSC-sEVs after partial patellar tendon resection surgery. All animals were anesthetized with 0.3% sodium pentobarbital (30 mg/kg) before surgery. The rat patellar tendon injury model was constructed as previously described [19]. Briefly, the median patellar tendon incision was made to remove the middle one-third of the patellar tendon tissue. GelMA (EFL-GM-60, 10% w/v) mixed with different ADSC-sEVs was then applied to the lesion and crosslinked into a gel state using ultraviolet light. Finally, the skin incision was closed using 4-0 sutures. Animals were euthanized by inhaling excessive carbon dioxide gas on days 7, 14, and 28, and patellar tendon tissue was extracted for subsequent studies. Histopathological and immunohistochemical analyses Patellar tendon tissues were fixed with 4% paraformaldehyde (Beyotime) and sectioned after paraffin embedding (0.4 μm). The tendon healing stage was evaluated using a light microscope after HE staining (Beyotime). For immunohistochemical analyses, tissue sections were stained with primary antibody: anti-SCXA (DF13293; Affinity), anti-TNMD (DF13293; Affinity), anti-collagen I (AF7001; Affinity), anti-CD146 (ab75769; Abcam), followed by incubation with a goat anti-rabbit IgG secondary antibody (ab6721; Abcam). For immunofluorescence analysis, tissue sections were incubated with primary antibodies: anti-CCR7 (ab32527; Abcam), anti-CD163 (ab182422; Abcam), anti-IL-6 (TA500067S; Origene), and anti-IL-10 ( ab33471; Abcam), followed by incubation with secondary antibodies (SA00013; ProteinTech, Chicago, IL, USA). The photos were taken with a DM4 B microscope (Leica). Three fields per section were selected randomly for statistical analysis. Positive signals were quantified with the ImageJ software. Biomechanical testing When obtaining rat patellar tendon tissue, the bones at both ends (patella-patellar tendon-tibia) were retained. The bones at both sides were fixed on a Zwick I Z010 (Bavaria,Germany) for mechanical testing. The load-displacement curve before patellar tendon rupture was recorded at a rate of 5 mm/min. Subsequently, the failure load (N) and stiffness (N/mm) were obtained by the testXpert software. Young’s modulus (N × 10 3 /mm 2 ) was calculated after measuring the cross-sectional area (mm 2 ) of the tendon with a vernier caliper. Collection and analysis of single-cell RNA sequencing (scRNA-seq) data The rat inguinal adipose tissue scRNA-seq data were obtained from the Gene Expression Omnibus database (GSM3717978), Sequence Read Archive database (SRR715485), and Array Express database (E-MTAB-6677). The unique molecular identifier (UMI) counting matrix was generated using R software’s Seurat package. Subsequent analysis was performed after filtering out cell data with a mitochondrial ratio greater than 20% and detecting over 6000 genes. Finally, 20764 single cells remained (2902 cells in GSM3717978, 9644 cells in SRR715485, and 8218 cells in E-MTAB-6677), and they were applied in downstream analyses. After quality control, the UMI count matrix was log normalized. Since sample from three samples were processed and sequenced in batches, sample was used to remove potential batch effect. In this process, top 2000 variable genes were used to create potential Anchors with FindIntgrationAnchors function of Seuart. Subsequently, IntegrateData function was used to integrate data and create a new matrix with 2000 features, in which potential batch effect was regressed out. To reduce the dimensionality of the scRNA-seq dataset, principal component analysis (PCA) was performed on an integrated data matrix. With Elbowplot function of Seurat, top 30 PCs were used to perform the downstream analysis. The main cell clusters were identified with the FindClusters function offered by Seurat with resolution set as default (res = 1.0). And then they were visualized with 2D tSNE or UMAP plots. Conventional markers described in a previous study were used to categorize every cell into a known biological cell type [22-24]. Firstly, 20764 cells were clustered into 10 major cell types. Subsequently, every major cell type was subset and further clustered into subclusters to detect heterogeneity within every cell type, respectively. The Seurat Findallmaker function was performed to identify preferentially expressed genes in clusters. Finally, the heterogeneity of different cell clusters in adipose tissue was detected based on the prioritized expression genes in the cell clusters. ADSC clusters’ scRNA-seq data were extracted for further heterogeneity analysis and screened for different ADSCs subgroups and specific marker genes. Flow cytometry sorting A total of 4×10 6 3rd generation ADSCs were collected and suspended in 2% FBS in PBS, followed by anti-TNFAIP6 for 1 h and FITC-conjugated anti-rabbit IgG secondary (S0008; Affinity) for 30 min, respectively. After filtering through a 300-mesh filter, TNFAIP6 - /TNFAIP6 + ADSCs were sorted using a flow cytometer (BD FACSMelody, Franklin Lakes, USA) and collected in a DMEM/F12 culture medium containing 20% FBS. After TNFAIP6 - /TNFAIP6 + ADSCs were amplified to 80%, the culture medium was discarded and replaced with an small extracellular vesicle-free medium for 24 h. Cell supernatant was collected, and TNFAIP6 - /TNFAIP6 + ADSC-sEVs were extracted using the above method. The adipogenic, osteogenic, and chondrogenic differentiation of various ADSC subpopulations was induced using assay kits from Cyagen, as described in our previous study [19]. Statistical analyses All values are expressed as means ± standard deviation. Quantitative data for each group were analyzed using a one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test in Graphpad Pism 9.5 software. Statistical significance was set at p < 0.05. Statement The work has been reported in line with the ARRIVE guidelines 2.0. Results Characteristics of ADSCs and internalization of ADSC-sEVs ADSCs grew adherent and showed a spindle-shaped morphology (Fig. S1 A, Supporting Information). ADSCs can be induced in vitro to differentiate into adipogenesis, osteogenic, and chondrogensis (Fig. S1 B, Supporting Information).Flow cytometric analysis of ADSC surface markers (CD90- and CD105-positive, and CD34-, CD45-, and CD11b-negative) (Fig. S1 C, Supporting Information). The PKH26 staining tracer experiment showed that ADSC-sEVs could be successfully uptaken by TSCs (Fig. S1 D, Supporting Information). Let-7c-5p was highly expressed in ADSC-sEVs In our previous investigations, we confirmed the efficacy of ADSC-sEVs in promoting the healing of tendon injuries [ 19 ]. To further explore the specific mechanism of action of ADSC-sEVs, we performed high-throughput sequencing of miRNA expression in ADSC-sEVs. The results showed that let-7c-5p was significantly upregulated in ADSC-sEVs (Fig. 1 A). A parallel study on the biological functions of miRNAs in TSC-sEVs demonstrated a pronounced enrichment of let-7c-5p (Fig. 1 B) [ 21 ]. This suggests that let-7c-5p in ADSC-sEVs has significant implications for TSCs. Characteristics of different ADSC-sEVs ADSC-sEVs, mimic-let-7c-5p ADSC-sEVs, and inhibitor-let-7c-5p ADSC-sEVs exhibited quasi-circular structures under TEM (Fig. 2 A). NTA showed that the diameters of these ADSC-sEVs were 110.3 nm, 112.7 nm, and 112.7 nm (Fig. 2 B). Western blot analysis confirmed the expression of CD9, TSG101, and HSP70 on the surface of all three ADSC-sEVs (Fig. 2 C). Regulation of let-7c-5p expression in ADSC-sEVs on the biological characteristics of TSCs ADSC-sEVs with different levels of let-7c-5p were obtained through transfection (Fig. 3 A). The expression of let-7c-5p in the let-7c-5p-mimic group was significantly increased, whereas its expression was not significantly suppressed in the let-7c-5p-inhibitor group. This result may be related to the fact that miRNA inhibitors act by competitive binding inhibition. NC-mimic and NC-inhibitor served as control groups. TSCs were treated with five different ADSC-sEVs. The EdU assay showed that let-7c-5p-mimic ADSC-sEVs significantly enhanced the proliferation-promoting effect on TSCs, whereas the let-7c-5p-inhibitor group attenuated this effect (Fig. 3 B, E). Transwell and scratch assays revealed that high let-7c-5p expression effectively promoted the TSCs migration (Fig. 3 C, D, F, G). Additionally, let-7c-5p expression alterations influenced the tenogenic differentiation of TSCs (Fig. 3 H-K). CRCT1 was identified as the target of let-7c-5p To explore downstream mechanisms, we predicted potential targets using the TargetScan, DIANA-microT, and miRanda databases. The results showed that CRCT1 was the only downstream target predicted by all three databases (Fig. 4 A). Therefore, we hypothesized that CRCT1 was the target gene of let-7c-5p. To verify this hypothesis, we constructed WT-CRCT1 and MUT-CRCT1 pmirGLO luciferase reporter plasmids and demonstrated targeting of let-7c-5p by CRCT1 using a dual-luciferase reporter assay (Fig. 4 B). We found that let-7c-5p mimics significantly reduced the fluorescence expression of WT-CRCT1 but had a lesser effect on MUT-CRCT1 (Fig. 4 C). In addition, we treated TSCs with let-7c-5p-inhibitor and NC-inhibitor ADSC-sEVs. qPCR results revealed that the expression of let-7c-5p was significantly correlated with that of CRCT1 (Fig. 4 D). Next, we predicted the CRCT1-related proteins using STRING (Fig. 4 E). KEGG analysis showed that the JAK/STAT signaling pathway was the most significantly enriched (Fig. 4 F). Let-7c-5p/CRCT1 affected the biological characteristics of TSCs through JAK2/STAT3 signaling pathway We introduced let-7c-5p-inhibitor ADSC-sEVs to TSCs transfected with si-CRCT1 or si-NC. Western blotting showed that a reduction in let-7c-5p content in ADSC-sEVs led to an increase in the expression of p-JAK2 and p-STAT3 in TSCs, suggesting that let-7c-5p/CRCT1 may exert its biological role through the JAK2/STAT3 signaling pathway (Fig. 5 A-C). Subsequently, we evaluated the effect of let-7c-5p/CRCT1/JAK2/STAT3 on TSCs using AG490. Western blotting showed that AG490 significantly inhibited the activation of the JAK2/STAT3 signaling pathway mediated by let-7c-5p-inhibitor ADSC-sEVs (Fig. 5 D–F). Pretreatment with AG490 also mitigated the enhancing effect of let-7c-5p-inhibitor ADSC-sEVs on the proliferation and migration abilities of TSCs (Fig. 5 G-L). Additionally, inhibition of the JAK2/STAT3 signaling pathway significantly reduced the effect of the let-7c-5p-inhibitor ADSC-sEVs on the tenogenic differentiation of TSCs (Fig. 5 M-P). Release characteristics of ADSC-sEVs in the GelMA We obtained different ADSC-sEVs using differential centrifugation (Fig. 6 A). The photoinitiator LAP was used to initiate GelMA polymerization reaction, and the sieve-like biological scaffold structure which could be loaded with ADSC-sEVs (Fig. 6 B, C). The ADSC-sEVs release analysis in vitro suggested that the GelMA-loaded ADSC-sEVs were gradually released in 7 days (Fig. 6 D). Similarly, the PKH26-labeled sEVs tracing experiments in vivo found that ADSC-sEVs were able to act locally for more than 7 days under GelMA loading (Fig. 6 E, F). These results demonstrate that GelMA-loaded ADSC-sEVs can achieve a sustained release effect, which helped ADSC-sEVs exert greater therapeutic efficacy in vivo. GelMA-loaded let-7c-5p-mimic ADSC-sEVs promoted tendon healing in vivo We subsequently administered ADSC-sEVs and let-7c-5p-mimic ADSC-sEVs to a rat patellar tendon injury model to evaluate their in vivo effects. HE staining revealed a more regular fibrous structure in the healing patellar tendon of the let-7c-5p-mimic ADSC-sEVs group compared to the ADSC-sEVs group at 2 and 4 weeks (Fig. 7 A). The let-7c-5p-mimic ADSC-sEVs group showed significantly higher expression of the tendon stem cell marker CD146 compared to the other two groups. Notably, the tendon stem cell marker CD146 displayed the highest expression in the let-7c-5p-mimic ADSC-sEVs group (Fig. 7 A-C). These results suggest that let-7c-5p effectively promotes the proliferation and tenogenic differentiation of TSCs in vivo. We evaluated the therapeutic effects of let-7c-5p on injured tendons using biomechanical tests. The healing patellar tendon tissue in the let-7c-5p-mimic ADSC-sEVs group showed significant advantages in terms of failure load, stiffness, and Young’s modulus (Fig. 7 D–G). GelMA-loaded let-7c-5p-mimic ADSC-sEVs reduced tissue inflammatory response in vivo Furthermore, we evaluated the effect of let-7c-5p on the early inflammatory response to tendon injury using immunofluorescence. The expression of inflammation-related factors in tendon tissues of the Control, ADSC-sEVs, and let-7c-5p-mimic ADSC-sEVs groups was assessed. The results indicated a significant decrease in the expression of IL-6 (a pro-inflammatory factor) in the let-7c-5p-mimic ADSC-sEVs group, while IL-10 (an anti-inflammatory factor) showed the opposite trend. Simultaneously, we found that the expression of CCR7 (an M1 macrophage marker) significantly decreased in the let-7c-5p-mimic ADSC-sEVs group, while CD163 (an M2 macrophage marker) significantly increased (Fig. 8 A, B). TNFAIP6 as a key marker gene for the functional and stemness subgroups of ADSCs Next, we explored the promoting effect of ADSCs on tendon healing from the perspective of cellular heterogeneity. To improve the accuracy of the analysis results, we used a multicenter joint analysis of scRNA-seq data (GSM3717978, SRR715485, and E-MTAB-6677) from subcutaneous adipocyte populations in the groin of rats. After normalizing the obtained UMI matrix, the FindIntegrationAnchors function of the Seurat package was used to integrate three sets of scRNA-seq data (Fig. 9 A). The results showed that the three cells group had very similar subgroups (Fig. 9 B, C). Based on relevant references [ 22 – 24 ], we integrated and re-annotated 10 cell groups, include Adipose derived stem cells, Pre-adipocytes, Aregs, Adipocytes, Endothelial cells, Smooth muscle cells, T cells, B cells, Macrophages, and Dendritic cells (Fig. 9 D-F). Subsequently, we extracted scRNA-seq data from ADSC cell populations for further analysis (Fig. 9 G). Based on the screening and analysis of maker genes, we categorized ADSCs into a stemness subgroup (maintaining stemness related genes, such as TNFAIP6, CEBPB, and MT1) and a functional subgroup (multidirectional differentiation ability and cytokine expression related genes, such as CXCL13, SFRP2, and BMP7) [ 25 – 30 ]. A significant difference was found in the expression of TNFAIP6 between the two ADSCs subgroups (Fig. 9 H–J). GelMA-loaded TNFAIP6- ADSC-sEVs promotes high-quality tendon healing through let-7c-5p Using flow cytometry sorting technology, we successfully classified ADSCs into TNFAIP6 + ADSCs and TNFAIP6 − ADSCs (Fig. 10 A–C). Our analysis results show that the stemness of TNFAIP6 + ADSCs is significantly stronger than that of TNFAIP6 − ADSCs (Fig. 10 D). Next, we extracted TNFAIP6 − /TNFAIP6 + ADSCs-sEVs and explored the expression levels of let-7c-5p in these small extracellular vesicles. As expected, let-7c-5p was significantly enriched in TNFAIP6 − ADSC-sEVs (Fig. 10 E). The regulatory ability of TNFAIP6 − ADSC-sEVs on tenogenic differentiation and biological characteristics (proliferation, migration, and tenogenic differentiation) in TSCs was also significantly better than that of TNFAIP6 + ADSC-sEVs (Fig. 10 F–O). Finally, we transplanted TNFAIP6 − /TNFAIP6 + ADSCs-sEVs into the rat patellar tendon injury model. The results showed that TNFAIP6 − ADSC-sEVs strongly promoted tendon repair in vivo (Fig. 10 P–T). Discussion Tendon injury is a common condition affecting the motor system. It poses challenges due to the slow and inefficient natural tendon repair process, leading to unsatisfactory long-term rehabilitation [ 31 ]. Restoring the original physiological structure and biological characteristics of tendons poses an ongoing challenge for medical professionals. TSCs are a cell population with self-renewal and multidirectional differentiation potential in tendon tissue [ 32 ]. Notably, Komatsu et al. observed that the transplantation of TSC sheets could significantly accelerate tendon healing and regeneration [ 33 ]. Recent studies have shown that MSCs are beneficial in tissue healing, particularly through the secretion of small extracellular vesicles. Using cell-free biological therapy with MSC-sEVs has emerged as a promising approach in clinical applications [ 34 , 35 ].We previously found that TSC-sEVs could significantly enhance the biological activity of tendon cell population and improve the quality of tendon healing [ 21 ]. However, it is not easy to extract TSCs from patients with preexisting tendon injuries. This makes it difficult for the application of TSC-sEVs to realize the transformation from basic research to clinical practice. Therefore, it is urgent to find other MSC-sEVs suitable for clinical application to optimize tendon healing. ADSCs are widely used in tissue engineering and regenerative medicine due to their abundant tissue sources, convenient cell separation, and strong cellular activity [ 36 ]. In a previous study, we introduced ADSC-sEVs for the first time to address tendon injuries, resulting in a notable improvement in tendon healing quality [ 19 ]. Given that ADSC-sEVs comprise a complex mixture of bioactive substances, this study explored the specific mechanisms and key components of ADSC-sEVs in treating tendon injuries. As key regulators in post-transcriptional gene expression, miRNAs play important roles in cell differentiation, biological development, and disease progression [ 37 ]. Liu et al. found that miRNA expression significantly impacts tendon growth and wound healing [ 38 ]. Therefore, in this study, we investigated miRNAs within ADSC-sEVs for the treatment of tendon injuries. We found that let-7c-5p was significantly enriched in ADSC-sEVs using high-throughput sequencing. Interestingly, in another study on TSC-sEVs, let-7c-5p also showed the most significant enrichment in TSC-sEVs [ 21 ]. This led us to hypothesize that ADSC-sEVs primarily promote tendon repair through let-7c-5p. The let-7 family, one of the earliest discovered miRNAs, is predominantly involved in cell differentiation and metabolic regulation [ 39 – 41 ]. Wang et al. demonstrated that let-7c-5p promotes the osteogenic differentiation of BMSCs via the TGF-β signaling pathway [ 42 ]. Additionally, Wang et al. found that let-7c-5p and miRNA-21-5p in BMSC-sEVs promote the proliferation and migration of endothelial cells treated with rapamycin [ 43 ]. In this study, we found that let-7c-5p effectively promoted the proliferation and migration of TSCs and induced their tenogenic differentiation. Pretreating ADSCs with the transfection method to increase the expression of let-7c-5p in ADSC-sEVs effectively improves the quality of tendon healing. These results suggest a significant role for let-7c-5p in the ADSC-sEVs treatment of tendon injuries. To further understand the role of let-7c-5p in the treatment of tendon injuries, we employed TargetScan, DIANA-microT, and miRanda databases to predict possible targets. CRCT1 was selected as the focus, as it was the sole target intersecting all three databases. CRCT1 is encoded by an epidermal differentiation complex and plays a vital role in epidermal differentiation [ 44 ]. In this study, we confirmed that let-7c-5p targets CRCT1 using a dual-luciferase reporter assay. PCR results confirmed that ADSC-sEVs with low expression of let-7c-5p could alleviate CRCT1 inhibition in TSCs. Therefore, we hypothesize that the biological efficacy of ADSC-sEVs depends on let-7c-5p/CRCT1. Subsequently, we predicted the downstream signaling pathways associated with let-7c-5p expression using bioinformatics. KEGG analysis showed that the JAK2/STAT3 pathway was potentially correlated with let-7c-5p/CRCT1. As a canonical signaling pathway present in various cells, the JAK2/STAT3 pathway is involved in numerous key biological processes in various diseases, such as tumors and inflammatory conditions [ 45 – 47 ]. In addition, Chen et al. showed that the JAK2/STAT3 signaling pathway plays a key role in regulating the aging process of TSCs [ 48 ]. Given the potential impact of the JAK2/STAT3 pathway on tendon healing, we explored its changes during ADSC-sEVs treatment. Western blot analyses showed a significant correlation between p-JAK2, p-STAT3, and CRCT1. Subsequently, pretreatment of TSCs with AG490 (a JAK2/STAT3 inhibitor) and incubation with let-7c-5p-inhibitor ADSC-sEVs showed that AG490 reversed the efficacy of ADSC-sEVs on the biological characteristics of TSCs. Therefore, the JAK2/STAT3 signaling pathway plays a significant role in let-7c-5p/CRCT1 mediated ADSC-sEVs promotion of tendon healing by ADSC-sEVs. Tendon repair is an intricate process where SCX serves as a pivotal molecule in tendon development, crucial for regulating TSC differentiation and proliferation [ 49 ]. TNMD is identified as a key regulatory factor influencing the biological characteristics of TSCs, including proliferation, differentiation, aging, and tendon maturation [ 50 ]. Dex et al. found that TNMD and collagen I are colocalized in the extracellular matrix, and the expression of collagen I positively affects the mechanical strength and function of tendons [ 51 ]. Building on our previous research, we increased the content of let-7c-5p in ADSC-sEVs to optimize the induction of TSCs tenogenic differentiation in this study. Furthermore, the expression of CD146 (TSCs maker) in tendons significantly and positively correlated with that of let-7c-5p in ADSC-sEVs. Similarly, let-7c-5p effectively promoted the proliferation and migration of TSCs in vitro, a process related to the let-7c-5p/CRCT1/JAK2/STAT3 signaling mechanism. In our previous study, ADSC-sEVs reduced early inflammatory responses by regulating macrophage polarization [ 19 ]. Interestingly, recent studies have shown that let-7c-5p inhibits inflammatory expression [ 52 – 54 ]. Consequently, we explored the effect of let-7c-5p on early inflammation in tendon injury. As expected, immunofluorescence analysis showed that the increased expression of let-7c-5p effectively inhibited inflammatory factors in the early stages of tendon injury, a process potentially related to the polarization of M2 macrophages. Finally, we evaluated the quality of tendon healing through biomechanical testing. Tendon load, stiffness, and Young’s modulus in the mimic group showed the most significant improvement, indicating that let-7c-5p plays a crucial role in ADSC-sEVs-mediated promotion of tendon healing. As the research progressed, we found that the expression of let-7c-5p and its therapeutic effect on tendon injury varied between batches of ADSC-sEVs. Additionally, current clinical trials show fluctuations in the efficacy of ADSC-sEVs [ 55 ]. Based on Wang et al.’s study on the heterogeneity of MSCs [ 56 ], we speculate that competition among different subgroups of ADSCs during culture leads to imbalanced proportions and changes in ADSC-sEVs function and efficacy. However, the current MSC biomarkers do not clearly define the heterogeneity of ADSC subgroups. Therefore, there is an urgent need to elucidate potential molecular markers for different ADSC subgroups and their heterogeneity. To enhance the accuracy of the analysis, we combined data from the Gene Expression Omnibus database (GSM3717978), the Sequence Read Archive database (SRR715485), and the Array Express database (E-MTAB-6677) to integrate and extract ADSCs scRNA-seq data from multiple centers for analysis. Finally, we categorized ADSCs into stemness and functional subgroups, with significant differential expression of TNFAIP6 observed between the two subgroups. TNFAIP6 is a glycoprotein with a molecular weight of 35–38 kDa, which plays an important role in maintaining the stemness and biological characteristics of MSCs [ 57 ]. Given the important role of the let-7 family in promoting stem cell differentiation [ 58 ], we hypothesize that the functional subgroup of ADSCs is the primary subgroup secreting let-7c-5p and that TNFAIP6 is the key marker for distinguishing ADSCs subgroups. In subsequent experiments, we successfully isolated the TNFAIP6 − ADSCs. As expected, the differentiation ability of TNFAIP6 − ADSCs was significantly weaker than that of TNFAIP6 + ADSCs, while the expression of let-7c-5p in TNFAIP6 − ADSC-sEVs was significantly higher than that in TNFAIP6 + ADSC-sEVs. In vivo and in vitro experiments also showed that TNFAIP6 − ADSC-sEVs significantly improved the quality of tendon healing through let-7c-5p. In previous studies, small extracellular vesicles have been found to exert their biological functions through local or intravenous injection. Currently, no practical carrier is available for in vivo treatment with small extracellular vesicles. GelMA, a photosensitive biohydrogel widely used in biomedicine due to its excellent biocompatibility and degradability, was selected as a carrier in this study [ 59 , 60 ]. GeIMA targeted the therapeutic position of ADSC-sEVs, and its sustained release effect enhanced the bioavailability of ADSC-sEVs. In addition, we significantly improved the original therapeutic effect by constructing TNFAIP6 − ADSC-sEVs with high let-7c-5p expression. Therefore, constructing conditional ADSC-sEVs to optimize their original therapeutic effects may be a potential strategy for small extracellular vesicle therapy in the future. Admittedly, this study had some limitations. First, the pathogenesis of tendon injury is complex. We validated the benefits of ADSC-sEVs only in a rat tendon injury model, which still needs to be confirmed in clinical trials. Second, we only observed short-term tendon healing quality, and long-term tendon treatment effects require further study. Finally, we successfully constructed and validated the superiority of TNFAIP6 − ADSC-sEVs in treating tendon injuries. However, further exploration is needed to determine the specific reasons for the high expression of let-7c-5p in TNFAIP6 − ADSC-sEVs. Conclusions In conclusion, this study explores the potential molecular mechanisms involved in tendon injury treatment using GelMA-loaded TNFAIP6 − ADSC-sEVs, focusing on the regulation of let-7c-5p expression. We combined scRNA-seq data to identify the key role of the TNFAIP6 − ADSCs subgroup in the treatment of tendon injury with ADSCs. Our results suggest that TNFAIP6 − ADSC-sEVs regulate the biological characteristics of TSCs through the let-7c-5p/CRCT1/JAK2/STAT3 signaling pathway, thereby promoting high-quality tendon healing. Additionally, this study provides a reference for constructing GelMA-loaded conditional ADSC-sEVs to optimize their therapeutic effects, which also provides new ideas for cell-free regenerative medicine. Declarations Acknowledgements We would like to acknowledge the reviewers for their helpful comments on this paper. The authors declare that they have not use AI-generated work in this manuscript. Author contributions HCL, ADZ, and MYS contributed to cytology experiment, animal experiments, data acquisition, and manuscript writing; JYZ and TTZ revised this manuscript and analyzed the data; WJL, ZMZ, and ZNZ revised and editing this manuscript; YW and YBM guided the experiment, conceptualization; SYW and LMH provided experimental technical support and final approval of manuscript; QBC and ZZL took part in the experimental design, text revision, and final approval of manuscript. Funding This study was supported by the Natural Science Foundation of Zhejiang Provincial (LQN25H170001), National Natural Science Foundation of China (81871837, 81572117), the Natural Science Foundation of Anhui Provincial (2308085QH259), and the Specialized Research Fund for Doctoral Programs in Colleges and Universities of China (20132307110007). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate This study received ethical approval from the Ethics Committee of Harbin Medical University (Mechanism of regulation of tendon stem cells differentiation through small extracellular vesicles) on Feb 23, 2018, with approval number Ky2018-135. 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Supplementary1GelMA.pdf Supplementary2WesternBlot.pdf Supplementary3ARRIVE.pdf GA.tif Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Editorial decision: Revision requested 17 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviews received at journal 27 Aug, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviewers agreed at journal 24 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 11 Aug, 2025 Submission checks completed at journal 10 Aug, 2025 First submitted to journal 02 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":false,"prefix":"","firstName":"Shuyao","middleName":"","lastName":"Wang","suffix":""},{"id":500700835,"identity":"b79ee18f-4586-4a16-9cb5-45efb4d2e04d","order_by":11,"name":"Limin Hou","email":"","orcid":"","institution":"The Sixth Hospital Affiliated to Harbin Medical University, HarbinMedical University","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Hou","suffix":""},{"id":500700837,"identity":"f1e1d682-e895-40da-8e4f-73ef2c1f8cba","order_by":12,"name":"Qingbo Cui","email":"","orcid":"","institution":"The Sixth Hospital Affiliated to Harbin Medical University, HarbinMedical University","correspondingAuthor":false,"prefix":"","firstName":"Qingbo","middleName":"","lastName":"Cui","suffix":""},{"id":500700839,"identity":"4c4de62c-7e45-418a-8728-bda50e60e43b","order_by":13,"name":"Zhaozhu Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDACCSBmbGCQY5z/+MCBDz9I0GLM3JCWeHBmDwlaEtsbcowPc7ARoUN+dvPDhz93HGbsbTjz4TADD4M8v9gB/FoY5xwzNpA8c5hZsrF3w+ECCwbDmbMT8GthlkgwkzBsO8xm2My74fAMHoYEg9sEtLBJpH+TSGw7zGN/jOfBYR42IrTwSOSYSRxsOyzB2MPDQJwWCYmcYsPGtnQDxhlsBsBAliDsF/kZ6Rsf/myzrm+cwfz4w4cfNvL80gS0YNhKmvJRMApGwSgYBdgBAFmYRsQ2sNr5AAAAAElFTkSuQmCC","orcid":"","institution":"The Sixth Hospital Affiliated to Harbin Medical University, HarbinMedical University","correspondingAuthor":true,"prefix":"","firstName":"Zhaozhu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-08-02 15:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7279057/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7279057/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04789-2","type":"published","date":"2025-12-30T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89393744,"identity":"6e663c6d-3993-41b1-a87a-6580df105ed5","added_by":"auto","created_at":"2025-08-19 13:26:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788285,"visible":true,"origin":"","legend":"\u003cp\u003eThe miRNA expression in different MSC-sEVs. (\u003cstrong\u003eA\u003c/strong\u003e) The high-throughput sequencing of miRNA expression in ADSC-sEVs, sorted by total read counts. (\u003cstrong\u003eB\u003c/strong\u003e) The high-throughput sequencing of miRNA expression in TSC-sEVs, sorted by total read counts. Data are presented as mean ± standard deviation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/f0a62e6450041b008d8f335f.png"},{"id":89396690,"identity":"953dce2c-fc05-4ac7-94b0-65add1e7c618","added_by":"auto","created_at":"2025-08-19 13:42:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":894500,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of different ADSC-sEVs. (\u003cstrong\u003eA\u003c/strong\u003e) Morphology of ADSC-sEVs, let-7c-5p-mimic ADSC-sEVs, and let-7c-5p-inhibitor ADSC-sEVs under a transmission electron microscope. (\u003cstrong\u003eB\u003c/strong\u003e) The particle size distribution of ADSC-sEVs, let-7c-5p-mimic ADSC-sEVs, and let-7c-5p-inhibitor ADSC-sEVs. (\u003cstrong\u003eC\u003c/strong\u003e) The surface markers of ADSC-sEVs, let-7c-5p-mimic ADSC-sEVs, and let-7c-5p-inhibitor ADSC-sEVs were detected by western blot. Bars 100nm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/dca232f8976134a35a9f9198.png"},{"id":89394217,"identity":"cc1b0b53-1e89-4509-9392-ccd0f91a83c5","added_by":"auto","created_at":"2025-08-19 13:34:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2880874,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ADSC-sEVs with different let-7c-5p expression on the biological characteristics of TSCs. (\u003cstrong\u003eA\u003c/strong\u003e) The expression of let-7c-5p in different ADSC-sEVs. (\u003cstrong\u003eB, E\u003c/strong\u003e) Effect of different ADSC-sEVs on the proliferation of TSCs by EdU assays. (\u003cstrong\u003eC, D, F, G\u003c/strong\u003e) Effect of different ADSC-sEVs on the migration of TSCs by transwell and scratch assays. (\u003cstrong\u003eH-K\u003c/strong\u003e) Western blot analysis of protein levels of SCXA, TNMD, and COL Ⅰ induced by different ADSC-sEVs. Bars, 100 μm. Data are represented as mean ± SD. *vs ADSC-sEVs group; \u003cem\u003en\u003c/em\u003e = 3. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/7acc469d2627a43ff2c0d92c.png"},{"id":89393749,"identity":"bb791c41-73eb-4ff8-92ae-c3932bf85c5d","added_by":"auto","created_at":"2025-08-19 13:26:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1453341,"visible":true,"origin":"","legend":"\u003cp\u003eLet-7c-5p targets the expression of CRCT1. (\u003cstrong\u003eA\u003c/strong\u003e) Venn diagram of let-7c-5p potential target genes in the TargetScan, DIANA-microT and miRanda databases. (\u003cstrong\u003eB\u003c/strong\u003e) Schematic diagram of dual luciferase reporter plasmid construction. WT, wild-type; MUT, mutant. (\u003cstrong\u003eC\u003c/strong\u003e) Columnar statistics for relative luciferase activity. (\u003cstrong\u003eD\u003c/strong\u003e) Expression level of CRCT1 after treatment with let-7c-5p-mimic/inhibitor. (\u003cstrong\u003eE, F\u003c/strong\u003e) Bioinformatics analysis of CRCT1-related proteins and signaling pathways.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/1d8c78244b15959e719cf899.png"},{"id":89393759,"identity":"83248c76-1866-41cb-8891-6cacbe52ebb4","added_by":"auto","created_at":"2025-08-19 13:26:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6570272,"visible":true,"origin":"","legend":"\u003cp\u003eADSC-sEVs promote the proliferation, migration, and tenogenic differentiation of TSCs via let-7c-5p/CRCT1/JAK2/STAT3 pathway. (\u003cstrong\u003eA-C\u003c/strong\u003e) Western blot analysis of protein levels of p-JAK2 and p-STAT3 expression in si-CRCT1 TSCs induced by let-7c-5p-inhibitor ADSC-sEVs. (\u003cstrong\u003eD-F\u003c/strong\u003e) AG490 inhibits the activation of p-JAK2 and p-STAT3 induced by let-7c-5p-inhibitor ADSC-sEVs. (\u003cstrong\u003eG, H\u003c/strong\u003e) EdU assay showed that AG490 enhanced the proliferation of let-7c-5p-inhibitor ADSC-sEVs on TSCs. (\u003cstrong\u003eI-L\u003c/strong\u003e) Transwell and scratch assays showed that AG490 enhanced the migration of let-7c-5p-inhibitor ADSC-sEVs on TSCs. (\u003cstrong\u003eM-P\u003c/strong\u003e) Western blot analysis of protein levels of SCXA, TNMD, and COL Ⅰ promoted by let-7c-5p-inhibitor ADSC-sEVs were enhanced by AG490. Bars, 100 μm. Data are represented as mean ± SD. *vs ADSC-sEVs group; \u003csup\u003e#\u003c/sup\u003evs let-7c-5p-inhibitor ADSC-sEVs; \u003cem\u003en\u003c/em\u003e=3. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/715fa41a3163d431aeda86a6.png"},{"id":89394216,"identity":"24c4cd66-b903-4f1d-824e-52164545236e","added_by":"auto","created_at":"2025-08-19 13:34:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6017214,"visible":true,"origin":"","legend":"\u003cp\u003eApplication and release characteristics of GelMA-loaded ADSC-sEVs in animal tendon injury model. (\u003cstrong\u003eA\u003c/strong\u003e) Steps for ultracentrifugal extraction of ADSC-sEVs. (\u003cstrong\u003eB\u003c/strong\u003e) The preparation method of GelMA. (\u003cstrong\u003eC\u003c/strong\u003e) The scanning electron microscopy image of GelMA-loaded ADSC-sEVs (ADSC-sEVs are shown by black arrow). Bars, 10 μm. (\u003cstrong\u003eD\u003c/strong\u003e) Profile of ADSC-sEVs released from the GelMA. (\u003cstrong\u003eE\u003c/strong\u003e) Treatment of GelMA-loaded ADSC-sEVs in animal tendon injury model. (\u003cstrong\u003eF\u003c/strong\u003e) In vivo imaging shew the retention time of PKH26-labeled ADSC-sEVs at the site of the tendon injury by loading GelMA.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/1f9ed10cf64d1fb1c7a43e31.png"},{"id":89394222,"identity":"0401ec20-b615-402d-ae07-e9b5da2ffb62","added_by":"auto","created_at":"2025-08-19 13:34:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5966853,"visible":true,"origin":"","legend":"\u003cp\u003eLet-7c-5p improved the healing of tendon injury. (\u003cstrong\u003eA\u003c/strong\u003e) The H\u0026amp;E staining and\u0026nbsp; immunohistochemistry assay (SCX, TNMD, COL Ⅰ, and CD146) of tendon injury at week 2 (n=6) and week 4 (n=6). (\u003cstrong\u003eB, C\u003c/strong\u003e) Quantitative analysis of tenogenic related factors at week 2 (n=6) and week 4 (n=6). (\u003cstrong\u003eD-G\u003c/strong\u003e) Results of biomechanical tests (failure load, stiffness, and Young’s modulus) at 4 weeks (n=6). Bars (H\u0026amp;E), 100 μm; bars (immunohistochemistry), 50 μm. Data are represented as mean ± SD. *vs Control group; \u003csup\u003e#\u003c/sup\u003evs ADSC-sEVs; n=6. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/5775c041767386ae453301af.png"},{"id":89394219,"identity":"7a868282-ff5b-4409-b969-1ff9c198c884","added_by":"auto","created_at":"2025-08-19 13:34:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1091835,"visible":true,"origin":"","legend":"\u003cp\u003eLet-7c-5p inhibits inflammatory expression in tendon injury. (\u003cstrong\u003eA\u003c/strong\u003e) The expression of CCR7\u003csup\u003e+\u003c/sup\u003e, CD163\u003csup\u003e+\u003c/sup\u003e, IL-6\u003csup\u003e+\u003c/sup\u003e, and IL-10\u003csup\u003e+\u003c/sup\u003e cells were detected by immunofluorescence at week 1. (\u003cstrong\u003eB\u003c/strong\u003e) Positive ratio of inflammation-related factors (n = 6). Bars 50 μm. Data are represented as mean ± SD. *vs Control group; \u003csup\u003e#\u003c/sup\u003evs ADSC-sEVs; n=6. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/05b6d6aa4f62fe2b70a5585d.png"},{"id":89394228,"identity":"ec9775dc-b7d5-4e20-b667-1946746070eb","added_by":"auto","created_at":"2025-08-19 13:34:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4727599,"visible":true,"origin":"","legend":"\u003cp\u003eThe scRNA-seq data analysis of ADSCs. (\u003cstrong\u003eA-C\u003c/strong\u003e) The multicenter joint analysis of scRNA-seq data from adipocyte populations. (\u003cstrong\u003eD, E\u003c/strong\u003e) tSNE 2D cell map displaying the clustering of scRNA-seq data (GSM3717978, SRR715485, and E-MTAB-6677) (\u003cstrong\u003eF\u003c/strong\u003e) tSNE 2D cell map colored by cell cluster identification. (\u003cstrong\u003eG\u003c/strong\u003e) Analysis of ADSCs subgroups (stemness and functional) based on scRNA-seq data. (\u003cstrong\u003eH-J\u003c/strong\u003e) Individual gene tSNE and violin plots showing the expression levels and distribution of representative marker genes.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/2e2cc5ca2a7aa436da2e34ec.png"},{"id":89394279,"identity":"8537d720-15f6-4ed7-863a-c1f1da2a5dd3","added_by":"auto","created_at":"2025-08-19 13:34:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":16126391,"visible":true,"origin":"","legend":"\u003cp\u003eTNFAIP6\u003csup\u003e- \u003c/sup\u003eADSC-sEVs improved the healing of tendon injury through let-7c-5p. (\u003cstrong\u003eA-D\u003c/strong\u003e) Isolation and characterization (adipogenesis, osteogenic, and chondrogenesis) of TNFAIP6\u003csup\u003e-\u003c/sup\u003e and TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs. (\u003cstrong\u003eE\u003c/strong\u003e) The expression levels of let-7c-5p in TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs and TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs. (\u003cstrong\u003eF-O\u003c/strong\u003e) The effect of TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs and TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs on the proliferation, migration, and tenogenic differentiation of TSCs. (\u003cstrong\u003eP-T\u003c/strong\u003e) The therapeutic effects of TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs and TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs on tendon injuries in vivo. Bars (cells), 100 μm; bars (H\u0026amp;E), 100 μm; bars (immunohistochemistry), 50 μm. Data are represented as mean ± SD. *vs TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs group; n (cell) = 3, \u003cem\u003en \u003c/em\u003e(immunohistochemistry) \u003cem\u003e= 6. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/2f16e4e2440ba6664dc7740c.png"},{"id":99545388,"identity":"dbea851b-2767-44ab-9feb-d27cb17e3f63","added_by":"auto","created_at":"2026-01-05 16:06:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":44557037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/a3acd158-c52d-4e0b-97f0-71087dc16468.pdf"},{"id":89394215,"identity":"183bdb01-6e72-4b02-82ff-da3355acb103","added_by":"auto","created_at":"2025-08-19 13:34:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12982,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/f2485560c2eafd61c4850e75.docx"},{"id":89393756,"identity":"7d1be3c7-9909-47fe-9d95-c242d8588d9e","added_by":"auto","created_at":"2025-08-19 13:26:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13329,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/bb4d75d9c8e93dafb96e7e74.docx"},{"id":89393762,"identity":"471ab2f8-f80d-4050-ae94-f8e7beedac6c","added_by":"auto","created_at":"2025-08-19 13:26:07","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1423448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 \u003c/strong\u003eThe characteristics of ADSCs and internalization of ADSC-sEVs. (\u003cstrong\u003eA\u003c/strong\u003e) The Morphology of ADSCs. (\u003cstrong\u003eB\u003c/strong\u003e) Adipogenesis, osteogenic and chondrogenesis differentiation of ADSCs. (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometry for detection of ADSC surface markers. (\u003cstrong\u003eD\u003c/strong\u003e) PKH26-labeled ADSC-sEVs internalization by TSCs. Bars, 100μm.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/aab76bd32f8acd541f2c1a71.tif"},{"id":89393752,"identity":"fd2eceba-9777-4335-b740-3f94ee76bc29","added_by":"auto","created_at":"2025-08-19 13:26:07","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":688755,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary1GelMA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/94a533f75f381a8538c1aa50.pdf"},{"id":89393750,"identity":"3a347636-aec0-4505-a5e6-a154961861cc","added_by":"auto","created_at":"2025-08-19 13:26:06","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3333133,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary2WesternBlot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/1c1e6c29b9bee351e9806a15.pdf"},{"id":89397767,"identity":"16331f16-51cb-4cd1-8528-9176f9e2f553","added_by":"auto","created_at":"2025-08-19 13:50:07","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":297005,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary3ARRIVE.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/0f7009a851088b5768ac35c0.pdf"},{"id":89393763,"identity":"94304d90-0cc5-4fb9-b8ea-99bfbb627d2e","added_by":"auto","created_at":"2025-08-19 13:26:07","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9527514,"visible":true,"origin":"","legend":"","description":"","filename":"GA.tif","url":"https://assets-eu.researchsquare.com/files/rs-7279057/v1/c36a2696538e8258ce6bd7f3.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSmall extracellular vesicles originating from TNFAIP6-ADSCs subpopoulation identified by single-cell RNA sequencing promote tendon healing\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTendons are integral structures within the musculoskeletal system, playing a crucial role in maintaining and regulating the biomechanics of the body [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The unique fiber structure of tendons contributes to their robust mechanical load performance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, certain sports activities or external forces can lead to tendon injuries, affecting approximately 30\u0026nbsp;million patients annually and resulting in a substantial medical expenditure burden of up to \u003cspan\u003e$\u003c/span\u003e180\u0026nbsp;billion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The management of tendon injuries has become a global health problem. Moreover, the sparse distribution of blood vessels and the low metabolic rate of cells in tendon tissues make restoring their original biological function difficult, rendering them susceptible to re-rupture even after healing [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite advancements in surgical techniques and rehabilitation methods for tendon repair, mitigating long-term complications and re-rupture remains challenging, leading to premature career terminations for many renowned athletes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, there is a pressing need to develop high-quality tendon repair methods.\u003c/p\u003e\u003cp\u003eTendon stem cells (TSCs) are unique cells with a remarkable capacity for self-renewal and differentiation and play a crucial role in tendon repair [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. TSCs undergo extensive proliferation within a short period following tendon injury and are highly prone to tenogenic differentiation during tendon healing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, adjusting the activity of TSCs is anticipated to enhance tendon healing and minimize the need for surgery. Mesenchymal stromal cell (MSC) transplantation shows promise as a cutting-edge biological therapy method, given MSC\u0026rsquo;s ability to regulate immune responses, improve the extracellular microenvironment, and activate endogenous stem cells [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For instance, Uysal et al. demonstrated that transplanting adipose-derived mesenchymal stromal cells (ADSCs) into injured tendons effectively promotes primary tendon healing [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, limitations such as heterogeneity between cell populations, low cell retention, and ectopic osteogenesis persist in MSC transplantation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, small extracellular vesicles, as the primary carriers of MSCs, have become a focal point in cell-free biotherapy research.\u003c/p\u003e\u003cp\u003eSmall extracellular vesicles (sEVs) are membrane-bound extracellular vesicles facilitating cellular communication through processes such as endocytosis, membrane fusion, or receptor-ligand interactions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. MSC-sEVs exhibit therapeutic potential across several diseases [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In a previous study, we utilized gelatin methacryloyl (GelMA) loaded ADSC-sEVs for the first time to treat tendon injuries [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our findings revealed that treatment with ADSC-sEVs loaded with GelMA effectively regulated the biological characteristics of TSCs (proliferation, migration, and tenogenic differentiation), significantly improving tendon healing quality. Small extracellular vesicles, being lipid-rich structures, harbor diverse bioactive molecules, such as proteins, mRNAs, miRNAs, and DNAs. While the specific mechanisms through which small extracellular vesicles endogenously regulate the biological characteristics of TSCs are not fully understood, miRNAs have been identified as key players. As integral components of gene regulatory networks, miRNAs mediate the effects of small extracellular vesicles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, as MSCs consist of a heterogeneous population of cells, there is limited research exploring the functions and therapeutic effects of ADSC-sEVs released by different ADSC subsets.\u003c/p\u003e\u003cp\u003eThis study aimed to identify critical subsets and miRNAs in ADSC-sEVs that contribute to tendon healing. Through second-generation sequencing technology and bioinformatics analysis, we predicted and verified that TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs regulate the biological characteristics of TSCs through the let-7c-5p-mediated CRCT1/JAK2/STAT3 signaling pathway. Overall, based on cellular heterogeneity, our study presents a concept of constructing GelMA-loaded conditioned ADSC-sEVs to enhance the treatment of tendon injury, which also provides new ideas for cell-free regenerative medicine.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eA total of 108 male SD rats (8\u0026ndash;10 weeks, 180\u0026ndash;230 g) were sourced from the Animal Experimental Center of Harbin Medical University. Experimental SD were given an adequate diet and maintained on a regular 12-h light/dark cycle. This study followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the ethics committee (No.ky2018-135).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and culture of ADSCs and TSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing methods employed in previous studies, TSCs and ADSCs were isolated from the patellar tendon and inguinal adipose tissues of SD rats using collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) digestion [19]. TSCs were cultured in DMEM (Invitrogen, Carlsbad, CA, USA), and ADSCs were cultured in DMEM/F12 (Invitrogen). Each culture medium was supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin (Beyotime, Shanghai, China). The cells were cultured in a cell incubator at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e, with cells from the 3rd to 5th passages selected for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and identification of ADSC-sEVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation method for ADSC-sEVs mirrored that of a previous study [19]. Briefly, ADSCs were incubated with an small extracellular vesicle-free medium (Biological Industries) for 24 h, and ADSC-sEVs in the supernatant were isolated through differential centrifugation (300 g, 10 min; 3000 g, 10 min; 10000 g, 30 min; 100000 g, 2 h). ADSC-sEVs were identified using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting. ADSC-sEVs were stained with the lipophilic membrane dye PKH26 (Sigma-Aldrich) for tracking experiments. GelMA loaded with 100 \u0026mu;g ADSC-sEVs was immersed in PBS for the ADSC-sEVs release analysis. The supernatant was collected every 24 h and the extent of release was performed using the BCA protein assay kit (Beyotime, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection of cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression in ADSCs was regulated through transfection using Lipofectamine 3000 (Invitrogen). Let-7c-5p mimic, let-7c-5p inhibitor, and their corresponding negative controls were designed by GenePharma. For transfection, 7.5 \u0026mu;L of Lipofectamine 3000 was incubated with 75 \u0026mu;L of RNAs (let-7c-5p mimics, let-7c-5p inhibitors, and their negative controls) in 250 \u0026mu;L of MEM (Invitrogen) for 15 min and then added to ADSCs for 48 h incubation period. Similarly, third-generation TSCs were transfected using Lipofectamine 3000 (small interfering RNAs against CRCT1 and negative control were purchased from GenePharma). The RNA sequences are presented in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential downstream target CRCT1 of let-7c-5p was predicted through TargetScan, DIANA-microT, and miRanda databases. We constructed the wild and mutant-type (WT/MUT-CRCT1) pmirGLO luciferase reporter plasmids for the CRCT1-3\u0026rsquo; UTR. Using Lipofectamine 3000, we transfected these plasmids along with let-7c-5p mimics and their respective negative controls into TSCs. After 24 h of transfection, fluorescence was detected using the Dual-Luciferase Reporter Assay Kit (Beyotime).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of TSCs with different ADSC-sEVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSCs were seeded in 6-well plates at a cell density of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e/well to investigate the impact of various ADSC-sEVs. Each group of TSCs was treated with 50 \u0026mu;g/mL of different ADSC-sEVs. The TSCs were randomly categorized into six groups as follows: (1) Control: TSC medium was replaced with an small extracellular vesicle-free medium. (2) ADSC-sEVs: normal ADSC-sEVs were added to the small extracellular vesicle-free medium. (3) NC-mimic ADSC-sEVs: mimic-negative controls transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (4) let-7c-5p-mimic ADSC-sEVs: let-7c-5p-mimic transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (5) NC-inhibitor ADSC-sEVs: inhibitor-negative controls transfected ADSCs derived small extracellular vesicles were added to the small extracellular vesicle-free medium. (6) let-7c-5p-inhibitor ADSC-sEVs: let-7c-5p-inhibitor transfected ADSCs-derived small extracellular vesicles were added to the small extracellular vesicle-free medium. Subsequently, we explored the regulation of let-7c-5p on downstream targets and introduced inhibitor-let-7c-5p ADSC-sEVs and AG490 (a JAK2/STAT3 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA) to TSCs transfected with si-CRCT1 or siNC. Additionally, we treated TSCs with TNFAIP6\u003csup\u003e+\u003c/sup\u003e and TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs to further explore the signaling pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proliferation capacity of TSCs was assessed by Edu Assay Kit (UE, China). TSCs in each group were pretreated with 50 \u0026mu;mol/L EdU for 4 h. Following fixation with 4% paraformaldehyde, the Click-iT EdU working solution was prepared according to the manufacturer\u0026rsquo;s instructions. Subsequently, 5 \u0026mu;g/mL Hoechst 33342 (UE) was used to label nuclei for 20 min, and the cell proliferation was counted using a fluorescent fiber microscope (Leica, Wetzlar, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the upper chamber of the Transwell plate, TSCs (1x10\u003csup\u003e4\u003c/sup\u003e) were seeded, and an small extracellular vesicle-free medium containing different ADSC-sEVs was added to the lower chamber based on the experimental groups. After incubation at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h, the TSCs in the upper chamber were stained with 0.1% crystal violet, and the extent of TSCs migration was counted under a light microscope (Leica).\u003c/p\u003e\n\u003cp\u003eAdditionally, a cell scratch assay was performed to evaluate TSCs migration. TSCs were seeded in 6-well plates with a cell density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well and cultured overnight at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. The assay involved scratching a straight line in the cultured cells and adding different ADSC-sEVs to an small extracellular vesicle-free medium. Changes in wound healing at 0 h and 24 h were recorded to evaluate the migration of TSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from TSCs using RIPA buffer (Beyotime), and 20 \u0026mu;g of protein from each group was selected for analyses. Immunoblotting was conducted with primary antibodies: anti-CD9 (ab92726; Abcam, Cambridge, UK), abti-TSG101 (ab125011; Abcam), anti-Hsp70 (ab2787; Abcam), anti-scleraxis (DF13293; Affinity Biologicals, Ancaster, ON, Canada), anti-tenomodulin (DF13715; Affinity), anti-collagen I (AF7001; Affinity), anti-phospho(p)-JAK2 (AF3024; Affinity), anti-JAK2 (AF6022; Affinity), anti-phospho(p)-STAT3 (AF3293; Affinity), anti-STAT3 (AF6294; Affinity), anti-TNFAIP6 (PA5-75332; Thermo Fisher, Massachusetts, USA), and a horseradish peroxidase-conjugated goat anti-rabbit IgG (BA1055; Boster, Wuhan, China) served as a secondary antibody. A chemiluminescence imaging system (ChemiScope 6200T, Clinx Science Instruments, Shanghai, China) was used for obtaining images. Protein bands were quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time polymerase chain reaction (qRT-PCR) analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA in TSCs was extracted using Trizol reagent (Beyotime), and total RNAs from small extracellular vesicles were extracted using a Total RNA Isolation Kit (Thermo). RNAs were reverse transcribed into cDNA using Premix Ex Taq II. The qRT-PCR primers were obtained from GenePharma (Table S2). RNA expression in the samples was assessed using real-time PCR with SYBR Green (Takara, Japan) on an ABI StepOnePlus system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental protocols and surgical procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 108 Sprague-Dawley rats were randomly divided into 5 groups: (1) Control (\u003cem\u003en\u003c/em\u003e = 36): Animals underwent partial patellar tendon resection surgery. (2) ADSC-sEVs (\u003cem\u003en\u003c/em\u003e = 24): Animals were treated with 200 \u0026mu;g ADSC-sEVs after partial patellar tendon resection surgery. (3) mimic-let-7c-5p ADSC-sEVs (\u003cem\u003en\u003c/em\u003e = 24): Animals were treated with 200 \u0026mu;g mimic-let-7c-5p ADSC-sEVs after partial patellar tendon resection surgery. (4) TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs (\u003cem\u003en\u003c/em\u003e = 12): Animals were treated with 200 \u0026mu;g TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs after partial patellar tendon resection surgery. (5) TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs (\u003cem\u003en\u003c/em\u003e = 12): Animals were treated with 200 \u0026mu;g TNFAIP6\u003csup\u003e-\u003c/sup\u003e ADSC-sEVs after partial patellar tendon resection surgery.\u003c/p\u003e\n\u003cp\u003eAll animals were anesthetized with 0.3% sodium pentobarbital (30 mg/kg) before surgery. The rat patellar tendon injury model was constructed as previously described [19]. Briefly, the median patellar tendon incision was made to remove the middle one-third of the patellar tendon tissue. GelMA (EFL-GM-60, 10% w/v) mixed with different ADSC-sEVs was then applied to the lesion and crosslinked into a gel state using ultraviolet light. Finally, the skin incision was closed using 4-0 sutures. Animals were euthanized by inhaling excessive carbon dioxide gas on days 7, 14, and 28, and patellar tendon tissue was extracted for subsequent studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological and immunohistochemical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatellar tendon tissues were fixed with 4% paraformaldehyde (Beyotime) and sectioned after paraffin embedding (0.4 \u0026mu;m). The tendon healing stage was evaluated using a light microscope after HE staining (Beyotime). For immunohistochemical analyses, tissue sections were stained with primary antibody: anti-SCXA (DF13293; Affinity), anti-TNMD (DF13293; Affinity), anti-collagen I (AF7001; Affinity), anti-CD146 (ab75769; Abcam), followed by incubation with a goat anti-rabbit IgG secondary antibody (ab6721; Abcam). For immunofluorescence analysis, tissue sections were incubated with primary antibodies: anti-CCR7 (ab32527; Abcam), anti-CD163 (ab182422; Abcam), anti-IL-6 (TA500067S; Origene), and anti-IL-10 ( ab33471; Abcam), followed by incubation with secondary antibodies (SA00013; ProteinTech, Chicago, IL, USA). The photos were taken with a DM4 B microscope (Leica). Three fields per section were selected randomly for statistical analysis. Positive signals were quantified with the ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiomechanical testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen obtaining rat patellar tendon tissue, the bones at both ends (patella-patellar tendon-tibia) were retained. The bones at both sides were fixed on a Zwick I Z010 (Bavaria,Germany) for mechanical testing. The load-displacement curve before patellar tendon rupture was recorded at a rate of 5 mm/min. Subsequently, the failure load (N) and stiffness (N/mm) were obtained by the testXpert software. Young\u0026rsquo;s modulus (N \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e2\u003c/sup\u003e) was calculated after measuring the cross-sectional area (mm\u003csup\u003e2\u003c/sup\u003e) of the tendon with a vernier caliper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection and analysis of single-cell RNA sequencing (scRNA-seq) data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rat inguinal adipose tissue scRNA-seq data were obtained from the Gene Expression Omnibus database (GSM3717978), Sequence Read Archive database (SRR715485), and Array Express database (E-MTAB-6677). The unique molecular identifier (UMI) counting matrix was generated using R software\u0026rsquo;s Seurat package. Subsequent analysis was performed after filtering out cell data with a mitochondrial ratio greater than 20% and detecting over 6000 genes. Finally, 20764 single cells remained (2902 cells in GSM3717978, 9644 cells in SRR715485, and 8218 cells in E-MTAB-6677), and they were applied in downstream analyses.\u003c/p\u003e\n\u003cp\u003eAfter quality control, the UMI count matrix was log normalized. Since sample from three samples were processed and sequenced in batches, sample was used to remove potential batch effect. In this process, top 2000 variable genes were used to create potential Anchors with FindIntgrationAnchors function of Seuart. Subsequently, IntegrateData function was used to integrate data and create a new matrix with 2000 features, in which potential batch effect was regressed out.\u003c/p\u003e\n\u003cp\u003eTo reduce the dimensionality of the scRNA-seq dataset, principal component analysis (PCA) was performed on an integrated data matrix. With Elbowplot function of Seurat, top 30 PCs were used to perform the downstream analysis. The main cell clusters were identified with the FindClusters function offered by Seurat with resolution set as default (res = 1.0). And then they were visualized with 2D tSNE or UMAP plots. Conventional markers described in a previous study were used to categorize every cell into a known biological cell type [22-24]. Firstly, 20764 cells were clustered into 10 major cell types. Subsequently, every major cell type was subset and further clustered into subclusters to detect heterogeneity within every cell type, respectively. The Seurat Findallmaker function was performed to identify preferentially expressed genes in clusters. Finally, the heterogeneity of different cell clusters in adipose tissue was detected based on the prioritized expression genes in the cell clusters. ADSC clusters\u0026rsquo; scRNA-seq data were extracted for further heterogeneity analysis and screened for different ADSCs subgroups and specific marker genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry sorting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 4\u0026times;10\u003csup\u003e6\u003c/sup\u003e 3rd generation ADSCs were collected and suspended in 2% FBS in PBS, followed by anti-TNFAIP6 for 1 h and FITC-conjugated anti-rabbit IgG secondary (S0008; Affinity) for 30 min, respectively. After filtering through a 300-mesh filter, TNFAIP6\u003csup\u003e-\u003c/sup\u003e/TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs were sorted using a flow cytometer (BD FACSMelody, Franklin Lakes, USA) and collected in a DMEM/F12 culture medium containing 20% FBS. After TNFAIP6\u003csup\u003e-\u003c/sup\u003e/TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs were amplified to 80%, the culture medium was discarded and replaced with an small extracellular vesicle-free medium for 24 h. Cell supernatant was collected, and TNFAIP6\u003csup\u003e-\u003c/sup\u003e/TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs were extracted using the above method. The adipogenic, osteogenic, and chondrogenic differentiation of various ADSC subpopulations was induced using assay kits from Cyagen, as described in our previous study [19].\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll values are expressed as means \u0026plusmn; standard deviation. Quantitative data for each group were analyzed using a one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test in Graphpad Pism 9.5 software. Statistical significance was set at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eCharacteristics of ADSCs and internalization of ADSC-sEVs\u003c/h2\u003e\u003cp\u003eADSCs grew adherent and showed a spindle-shaped morphology (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, Supporting Information). ADSCs can be induced in vitro to differentiate into adipogenesis, osteogenic, and chondrogensis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, Supporting Information).Flow cytometric analysis of ADSC surface markers (CD90- and CD105-positive, and CD34-, CD45-, and CD11b-negative) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, Supporting Information). The PKH26 staining tracer experiment showed that ADSC-sEVs could be successfully uptaken by TSCs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, Supporting Information).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eLet-7c-5p was highly expressed in ADSC-sEVs\u003c/h2\u003e\u003cp\u003eIn our previous investigations, we confirmed the efficacy of ADSC-sEVs in promoting the healing of tendon injuries [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To further explore the specific mechanism of action of ADSC-sEVs, we performed high-throughput sequencing of miRNA expression in ADSC-sEVs. The results showed that let-7c-5p was significantly upregulated in ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A parallel study on the biological functions of miRNAs in TSC-sEVs demonstrated a pronounced enrichment of let-7c-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This suggests that let-7c-5p in ADSC-sEVs has significant implications for TSCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eCharacteristics of different ADSC-sEVs\u003c/h2\u003e\u003cp\u003eADSC-sEVs, mimic-let-7c-5p ADSC-sEVs, and inhibitor-let-7c-5p ADSC-sEVs exhibited quasi-circular structures under TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). NTA showed that the diameters of these ADSC-sEVs were 110.3 nm, 112.7 nm, and 112.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Western blot analysis confirmed the expression of CD9, TSG101, and HSP70 on the surface of all three ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eRegulation of let-7c-5p expression in ADSC-sEVs on the biological characteristics of TSCs\u003c/h2\u003e\u003cp\u003eADSC-sEVs with different levels of let-7c-5p were obtained through transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The expression of let-7c-5p in the let-7c-5p-mimic group was significantly increased, whereas its expression was not significantly suppressed in the let-7c-5p-inhibitor group. This result may be related to the fact that miRNA inhibitors act by competitive binding inhibition. NC-mimic and NC-inhibitor served as control groups. TSCs were treated with five different ADSC-sEVs. The EdU assay showed that let-7c-5p-mimic ADSC-sEVs significantly enhanced the proliferation-promoting effect on TSCs, whereas the let-7c-5p-inhibitor group attenuated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, E). Transwell and scratch assays revealed that high let-7c-5p expression effectively promoted the TSCs migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, F, G). Additionally, let-7c-5p expression alterations influenced the tenogenic differentiation of TSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-K).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eCRCT1 was identified as the target of let-7c-5p\u003c/h2\u003e\u003cp\u003eTo explore downstream mechanisms, we predicted potential targets using the TargetScan, DIANA-microT, and miRanda databases. The results showed that CRCT1 was the only downstream target predicted by all three databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Therefore, we hypothesized that CRCT1 was the target gene of let-7c-5p. To verify this hypothesis, we constructed WT-CRCT1 and MUT-CRCT1 pmirGLO luciferase reporter plasmids and demonstrated targeting of let-7c-5p by CRCT1 using a dual-luciferase reporter assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We found that let-7c-5p mimics significantly reduced the fluorescence expression of WT-CRCT1 but had a lesser effect on MUT-CRCT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, we treated TSCs with let-7c-5p-inhibitor and NC-inhibitor ADSC-sEVs. qPCR results revealed that the expression of let-7c-5p was significantly correlated with that of CRCT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Next, we predicted the CRCT1-related proteins using STRING (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). KEGG analysis showed that the JAK/STAT signaling pathway was the most significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eLet-7c-5p/CRCT1 affected the biological characteristics of TSCs through JAK2/STAT3 signaling pathway\u003c/h2\u003e\u003cp\u003eWe introduced let-7c-5p-inhibitor ADSC-sEVs to TSCs transfected with si-CRCT1 or si-NC. Western blotting showed that a reduction in let-7c-5p content in ADSC-sEVs led to an increase in the expression of p-JAK2 and p-STAT3 in TSCs, suggesting that let-7c-5p/CRCT1 may exert its biological role through the JAK2/STAT3 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequently, we evaluated the effect of let-7c-5p/CRCT1/JAK2/STAT3 on TSCs using AG490. Western blotting showed that AG490 significantly inhibited the activation of the JAK2/STAT3 signaling pathway mediated by let-7c-5p-inhibitor ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;F). Pretreatment with AG490 also mitigated the enhancing effect of let-7c-5p-inhibitor ADSC-sEVs on the proliferation and migration abilities of TSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-L). Additionally, inhibition of the JAK2/STAT3 signaling pathway significantly reduced the effect of the let-7c-5p-inhibitor ADSC-sEVs on the tenogenic differentiation of TSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-P).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eRelease characteristics of ADSC-sEVs in the GelMA\u003c/h2\u003e\u003cp\u003eWe obtained different ADSC-sEVs using differential centrifugation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The photoinitiator LAP was used to initiate GelMA polymerization reaction, and the sieve-like biological scaffold structure which could be loaded with ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). The ADSC-sEVs release analysis in vitro suggested that the GelMA-loaded ADSC-sEVs were gradually released in 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similarly, the PKH26-labeled sEVs tracing experiments in vivo found that ADSC-sEVs were able to act locally for more than 7 days under GelMA loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). These results demonstrate that GelMA-loaded ADSC-sEVs can achieve a sustained release effect, which helped ADSC-sEVs exert greater therapeutic efficacy in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eGelMA-loaded let-7c-5p-mimic ADSC-sEVs promoted tendon healing in vivo\u003c/h2\u003e\u003cp\u003eWe subsequently administered ADSC-sEVs and let-7c-5p-mimic ADSC-sEVs to a rat patellar tendon injury model to evaluate their in vivo effects. HE staining revealed a more regular fibrous structure in the healing patellar tendon of the let-7c-5p-mimic ADSC-sEVs group compared to the ADSC-sEVs group at 2 and 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The let-7c-5p-mimic ADSC-sEVs group showed significantly higher expression of the tendon stem cell marker CD146 compared to the other two groups. Notably, the tendon stem cell marker CD146 displayed the highest expression in the let-7c-5p-mimic ADSC-sEVs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). These results suggest that let-7c-5p effectively promotes the proliferation and tenogenic differentiation of TSCs in vivo. We evaluated the therapeutic effects of let-7c-5p on injured tendons using biomechanical tests. The healing patellar tendon tissue in the let-7c-5p-mimic ADSC-sEVs group showed significant advantages in terms of failure load, stiffness, and Young\u0026rsquo;s modulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eGelMA-loaded let-7c-5p-mimic ADSC-sEVs reduced tissue inflammatory response in vivo\u003c/h2\u003e\u003cp\u003eFurthermore, we evaluated the effect of let-7c-5p on the early inflammatory response to tendon injury using immunofluorescence. The expression of inflammation-related factors in tendon tissues of the Control, ADSC-sEVs, and let-7c-5p-mimic ADSC-sEVs groups was assessed. The results indicated a significant decrease in the expression of IL-6 (a pro-inflammatory factor) in the let-7c-5p-mimic ADSC-sEVs group, while IL-10 (an anti-inflammatory factor) showed the opposite trend. Simultaneously, we found that the expression of CCR7 (an M1 macrophage marker) significantly decreased in the let-7c-5p-mimic ADSC-sEVs group, while CD163 (an M2 macrophage marker) significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTNFAIP6 as a key marker gene for the functional and stemness subgroups of ADSCs\u003c/h3\u003e\n\u003cp\u003eNext, we explored the promoting effect of ADSCs on tendon healing from the perspective of cellular heterogeneity. To improve the accuracy of the analysis results, we used a multicenter joint analysis of scRNA-seq data (GSM3717978, SRR715485, and E-MTAB-6677) from subcutaneous adipocyte populations in the groin of rats. After normalizing the obtained UMI matrix, the FindIntegrationAnchors function of the Seurat package was used to integrate three sets of scRNA-seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The results showed that the three cells group had very similar subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, C). Based on relevant references [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we integrated and re-annotated 10 cell groups, include Adipose derived stem cells, Pre-adipocytes, Aregs, Adipocytes, Endothelial cells, Smooth muscle cells, T cells, B cells, Macrophages, and Dendritic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-F). Subsequently, we extracted scRNA-seq data from ADSC cell populations for further analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). Based on the screening and analysis of maker genes, we categorized ADSCs into a stemness subgroup (maintaining stemness related genes, such as TNFAIP6, CEBPB, and MT1) and a functional subgroup (multidirectional differentiation ability and cytokine expression related genes, such as CXCL13, SFRP2, and BMP7) [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A significant difference was found in the expression of TNFAIP6 between the two ADSCs subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eH\u0026ndash;J).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eGelMA-loaded TNFAIP6- ADSC-sEVs promotes high-quality tendon healing through let-7c-5p\u003c/h2\u003e\u003cp\u003eUsing flow cytometry sorting technology, we successfully classified ADSCs into TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs and TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u0026ndash;C). Our analysis results show that the stemness of TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs is significantly stronger than that of TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eD). Next, we extracted TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e/TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs-sEVs and explored the expression levels of let-7c-5p in these small extracellular vesicles. As expected, let-7c-5p was significantly enriched in TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eE). The regulatory ability of TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs on tenogenic differentiation and biological characteristics (proliferation, migration, and tenogenic differentiation) in TSCs was also significantly better than that of TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eF\u0026ndash;O). Finally, we transplanted TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e/TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs-sEVs into the rat patellar tendon injury model. The results showed that TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs strongly promoted tendon repair in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eP\u0026ndash;T).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTendon injury is a common condition affecting the motor system. It poses challenges due to the slow and inefficient natural tendon repair process, leading to unsatisfactory long-term rehabilitation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Restoring the original physiological structure and biological characteristics of tendons poses an ongoing challenge for medical professionals. TSCs are a cell population with self-renewal and multidirectional differentiation potential in tendon tissue [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, Komatsu et al. observed that the transplantation of TSC sheets could significantly accelerate tendon healing and regeneration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies have shown that MSCs are beneficial in tissue healing, particularly through the secretion of small extracellular vesicles. Using cell-free biological therapy with MSC-sEVs has emerged as a promising approach in clinical applications [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].We previously found that TSC-sEVs could significantly enhance the biological activity of tendon cell population and improve the quality of tendon healing [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, it is not easy to extract TSCs from patients with preexisting tendon injuries. This makes it difficult for the application of TSC-sEVs to realize the transformation from basic research to clinical practice. Therefore, it is urgent to find other MSC-sEVs suitable for clinical application to optimize tendon healing. ADSCs are widely used in tissue engineering and regenerative medicine due to their abundant tissue sources, convenient cell separation, and strong cellular activity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In a previous study, we introduced ADSC-sEVs for the first time to address tendon injuries, resulting in a notable improvement in tendon healing quality [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given that ADSC-sEVs comprise a complex mixture of bioactive substances, this study explored the specific mechanisms and key components of ADSC-sEVs in treating tendon injuries. As key regulators in post-transcriptional gene expression, miRNAs play important roles in cell differentiation, biological development, and disease progression [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Liu et al. found that miRNA expression significantly impacts tendon growth and wound healing [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, in this study, we investigated miRNAs within ADSC-sEVs for the treatment of tendon injuries.\u003c/p\u003e\u003cp\u003eWe found that let-7c-5p was significantly enriched in ADSC-sEVs using high-throughput sequencing. Interestingly, in another study on TSC-sEVs, let-7c-5p also showed the most significant enrichment in TSC-sEVs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This led us to hypothesize that ADSC-sEVs primarily promote tendon repair through let-7c-5p. The let-7 family, one of the earliest discovered miRNAs, is predominantly involved in cell differentiation and metabolic regulation [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Wang et al. demonstrated that let-7c-5p promotes the osteogenic differentiation of BMSCs via the TGF-β signaling pathway [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, Wang et al. found that let-7c-5p and miRNA-21-5p in BMSC-sEVs promote the proliferation and migration of endothelial cells treated with rapamycin [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this study, we found that let-7c-5p effectively promoted the proliferation and migration of TSCs and induced their tenogenic differentiation. Pretreating ADSCs with the transfection method to increase the expression of let-7c-5p in ADSC-sEVs effectively improves the quality of tendon healing. These results suggest a significant role for let-7c-5p in the ADSC-sEVs treatment of tendon injuries.\u003c/p\u003e\u003cp\u003eTo further understand the role of let-7c-5p in the treatment of tendon injuries, we employed TargetScan, DIANA-microT, and miRanda databases to predict possible targets. CRCT1 was selected as the focus, as it was the sole target intersecting all three databases. CRCT1 is encoded by an epidermal differentiation complex and plays a vital role in epidermal differentiation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we confirmed that let-7c-5p targets CRCT1 using a dual-luciferase reporter assay. PCR results confirmed that ADSC-sEVs with low expression of let-7c-5p could alleviate CRCT1 inhibition in TSCs. Therefore, we hypothesize that the biological efficacy of ADSC-sEVs depends on let-7c-5p/CRCT1.\u003c/p\u003e\u003cp\u003eSubsequently, we predicted the downstream signaling pathways associated with let-7c-5p expression using bioinformatics. KEGG analysis showed that the JAK2/STAT3 pathway was potentially correlated with let-7c-5p/CRCT1. As a canonical signaling pathway present in various cells, the JAK2/STAT3 pathway is involved in numerous key biological processes in various diseases, such as tumors and inflammatory conditions [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In addition, Chen et al. showed that the JAK2/STAT3 signaling pathway plays a key role in regulating the aging process of TSCs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Given the potential impact of the JAK2/STAT3 pathway on tendon healing, we explored its changes during ADSC-sEVs treatment. Western blot analyses showed a significant correlation between p-JAK2, p-STAT3, and CRCT1. Subsequently, pretreatment of TSCs with AG490 (a JAK2/STAT3 inhibitor) and incubation with let-7c-5p-inhibitor ADSC-sEVs showed that AG490 reversed the efficacy of ADSC-sEVs on the biological characteristics of TSCs. Therefore, the JAK2/STAT3 signaling pathway plays a significant role in let-7c-5p/CRCT1 mediated ADSC-sEVs promotion of tendon healing by ADSC-sEVs.\u003c/p\u003e\u003cp\u003eTendon repair is an intricate process where SCX serves as a pivotal molecule in tendon development, crucial for regulating TSC differentiation and proliferation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. TNMD is identified as a key regulatory factor influencing the biological characteristics of TSCs, including proliferation, differentiation, aging, and tendon maturation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Dex et al. found that TNMD and collagen I are colocalized in the extracellular matrix, and the expression of collagen I positively affects the mechanical strength and function of tendons [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Building on our previous research, we increased the content of let-7c-5p in ADSC-sEVs to optimize the induction of TSCs tenogenic differentiation in this study. Furthermore, the expression of CD146 (TSCs maker) in tendons significantly and positively correlated with that of let-7c-5p in ADSC-sEVs. Similarly, let-7c-5p effectively promoted the proliferation and migration of TSCs in vitro, a process related to the let-7c-5p/CRCT1/JAK2/STAT3 signaling mechanism. In our previous study, ADSC-sEVs reduced early inflammatory responses by regulating macrophage polarization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Interestingly, recent studies have shown that let-7c-5p inhibits inflammatory expression [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Consequently, we explored the effect of let-7c-5p on early inflammation in tendon injury. As expected, immunofluorescence analysis showed that the increased expression of let-7c-5p effectively inhibited inflammatory factors in the early stages of tendon injury, a process potentially related to the polarization of M2 macrophages. Finally, we evaluated the quality of tendon healing through biomechanical testing. Tendon load, stiffness, and Young\u0026rsquo;s modulus in the mimic group showed the most significant improvement, indicating that let-7c-5p plays a crucial role in ADSC-sEVs-mediated promotion of tendon healing.\u003c/p\u003e\u003cp\u003eAs the research progressed, we found that the expression of let-7c-5p and its therapeutic effect on tendon injury varied between batches of ADSC-sEVs. Additionally, current clinical trials show fluctuations in the efficacy of ADSC-sEVs [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Based on Wang et al.\u0026rsquo;s study on the heterogeneity of MSCs [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], we speculate that competition among different subgroups of ADSCs during culture leads to imbalanced proportions and changes in ADSC-sEVs function and efficacy. However, the current MSC biomarkers do not clearly define the heterogeneity of ADSC subgroups. Therefore, there is an urgent need to elucidate potential molecular markers for different ADSC subgroups and their heterogeneity. To enhance the accuracy of the analysis, we combined data from the Gene Expression Omnibus database (GSM3717978), the Sequence Read Archive database (SRR715485), and the Array Express database (E-MTAB-6677) to integrate and extract ADSCs scRNA-seq data from multiple centers for analysis. Finally, we categorized ADSCs into stemness and functional subgroups, with significant differential expression of TNFAIP6 observed between the two subgroups. TNFAIP6 is a glycoprotein with a molecular weight of 35\u0026ndash;38 kDa, which plays an important role in maintaining the stemness and biological characteristics of MSCs [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Given the important role of the let-7 family in promoting stem cell differentiation [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], we hypothesize that the functional subgroup of ADSCs is the primary subgroup secreting let-7c-5p and that TNFAIP6 is the key marker for distinguishing ADSCs subgroups. In subsequent experiments, we successfully isolated the TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs. As expected, the differentiation ability of TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs was significantly weaker than that of TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSCs, while the expression of let-7c-5p in TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs was significantly higher than that in TNFAIP6\u003csup\u003e+\u003c/sup\u003e ADSC-sEVs. In vivo and in vitro experiments also showed that TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs significantly improved the quality of tendon healing through let-7c-5p.\u003c/p\u003e\u003cp\u003eIn previous studies, small extracellular vesicles have been found to exert their biological functions through local or intravenous injection. Currently, no practical carrier is available for in vivo treatment with small extracellular vesicles. GelMA, a photosensitive biohydrogel widely used in biomedicine due to its excellent biocompatibility and degradability, was selected as a carrier in this study [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. GeIMA targeted the therapeutic position of ADSC-sEVs, and its sustained release effect enhanced the bioavailability of ADSC-sEVs. In addition, we significantly improved the original therapeutic effect by constructing TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs with high let-7c-5p expression. Therefore, constructing conditional ADSC-sEVs to optimize their original therapeutic effects may be a potential strategy for small extracellular vesicle therapy in the future.\u003c/p\u003e\u003cp\u003eAdmittedly, this study had some limitations. First, the pathogenesis of tendon injury is complex. We validated the benefits of ADSC-sEVs only in a rat tendon injury model, which still needs to be confirmed in clinical trials. Second, we only observed short-term tendon healing quality, and long-term tendon treatment effects require further study. Finally, we successfully constructed and validated the superiority of TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs in treating tendon injuries. However, further exploration is needed to determine the specific reasons for the high expression of let-7c-5p in TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study explores the potential molecular mechanisms involved in tendon injury treatment using GelMA-loaded TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs, focusing on the regulation of let-7c-5p expression. We combined scRNA-seq data to identify the key role of the TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs subgroup in the treatment of tendon injury with ADSCs. Our results suggest that TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs regulate the biological characteristics of TSCs through the let-7c-5p/CRCT1/JAK2/STAT3 signaling pathway, thereby promoting high-quality tendon healing. Additionally, this study provides a reference for constructing GelMA-loaded conditional ADSC-sEVs to optimize their therapeutic effects, which also provides new ideas for cell-free regenerative medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the reviewers for their helpful comments on this paper. The authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCL, ADZ, and MYS contributed to cytology experiment, animal experiments, data acquisition, and manuscript writing; JYZ and TTZ revised this manuscript and analyzed the data; WJL, ZMZ, and ZNZ revised and editing this manuscript; YW and YBM guided the experiment, conceptualization; SYW and LMH provided experimental technical support and final approval of manuscript; QBC and ZZL took part in the experimental design, text revision, and final approval of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Zhejiang Provincial (LQN25H170001), National Natural Science Foundation of China (81871837, 81572117), the Natural Science Foundation of Anhui Provincial (2308085QH259), and the Specialized Research Fund for Doctoral Programs in Colleges and Universities of China (20132307110007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received ethical approval from the Ethics Committee of Harbin Medical University (Mechanism of regulation of tendon stem cells differentiation through small extracellular vesicles) on Feb 23, 2018, with approval number Ky2018-135. All animal experiments were conducted in accordance with the ARRIVE guidelines and adhered to the applicable regulations, such as the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYoshimoto Y, Oishi Y. 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Bioact Mater. 2021;8:267-95. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TNFAIP6, Adipose-derived mesenchymal stromal cells, Tendon stem cells, Small extracellular vesicles, Tendon healing","lastPublishedDoi":"10.21203/rs.3.rs-7279057/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7279057/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSmall extracellular vesicles originating from adipose-derived mesenchymal stromal cells (ADSC-sEVs) have excellent therapeutic value in tendon injury healing, but its mechanism and effect have not been fully elucidated. This study aimed to identify the key subsets and mechanisms involved in ADSC-sEVs contributing to tendon healing.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBased on our previous research of ADSC-sEVs improving the quality of tendon healing, we utilizing second-generation sequencing and bioinformatics methods to predict the key role of the TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSCs subgroup in the treatment of tendon injury. We constructed different ADSC-sEVs through transfection ADSCs and treated to tendon stem cells (TSCs) for further exploration. The EdU, cell scratch, and transwell assays were used for proliferation proliferation and migration assays. Western blot and qRT-PCR analyses were used for qualitative analysis. Histopathological, immunohistochemical and, biomechanical testing were used for in vitro validation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs significantly improves the therapeutic effect of ADSC-sEVs on tendon injury, which is related to the high expression of let-7c-5p. Based on application of different ADSC-sEVs in vitro and vivo, we identified CRCT1/JAK2/STAT3 as a key downstream signaling pathway regulated by let-7c-5p.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings contributed to deeper understanding of how TNFAIP6\u003csup\u003e\u0026minus;\u003c/sup\u003e ADSC-sEVs promote tendon healing through the let-7c-5p/CRCT1/JAK2/STAT3 signaling pathway. Furthermore, this study proposes a concept for constructing conditional ADSC-sEVs to enhance its inherent therapeutic effect.\u003c/p\u003e","manuscriptTitle":"Small extracellular vesicles originating from TNFAIP6-ADSCs subpopoulation identified by single-cell RNA sequencing promote tendon healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 13:26:01","doi":"10.21203/rs.3.rs-7279057/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-17T06:24:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T06:24:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T11:29:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331177547163986021872924773274935316187","date":"2025-09-13T11:10:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124519308856058479964790377564544189930","date":"2025-09-08T05:39:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T05:11:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315943255563317241927847050716613876643","date":"2025-08-26T12:02:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35068522184764934910829053631784616587","date":"2025-08-26T12:02:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168681183181070991525440360921067606579","date":"2025-08-24T15:31:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98321229123173128398671195630177423205","date":"2025-08-14T02:57:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207341800511589250321632847873963493026","date":"2025-08-14T02:44:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339820111613921027399832584981350576069","date":"2025-08-13T07:01:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-12T00:57:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T15:35:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T00:34:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-08-02T15:04:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"882edad8-c382-4740-8535-0124caefc805","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:01:53+00:00","versionOfRecord":{"articleIdentity":"rs-7279057","link":"https://doi.org/10.1186/s13287-025-04789-2","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-12-30 15:58:19","publishedOnDateReadable":"December 30th, 2025"},"versionCreatedAt":"2025-08-19 13:26:01","video":"","vorDoi":"10.1186/s13287-025-04789-2","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04789-2","workflowStages":[]},"version":"v1","identity":"rs-7279057","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7279057","identity":"rs-7279057","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}