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Bone marrow mesenchymal stem cells-derived exosomes promote tendon regeneration via facilitating the proliferation and migration of endogenous tendon stem/progenitor cells Huilei Yu , Jin Cheng , Weili Shi , Bo Ren , Fengyuan Zhao , Yuanyuan Shi , Peng Yang , Xiaoning Duan , Jiying Zhang , Xin Fu , Xiaoqing Hu , Yingfang Ao PII: DOI: Reference:

S1742-7061(20)30069-6 https://doi.org/10.1016/j.actbio.2020.01.051 ACTBIO 6574

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Acta Biomaterialia

Received date: Revised date: Accepted date:

16 September 2019 15 January 2020 31 January 2020

Please cite this article as: Huilei Yu , Jin Cheng , Weili Shi , Bo Ren , Fengyuan Zhao , Yuanyuan Shi , Peng Yang , Xiaoning Duan , Jiying Zhang , Xin Fu , Xiaoqing Hu , Yingfang Ao , Bone marrow mesenchymal stem cells-derived exosomes promote tendon regeneration via facilitating the proliferation and migration of endogenous tendon stem/progenitor cells, Acta Biomaterialia (2020), doi: https://doi.org/10.1016/j.actbio.2020.01.051

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Bone marrow mesenchymal stem cells-derived exosomes promote tendon regeneration via facilitating the proliferation and migration of endogenous tendon stem/progenitor cells Huilei Yu†, Jin Cheng†, Weili Shi, Bo Ren, Fengyuan Zhao, Yuanyuan Shi, Peng Yang, Xiaoning Duan, Jiying Zhang, Xin Fu, Xiaoqing Hu*, Yingfang Ao*

Institute of Sports Medicine, Beijing Key laboratory of Sports Injuries, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, P.R.China †

Huilei Yu and Jin Cheng contributed equally to this work.

*Corresponding

author:

Yingfang

Ao

and

Xiaoqing

Hu;

E-mail:

[email protected] (Yingfang Ao), [email protected] (Xiaoqing Hu); Tel.: (86) 10-82267390, fax: (86) 10-62010440. Address: Institute of Sports Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, P. R. China

Abstract Mesenchymal stem cells (MSCs)-derived exosomes are being increasingly focused as the new biological pro-regenerative therapeutic agents for various types of tissue injury. Here, we explored the potential of a novel exosome-based therapeutic application combined with a local fibrin delivery strategy for tendon repair. After discovering that bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exos) promoted the proliferation, migration and tenogenic differentiation of tendon stem/progenitor cells (TSPCs) in vitro, we embedded BMSCs-exos in fibrin and injected it into the defect area of rat patellar tendon, and the results showed that the exosomes could be controlled-released from the fibrin, retained within the defect area, and internalized by TSPCs. BMSCs-exos embedded in fibrin significantly improved the histological scores, enhanced the expression of mohawk, tenomodulin, and type I collagen, as well as the mechanical properties of neo-tendon, and also 1

promoted the proliferation of local TSPCs in vivo. Overall, we demonstrated the beneficial role of BMSCs-exos in tendon regeneration, and that fibrin-exosomes delivery system represents a successful local treatment strategy of exosomes. This study brings prospects in the potential application of exosomes in novel therapies for tendon injury. Statement of Significance Mesenchymal stem cells have been identified as a preferred approach in tissue regeneration. In this study, we reported bone marrow mesenchymal stem cells (BMSCs) promote the proliferation and migration of tendon stem/progenitor cells (TSPCs) via the paracrine signaling effect of the nanoscale exosomes. We also demonstrated that the application of BMSCs-derived exosomes might be a promising approach to activate the regenerative potential of endogenous TSPCs in tendon injured region, and fibrin-exosomes delivery system represents a successful local treatment strategy of exosomes.

Keywords: Mesenchymal stem cells; Exosomes; TSPCs; Fibrin glue; Tendon regeneration 1. Introduction Tendon injuries occur frequently during sports and other rigorous activities, the naturally healed tendon often has inferior mechanical properties and is susceptible to reinjury, and patients often suffer from long-term pain, discomfort, and even disability[1]. To date, functional healing of tendon injuries has been a great challenge, and effective therapeutic techniques for tendon repair are needed[2]. Recently, mesenchymal stem cells (MSCs)-derived exosomes (MSCs-exos) have been identified as a new therapeutic strategy in tissue regeneration and employed in myocardial infarction, stroke, limb ischemia, peri-natal hypoxic-ischemic brain injury, kidney injury and osteochondral injury [3, 4]. Exosomes are small, secreted 2

membrane vesicles with diameter of 30-150 nm, which are primarily thought to function as intercellular communication vehicles to transfer bioactive lipids, nucleic acids and proteins between cells to elicit biological responses in recipient cells [5]. MSCs-exos are able to function as paracrine mediators of parental MSCs in tissue regeneration and exert a series of therapeutic effects including promoting regeneration, modulating immunoreaction and alleviating degeneration[6]. MSCs-based therapy often accompanies with cell-related disadvantages including phenotype drifting and uncontrolled action of implanted cells, both of which are obstacles in clinical translation[1]. Therefore, the utilization of MSCs-exos might be a promising approach to avoid cell-related shortages and preserve the regenerative property of MSCs[7, 8]. MSCs have been exploited in improving the regeneration of injured tendon[9, 10], however, few researches focused on the effect of MSCs-exos on tendon repair and the related mechanism. A previous study demonstrated that the combination of co-cultured BMSCs and TSPCs is a better choice of cell source for tendon repair than BMSCs or TSPCs alone[11], and the cultural supernatant of BMSCs could increase the viability of tendon cells in vitro and improve the repair effect of rotator cuff tear[8], which is also a kind of tendon injury. Thus, the role of BMSCs in promoting tendon regeneration might be contributed by paracrine secretome, in which exosomes are involved. MSCs-exos were mostly injected into the circulation system or body cavities directly to play their regenerative role[6]; however, it is not feasible to apply exosomes in the same way for tendon injury. The free exosomes in aqueous solution is difficult to retain in the tendon injury site and would flow away or undergo rapid clearance[12]. Thus, a carrier of exosomes for tendon repair is required and should be provided with properties including sustained-release of exosomes, no side effect for internalization of exosomes, and appropriate degradation rate. Fibrin glue has been used as a cell carrier with well biocompatibility in tendon regeneration[13] and as an exosome carrier for the treatment of incisional hernia[14], but whether fibrin glue can be used as a carrier for BMSCs-exos in tendon regeneration has not been thoroughly explored yet. Hence, the aim of the present study is to explore the effect of BMSCs on TSPCs via exosomes, and the therapeutic effect of BMSCs-exos for tendon injury in vivo. Specifically, we investigated the following: (1) the effect of BMSCs-exos on the proliferation, migration and tenogenic differentiation of TSPCs; (2) the release of 3

BMSCs-exos embedded in fibrin glue and their internalization by TSPCs; (3) the therapeutic effect of BMSCs-exos embedded in fibrin glue in an established rat model of patellar tendon window defect. Our data suggested that BMSCs-exos embedded in fibrin glue represented a novel approach with great potential for promoting tendon regeneration after injury. 2. Materials and methods All animal experimental protocols were approved by the local Institutional Animal Care and Use Committee, complying with the “Guide for the Care and Use of Laboratory Animals” published by the National Academy Press (NIH Publication No. 85-23, revised 1996). 2.1. Isolation and identification of BMSCs and TSPCs Rat BMSCs were isolated according to the protocol previously reported [15]. Briefly, the bone marrow was isolated from the femur and tibia of Sprague-Dawley rat weighing 80 g. The cells from the bone marrow were incubated in α-minimal essential medium (α-MEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C with 5% humidified CO2. Non-adherent cells were removed by changing the culture medium after 3 days of incubation. After culturing for 4 to 5 days, adherent cells reached confluence and were defined as passage 0. Rat TSPCs

were

isolated

and

cultured

as

previously described

with

slight

modification[16]. Briefly, patellar tendon was excised from rat knees, the tendon sheath and surrounding paratenon were carefully removed, and tendon tissues were cut into small pieces and followed by digestion with 0.2% collagenase type I (Invitrogen, Carlsbad, CA, USA) in low-glucose Dulbecco's Modified Eagle's Medium (LG-DMEM) for 1 h at 37 °C. The digested cells were then suspended in LG-DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Fresh medium was replaced every 3 days. The specific cell surface antigen markers of BMSCs and TSPCs were examined via flow cytometry (FCM). The cells of passage 2 were used and the primary antibodies included anti-CD44 (eBioscience, 12-0444-82), anti-CD90 (BD, 561973), anti-CD34 (Abcam, ab81289), anti-CD45 (BD, 561867) and anti-CD146(Abcam, ab75769). A trilineage-induced differentiation assay was also performed to identify the adipogenic, osteogenic, and chondrogenic differentiation potential of BMSCs and TSPCs. The cells of passage 2 were used in all experiments. Briefly, the cells were incubated in a six-well plate at a density of 1 × 105 cells/well with Rat MSC 4

Adipogenic and Osteogenic Differentiation Medium (Cyagen Biosciences) for adipogenesis and osteogenesis induction, respectively. After 3 weeks of culture, adipogenesis of cells was examined through oil red O staining, and osteogenesis through alizarin red staining. For chondrogenesis, pellet culture was performed. Briefly, the cells were digested with trypsin, a total of 1 × 106 cells/tube were washed with LG-DMEM twice, resuspended in 0.5 mL of Rat MSC Chondrogenic Differentiation Medium (Cyagen Biosciences) in a 15 mL polypropylene centrifuge tube, and centrifuged at 150 g for 5 min. The pellet was incubated at the bottom of the tube with the supernatant at 37℃ in 5% CO2 for 24 h, then the tube was gently flicked to ensure the pellet was free-floating. The medium was changed every 2-3 days. After 3 weeks of incubation, the pellet was fixed in 4% (m/v) paraformaldehyde, and embedded in paraffin. Alcian Blue staining was then performed to assess the glycosaminoglycan formation in the ECM of the pellet. 2.2. Preparation and identification of MSC exosomes For exosome preparation, the cells were cultured in exosome-depleted medium, which was prepared by centrifuging at 100,000 g for 16 h. The cells were cultured for 48 h and then the conditioned medium (CM) was collected for exosome isolation by differential centrifugation at 4℃ as follows: 300 g for 5 min, 2000 g for 30 min, 10,000 g for 30 min, 100,000 g for 70 min, and then the pellet was washed with PBS and centrifuged at 100,000 g for 70 min. Exosomes were re-suspended in PBS and then preserved at -80℃. The particle size of the final exosomes was measured by Nanoparticle tracking analysis (NTA) measurements using ZetaView (Particle Metrix). The morphology of the exosomes was verified by transmission electron microscopy (TEM, JEM1400PLUS). A drop of exosome pellet (20 μL) was absorbed by a carbon film, followed by incubation for 5 min at room temperature and 2% phosphotungstic acid was used for negative staining, and samples were then air-dried for image capturing by TEM. To quantify the final exosome pellet suspended in PBS, a BCA Protein Assay Kit (Applygen) was used to measure the protein concentration. Exosome-associated markers were detected by western blot assay, and the primary antibodies included anti-CD9 (Abcam, ab92726, 1:2000), anti-ALIX (Abcam, ab186429, 1:5000) and anti-TSG101 (Proteintech,14497-1-AP, 1:1000). 2.3. Cellular internalization and cargo delivery of exosomes The mRNA inside exosomes were labelled using ExoGlow-RNA EV Labeling kit 5

(System Biosciences) to trace the cargoes inside exosomes according to the manufacturer’s instruction. Briefly, every 100 µg exosomes were incubated with 200 µL reaction mixture of 10 µL probe and 190 µL reaction buffer for 1 h at 37℃ in dark and then centrifuged at 100,000 g for 70 min to remove the free dye. TSPCs were incubated with designated amounts (0.1 µg/mL, 1 µg/mL and 10 µg/mL) of labelled exosomes for 12 h, fixed in 4% paraformaldehyde, and subsequently stained by Phalloidin conjugated with Rhodamine and Hoechst 33258. Internalization of the labelled exosomes by TSPCs was determined by fluorescent microscopy, besides, the 3D projection of exosomes and TSPCs were also obtained to determine whether exosomes was located inside the TSPCs or bounded to cell membrane. 2.4. EdU staining assay To evaluate the influence of BMSCs on the proliferation of TSPCs, 1 × 104 TSPCs/well were seeded in the lower chambers of 24-well transwell plates and cultured in serum-free LG-DMEM. 12 hours later, to ensure the ratio of BMSCs/TSPCs be 0, 1 and 2, designated amount of BMSCs (0, 1 × 104 and 2 × 104 cells/well for group 0, group 1, and group 2 respectively) was seeded in the upper chambers with 0.4 µm diameter pores, and the medium was replaced by low serum LG-DMEM (0.5% exosome-free FBS) and 100 U/mL penicillin and 100 mg/mL streptomycin (PS). After 24 h of co-culture, EdU (5-ethynyl-2’-deoxyuridine, RiboBio) was added to the medium and incubated for 6 h. The TSPCs were then fixed and EdU staining was performed according to the instructions. To observe the effect of BMSCs on TSPCs when the production of exosomes is inhibited, BMSCs were pre-treated with 10 µM GW4869 (an inhibitor of neutral sphingomyelinase) (Sigma) for 24 h before seeded in the co-culture system [17], and the same operation was conducted as above. To evaluate the influence of BMSCs-exos on the proliferation of TSPCs, 2 × 103 cells/well were seeded in 96-well plates, 12 h later, the medium was replaced by low serum LG-DMEM, and designated amount of BMSCs-exos (0.1 µg/mL, 1 µg/mL and 10 µg/mL) or vehicle control (PBS) were added into the medium. After 24 h of culture, EdU was added into the medium and incubated for 6 h. The TSPCs were then fixed, and EdU staining was performed according to the instructions. The quantification of EdU positive cells was performed based on the photos taken by the fluorescent microscope. Cells in five randomly-selected fields at 100x 6

magnification were counted. The percentage of proliferating cells was calculated by dividing the number of EdU positive cells by total cells. 2.5 CCK-8 assay To evaluate the influence of BMSCs on the proliferation of TSPCs, 1 × 10 4 TSPCs/well were seeded in the lower chambers of 24-well transwell plates and cultured in serum-free LG-DMEM. 12 hours later, to ensure the ratio of BMSCs/TSPCs be 0, 1 and 2, designated amount of BMSCs (0, 1 × 104 and 2 × 104 cells/well for group 0, group 1, and group 2 respectively) was seeded in the upper chambers with 0.4 µm diameter pores, and the medium was replaced by low serum LG-DMEM. After 12, 24 and 48 h of co-culture, the medium was replaced with low serum LG-DMEM containing 10% CCK-8 reagent and incubated for 2 h, then the medium was transferred to 96-well plate and the optical density (OD value) of the medium was measured with a microplate reader at 450 nm. To observe the effect of BMSCs on TSPCs when the production of exosomes is inhibited, BMSCs were pre-treated with 10 µM GW4869 (an inhibitor of neutral sphingomyelinase) (Sigma) for 24 h before seeded in the co-culture system [17], and the same operation was conducted as above. To evaluate the influence of BMSCs-exos on the proliferation of TSPCs, 2 × 103 cells/well were seeded in 96-well plates, 12 h later, the medium was replaced by low serum LG-DMEM, and designated amount of BMSCs-exos (0.1 µg/mL, 1 µg/mL and 10 µg/mL) or vehicle control (PBS) were added into the medium. After 12, 24, and 48 h of culture, the medium was replaced with low serum LG-DMEM containing 10% CCK-8 reagent and incubated for 2 h. The medium was then transferred to 96-well plate, and the optical density (OD value) of the medium was measured with a microplate reader at 450 nm. 2.5. Transwell assay Designated amount of BMSCs (0, 1 × 104 and 2 × 104 cells/well for group 0, group 1, and group 2 respectively) were seeded in the lower chamber of 24-well transwell plates, and 12 h later, 1 × 104 TSPCs in low serum LG-DMEM were seeded in the upper chamber with 8 µm diameter pores. After designated time (12 h and 24 h) of co-culture, cells on the upper surface of the transwell filters was swabbed off. To observe the effect of BMSCs on TSPCs when the production of exosomes is inhibited, BMSCs were pre-treated with 10 µM GW4869 (an inhibitor of neutral sphingomyelinase) (Sigma) for 24 h before seeded in the co-culture system, and the 7

same operation was conducted as above. To evaluate the influence of BMSCs-exos on the migration of TSPCs, 1 × 104 TSPCs in low serum LG-DMEM were seeded in the upper chamber with 8 µm diameter pores of 24-well transwell plates and designated amount of BMSCs-exos (0.1 µg/mL, 1 µg/mL and 10 µg/mL) or vehicle control (PBS) was added in the lower chamber. 24 h later, cells on the upper surface of the transwell filters was swabbed off. Cells on the bottom of the filters were then fixed in 4% paraformaldehyde and stained with crystal violet. Cells in five randomly-selected fields at 100x magnification were counted, and cell migration rate was calculated by dividing the number of cells left on the filter by the initial number of cells seeded. 2.6. Wound healing assay The wound healing assay was performed as previously reported[18]. Briefly, TSPCs were plated in 6-well plates, the medium was replaced by FBS-free LG-DMEM after the cells reach confluence to block their further proliferation. A sterile 200 μL pipette tip was used to scratch in the middle of the well bottom and then designated amounts of BMSCs-exos (0.1 µg/mL, 1 µg/mL and 10 µg/mL) were added into the medium. Photos were taken under optical microscope at 0, 24, and 48 h after scratch. 2.7. Fabrication of fibrin-exos To fabricate fibrin gel containing BMSCs-exos, 5 µL of BMSCs-exos (4 µg/µL) was mixed with 1 μL thrombin (500 IU/mL) and 4 µL fibrinogen (50 mg/mL) to constitute 10 µL gel; the mixture was performed quickly and thoroughly in about 8 s before the gelation was completed. The formed gel was termed as “fibrin-exos” as the experimental group. For the control group, BMSCs-exos were replaced by PBS during the same process, and the formed exosomes-free gel was termed as “fibrin-vehicle”. 2.8. Release and uptake dynamics of fibrin-exos The release ratio of BMSCs-exos in fibrin gel was tested based on the fluorescent signal similar to a previously reported method[19]. Briefly, 5 µL of BMSCs-exos (4 µg/µL) labeled by ExoGlow-RNA EV Labeling kit (System Biosciences) was mixed with 1 μL thrombin (500 IU/mL) and 4 µL fibrinogen (50 mg/mL) and gelated in a 96-well plate. Then, 100 μL PBS was added into each well to submerge the fibrin-exos. After incubating in a 37 °C incubator for 0, 1, 3, 6, 12, and 24 h, the supernatant was collected and moved to another 96-well plate for fluorescence 8

analysis. A gradient concentration of labelled BMSCs-exos from 0.15-10 µg in 100 µL PBS was used to establish the standard curve of exosome concentration based on the linear dependent relationship between the quantity of exosomes and the fluorescent signal. The amount of leaching exosomes was measured via fluorescent signal according to the standard curve. To verify the internalization of the leaching BMSCs-exos from fibrin gel by TSPCs, 10 µL fibrin-exos containing 20 μg BMSCs-exos labelled by ExoGlow-RNA EV Labeling kit was added into the medium (500 µL) of the TSPCs (5 × 104 cells/well in a 24-well plate). After incubating for 24 h, the TSPCs were washed by PBS, fixed in 4% paraformaldehyde, and stained by Rhodamine conjugated with Phalloidin and Hoechst 33258. Internalization of the labelled exosomes by TSPCs were determined by fluorescent microscopy. 2.9. Real-time quantitative PCR Total RNA was isolated from TSPCs using TRIZOL (Invitrogen), 1 µg RNA was reverse transcribed using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo) and then amplified using real-time PCR system (applied biosystems) with SYBR® Select Master Mix (applied biosystems). The primers used for Mohawk (Mkx), tenomodulin (Tnmd), type I collagen alpha 1 (Col 1a1) and proliferating cell nuclear antigen (PCNA) were shown in Supplementary Table 1. The PCR cycling condition comprised an initial denaturation at 95℃ for 30 s followed by 40 cycles of amplification consisting of 15 s of denaturation at 95℃ and 30 s of extension at 60℃. Relative mRNA expression levels of target genes were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and calculated using the comparative CT method. 2.10. Rat patellar tendon defect model To create tendon defects, the central one-third of the patellar tendon (0.8 mm in width) of Sprague-Dawley male adult rats (body weight of 200g) was removed from the distal apex of the patella to the insertion of the tibia tuberosity with two stacked sharp blades according to a well-established protocol[13, 20]. Next, 5 µL of BMSCs-exos (4 µg/µL) was mixed with 1 μL thrombin (500 IU/mL) and 4 µL fibrinogen (50 mg/mL) and injected into the defect area of patellar tendon for gelation. The time between mixing and injecting should be 5-8 s to ensure optimized gelation. Six rats were used for exosome retention evaluation in vivo. Exosomes were 9

incubated with 100 µL DiR dye (Biotium) for 30 min at room temperature and centrifuged at 100, 000 g for 70 min to remove the free dye. The right knees of the rats were then implanted with fibrin-DiR-exos and the left knees with fibrin-vehicle, the signal of DiR-exos in the knees was measured by IVIS Spectrum Imaging System when rats were under anesthesia after 3 days and 2 weeks respectively. To evaluate the repair effects, fifty-two rats were randomly divided into 2 groups: (a) fibrin-exos and (b) fibrin-vehicle. The animals were allowed for free cage activity until euthanasia. 5 rats in each group were euthanized 3 days and 1, 2, and 4 weeks after surgery respectively, and the patellar tendons were harvested for histologic examination. At 4 weeks after surgery, 6 rats in each group were sacrificed for mechanical test. Another three rats were used for EdU labelling in vivo, 5 mg/kg EdU in PBS was injected into the rats intra-abdominally and repeated every 24 h at day 1-3, 4-7 and 8-14 post-surgery respectively. 2.11. Histology and immunohistochemistry Specimens for H&E staining, Sirius red staining and immunohistochemistry were immediately fixed in 10% neutral buffered formalin, dehydrated through an alcohol gradient, cleaned, and embedded in paraffin blocks. Histologic sections (5 mm) were prepared using a microtome and stained with hematoxylin and eosin (H&E). General histological scoring was performed using a blinded semi-quantitative scoring system based on six parameters of H&E staining, which was utilized previously (Supplementary table 2)[21]. All sections were analyzed by 2 pathologists blinded to the treatment groups. Sirius red staining was performed according to standard procedures to examine the general appearance of the collagen fibers. Polarizing microscopy was used to detect mature collagen fibrils. Immunohistochemistry were performed as previously reported. Briefly, endogenous peroxidase was blocked by incubating the sections with 3% hydrogen peroxide in methanol for 10 min, nonspecific protein binding was blocked by incubating with 10% goat serum. After overnight incubation at 4°C with antibodies against type I collagen (Col I, Sigma, C2456, 1:400) and tenomodulin (Tnmd, Abcam, ab203676, 1:400), sections were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Zsjqbio) for 30 min at room temperature. The 3,39-diaminobenzidine substrate system (DAB, Zsjqbio) was used for color development. Hematoxylin staining was performed to reveal the nuclei. Semi-quantitative analysis of Col I and 10

Tnmd was performed by measuring the percentage of positively-stained area in the defect area using Image Pro Plus software. 2.12. Immunofluorescence Rats under anesthesia were transcardially perfused with 4% PFA, and then the patellar tendon was removed, post-fixed and infiltrated with 20-30% sucrose. For immunostaining, tendons were cut into sections with a thickness of 15 μm in a cryostat, blocked with the goat serum, treated with primary antibodies against CD146 (Abcam, ab75769), and then incubated with FITC-conjugated secondary antibodies. Hoechst 33258 staining was performed to reveal the cell nuclei. For EdU staining, tissue sections were stained according to the instructions. Fluorescence images of sections were captured using a confocal microscope. The CD146+ cells and EdU+ cells were counted in five randomly selected fields. 2.13. Mechanical testing The healing tendon tissues of 6 rats in each group at 4 weeks post-surgery were harvested for biomechanical tests according to the methods described in previous studies[9, 11, 13, 20]. Briefly, the tendon samples were tested on a universal tensile testing machine (AGS-X, SHIMADZU) with a load cell of 50 N using the protocols as follows: 1) the tendon-patellar and tendon-tibia composite was harvested and surrounding muscles were carefully removed; 2) two parallel blades with an interval of 0.6 mm were used to separate the neo-tendon from the adjacent normal tendon; 3) the tibia shaft and proximal patella were fixed onto the testing clamp; 4) 0.1 N was preloaded and cyclic elongation of 0-0.5 mm for 20 cycles at 5 mm/min was conducted; 5) the cross-sectional area (S) and initial length (L) were measured with a Vernier caliper; 6) the load-displacement curve was recorded at an elongation rate of 5 mm/min.

The stress at failure (MPa) was calculated based on the ultimate load

divided by the cross-sectional area. The Young’s modulus (MPa) was calculated according to the linear slope of a stress-strain curve. 2.14. Statistical analysis All quantitative data were presented as mean ± SD. Student’s t test was used for the comparison between two groups and one-way ANOVA was used for the comparison among three or more groups. A P value of <0.05 was considered to be statistically significant. 3. Results 3.1 BMSCs promoted the proliferation and migration of TSPCs 11

We first evaluated the multi-differentiation potential of TSPCs and BMSCs toward osteogenesis, adipogenesis and chondrogenesis, and the results showed that both TSPCs and BMSCs were able to undergo multi-differentiation (Fig. S1A-C and Fig. S2A-C), while the adipogenesis potential of TSPCs was slightly weaker than BMSCs (Fig. S1A and Fig. S2A). We used flow cytometric analysis to measure the surface markers and observed that over 97.5% of BMSCs and TSPCs were positive for CD44 and CD90, and less than 5.3% were positive for CD34 and CD45, besides, CD146 was positive in 90.6% of TSPCs (Fig. S1D and Fig. S2D), consistent with previous reports[16]. To investigate the influence of BMSCs on the proliferation and migration of TSPCs, 3 groups (group 0, 1, and 2, defined in the Methods section) of co-culture system were set by different ratios of BMSCs/TSPCs. The proliferation ability of TSPCs co-cultured with BMSCs was measured by EdU staining, CCK-8 assay and mRNA expression of PCNA. Compared with group 0, the ratio of EdU+ TSPCs in group 1 (10% ± 3% vs 4.7% ± 0.6%; P=0.015) and group 2 (11% ± 2% vs 4.7% ± 0.6%; P=0.003) both increased significantly (Fig. 1A). The CCK-8 results also revealed significantly enhanced cell viability in group 1 and group 2 at 24 h and 48 h compared to group 0 (Fig. 1B). Moreover, the higher mRNA level of PCNA in group 1 and group 2 at 12 h and 24 h further indicated that BMSCs promoted the proliferation of TSPCs (Fig. 1C). The migration ability of TSPCs was measured by crystal violet staining. Compared with group 0, the migration rate of group 1 increased by 1.50-fold (15% ± 2% vs 10% ± 1%; P=0.01) at 12 h and 1.79-fold (59% ± 9% vs 33% ± 6%; P=0.01) at 24 h, and that of group 2 increased by 1.80-fold (18 ± 4 % vs 10 ± 1 %; P=0.03) at 12 h and 2.21-fold (73% ± 10% vs 33% ± 6%; P=0.01) at 24 h (Fig. 1D). These results revealed the promoting effect of BMSCs on the proliferation and migration of TSPCs. 3.2 Inhibition of exosome secretion impeded the function of BMSCs on TSPCs To further examine whether exosomes play a role during the regulation of BMSCs on TSPCs, we first utilized the exosome inhibitor GW4869 to block exosome production and assessed the impact of BMSCs on TSPCs. We found that the increased proportion of EdU+ TSPCs by BMSCs was significantly inhibited by GW4869 (7% ± 1% vs 11% ± 3%; P=0.046), and it also suppressed enhanced cell viability and elevated expression of PCNA by BMSCs (Fig. 1E-G). The accelerated migration of TSPCs by BMSCs was also partly inhibited by GW4869 (56% ± 4% vs 68% ± 5%; P=0.034) (Fig.1H). Thus, it is possible that the paracrine effect of BMSCs, partly at 12

least, was contributed by exosomes. 3.3 BMSCs-exos enhanced proliferation, migration and tenogenic differentiation of TSPCs Considering exosomes are involved in intercellular communication and MSCs-exos have beneficial effects on various cell types, we next explored whether BMSCs-exos could exert the same effects on TSPCs as BMSCs. The BMSCs-exos isolated by ultracentrifugation possessed a cup-shaped morphology, a diameter distribution of 101.1±50.6 nm, and the expression of exosome-specific protein markers ALIX, CD9 and TSG101 (Fig. 2A). When co-cultured with TSPCs by the concentration of 0.1, 1 and 10 µg/mL for 12 h, the RNA cargoes inside BMSCs-exos could be delivered into TSPCs (Fig. 2B and Supplementary Video). The proliferation and migration ability of TSPCs treated with BMSCs-exos was then detected. The EdU+ TSPCs increased in the exos-treated groups with the concentration of 1 µg/mL (11% ± 1% vs 4.8% ± 0.8%; P<0.001) and 10 µg/mL (11% ± 2% vs 4.8% ± 0.8%; P<0.001) compared with vehicle group (Fig. 2C). The results of the CCK-8 assay and the mRNA level of PCNA also indicated the same trend (Fig. 2D-E). The migration ability of TSPCs was significantly promoted in the exos-treated groups with the concentration of 0.1 µg/mL (52% ± 5% vs 40% ± 3%; P=0.042), 1 µg/mL (63% ± 4% vs 40% ± 3%; P=0.007) and 10 µg/mL (61% ± 3% vs 40% ± 3%; P=0.003) compared with vehicle group at 24 h (Fig. 2F). Consistent results were observed in the wound healing assay (Fig. S3). BMSCs-exos led to the induction of tendon-related genes including Tnmd (1 µg/mL: 2.69-fold, P=0.001; 10 µg/mL: 2.91-fold, P=0.007), Mkx (1 µg/mL: 1.65-fold, P=0.016; 10 µg/mL: 2.06-fold, P=0.012), and the extracellular matrix gene Col1a1 (1 µg/mL: 1.46-fold, P=0.047; 10 µg/mL: 1.88-fold, P=0.039) (Fig. 2G). These results indicated that BMSCs-exos could promote the proliferation, migration and tenogenic differentiation of TSPCs. 3.4 The dynamics of BMSCs-exos in fibrin hydrogel Due to the difficulty to immobilize the aqueous exosome in the tendon injury area, we embedded the exosomes into the fibrin to exert the therapeutic function of BMSCs-exos for tendon regeneration precisely and efficiently. First, we measured the release and internalization of exosomes embedded in the fibrin (Fig. 3A). ExoGlow RNA probe labelled BMSCs-exos were used to monitor exosomes in vitro and a linear dependent relationship between fluorescent signal and exosome quantity was observed with the regression coefficient of 0.995 (Fig. 3B), the amount of 13

BMSCs-exos released from the fibrin gradually increased with the extension of incubating time. Within 24 h, an average of 0.07 μg BMSCs-exos could be released from fibrin hydrogel per hour (Fig. 3C). Then 10 µL of fibrin-exos containing 20 µg labelled BMSCs-exos was added into the cultural medium of TSPCs. After incubating for 24 h, green fluorescence was observed in TSPCs, suggesting the internalization of BMSCs-exos released from fibrin hydrogel by TSPCs (Fig. 3D). We proposed that fibrin hydrogel could improve the retention and stability of BMSCs-exos in vivo. To detect the retention of exosomes in vivo, 20 μg of DiR-labeled BMSCs-exos incorporated with fibrin hydrogel was injected into the patellar tendon defect area of rats at a total volume of 10 μL (Fig. 3E), and the rats were imaged using an IVIS Spectrum Imaging System at indicated time points. The fluorescent signals in the fibrin-exos group could be acquired at 3 days and 2 weeks after injection (Fig. 3F). These observations demonstrated that BMSCs-exos embedded in fibrin hydrogel could be retained with the injury site in vivo stably up to 2 weeks. 3.5 Fibrin-exos mediated a dynamic remodeling process to accelerate tendon repair Next, we evaluated whether the BMSCs-exos embedded in fibrin hydrogel had beneficial effect on tendon repair by treating rat patellar tendon defect with fibrin-exos or fibrin-vehicle control. Macroscopically, we observed that the defect was clearly visible and filled with transparent tissue at week 1, while the fibrin-exos group showed a slightly blurry margin of the defect compared to control group (Fig. 4A). The filled tissue was granulated with more intensive cells inside than the native tendon, and the granulated tissue of fibrin-exos group showed more regular alignment (Fig. 4B and Fig. S4) and more deposition of extracellular matrix type I collagen (Fig. 4C). The enhanced tissue repair in the fibrin-exos group at week 1 persisted and extended to week 2 and 4. At week 2, the fibrin-exos group showed improved integration of the healing tissue with the host tendon while the defect area of the control group displayed poor regularity with structure disruptions (Fig. 4A). Histologically, the fibrin-exos group showed considerably more type I collagen deposition than fibrin-vehicle group (Fig. 4C). At week 4, the macroscopic view of the fibrin-exos group showed more approximate appearance including color and transparency to the native tendon than the control group (Fig. 4A). The cell density and alignment in the defect region of fibrin-exos 14

group were also much closer to the native tendon (Fig. 4B). Abundant extracellular matrix type I collagen was deposited with orderly alignment in the fibrin-exos group, while the fibrin-vehicle group showed less and disordered type I collagen deposition (Fig. 4C). Histological scores of fibrin-exos-treated tendons at week 4 were also lower than those of fibrin-vehicle-treated tendons (7 ± 2 vs 10 ± 2, P = 0.021, Fig. 4D), indicating the regeneration of tendon at 4 weeks post-surgery in fibrin-exos group was better than control group. Immunohistochemical staining of Col I and Tnmd was conducted, and the fibrin-exos group displayed significantly higher expression of Col I (week 1: 9% ± 2% vs 5% ± 3%, P = 0.039; week 2: 44% ± 5% vs 33% ± 4%, P = 0.008; week 4: 81% ± 8% vs 52% ± 7%, P = 0.002) (Fig. 4E) and higher expression of Tnmd at week 1 (48% ± 10% vs 34% ± 4%, P = 0.034) (Fig. 4F). These results indicated that fibrin-exos significantly enhanced the expression of tendon related protein during the repair process. Moreover, the mechanical properties including modulus and stress at failure were evaluated. The stress at failure of the healing tendons and modulus were 1.84-fold (13 ± 5 MPa vs 7 ± 2 MPa, P = 0.034) and 1.86-fold (39 ± 8 MPa vs 21 ± 4 MPa, P = 0.012) higher in fibrin-exos group compared to fibrin-vehicle group respectively (Fig. 4G), suggesting that the repaired tendons of fibrin-exos group had superior mechanical properties. 3.6 BMSCs-exos enhanced the proliferation of TSPCs during tendon repair To further investigate the mechanism for the improved tendon regeneration and evaluate whether BMSCs-exos can affect TSPCs in vivo, we measured the amount of TSPCs in the neo-tissue at different time points. Here we used immunostaining of CD146 to detect TSPCs in vivo as it has been used as the marker of TSPCs as reported previously [22]. Three days post-surgery, we found CD146+ TSPCs emerged in the defect region with residual fibrin glue surrounded (Fig. 5), and the amount of CD146+ TSPCs in the defect region elevated at week 1 post-surgery and decreased at week 2 and 4 post-surgery. Compared with fibrin-vehicle group, the amount of TSPCs in fibrin-exos group elevated at day 3 and week 1 post-surgery (day 3: 10% ± 2% vs 6% ± 2%, P = 0.035; week 1: 25% ± 4% vs 18% ± 3%, P = 0.022) (Fig. 5). To further investigate the effect of BMSCs-exos on TSPCs in vivo, we detected the DiR-exos and CD146+ TSPCs in the fibrin-DiR-exos implanted group 3 days post-surgery and found that there were DiR-labelled exosomes inside the TSPCs, indicating that DiR-exos were located in the defect region where they were injected, and the CD146+ 15

TSPCs were mainly presented in the neo-tissue and the adjacent regions (Fig. 6A), and the exosomes implanted in the tendon defect area were internalized by the local TSPCs (Fig. 6B). To investigate whether the proliferation of CD146+ TSPCs was affected by fibrin-exos in vivo, we utilized EdU to label the proliferating cells of 1-3d, 4-7d and 8-14d post-surgery. The results showed that the ratio of proliferating CD146+ cells to total CD146+ cells was 1.73-fold higher (71% ± 12% vs 41% ± 17%, P = 0.048) in fibrin-exos group compared with fibrin-vehicle group in 1-3 d, while in 4-7 d and 8-14 d, it did not have significant difference between the two groups (Fig. 7), which might be due to the fact that cell proliferation usually occurs in the early phase of tendon regeneration[23]. 4. Discussion The regenerative potential of MSCs has been introduced in tendon tissue engineering for a long time and the improved tendon regeneration by MSCs has been demonstrated by various studies[9, 24, 25]. However, the strategies utilizing MSCs directly are still facing with inevitable challenges including operation costs, preservation of the cell viability, and phenotype drift from cell harvest, expansion, storage to finally delivery to patients[26]. To explore new strategies following the benefit of MSCs, further understanding of the underlying functional mechanism would be helpful. Exosomes have been recognized as an approach for intercellular communication and crosstalk between cells, and play an important role in the paracrine function of MSCs[17, 27, 28]. Previous studies have found that exosomes could play a beneficial role in the process of tendon injury and repair [29-31]. Shen and his colleagues reported that a collagen sheet system containing exosomes from adipose-derived stem cells improved tendon regeneration via regulation of macrophage inflammatory response [32]. A recent study has reported that BMSCs-derived extracellular vesicles could regulate inflammation and enhance tendon healing in vivo [33]. However, it has several limitations: First, this study showed the increased proportion of tendon-resident stem/progenitor cells in the defect area, but no further evidences for the underlying mechanism such as cell proliferation or migration were provided for this phenomenon. Second, functional assessment for BMSC-derived extracellular vesicles in vitro only included CCK-8 assay and real-time PCR for collagen type I, which might not be adequate enough. Besides, the exosome-releasing characteristics of the fibrin-exosomes complex were not evaluated in this study. 16

In this study, first, we found that BMSCs promoted the proliferation and migration of TSPCs via its paracrine of exosomes, and BMSCs-exos improved the tenogenic differentiation of TSPCs. Second, we demonstrated that BMSCs-exos embedded in fibrin glue represented a feasible approach which allows the sustained-release of BMSCs-exos and internalization of BMSCs-exos by TSPCs. Moreover, the BMSCs-exos embedded in fibrin glue significantly improved tendon regeneration and promoted the proliferation of TSPCs in vivo. TSPCs located in tendon were identified in 2007 [16] and a controversy gradually emerged that whether the regenerative role of BMSCs is based on its proliferation and differentiation capacity or via an indirect way through the activation of the local stem/ progenitor cells. With regard to the seeding cells in tendon tissue engineering, Pietschmann, M. F., et al. reported that TSPCs is better than BMSCs[34], and later Wu, T., et al. reported that the combination of co-cultured BMSCs and TSPCs is better than BMSCs or TSPCs alone[11]. Thus, we may deduce that the inter-cellular interaction of BMSCs and TSPCs has a beneficial role in promoting tendon repair. The conditioned medium of BMSCs promoted the proliferation, migration and viability of tenocytes in vitro and improved the repair effect of rotator cuff tear[7, 8]. Therefore, we proposed a hypothesis that BMSCs might have a beneficial effect on TSPCs via indirect way such as paracrine signaling. Herein, the results from indirect co-culture system demonstrated that BMSCs indeed promoted the proliferation and migration of TSPCs via paracrine approach (Fig. 1). BMSCs-exos could be internalized by TSPCs in vitro and in vivo, and promoted the proliferation, migration, and tenogenic differentiation of TSPCs in a dose-dependent manner. When delivered to the defect area of patellar tendon, BMSCs-exos induced higher expression of Col I and Tnmd at week 1 post-surgery and more ordered fibril orientation at week 2 and 4 post-surgery, and improve the mechanical properties at week 4 post-surgery (Fig. 4). Tnmd is a tendon-specific marker important for tendon maturation and exerts a positive effect on TSPCs by supporting their self-renewal and preventing senescences[35], and it is also essential for the prevention of fibrovascular scar formation during early tendon healing[36]. The expression of Tnmd was reported to be elevated at week 1 and 2 in the tendon healing process[37], and in the present study, we found that the expression of Tnmd at week 1 was increased by fibrin-exos, suggesting that fibrin-exos contributed to tendon regeneration via promoting Tnmd expression in the early stage. 17

Considering the effect of BMSCs-exos on TSPCs in vitro, the beneficial role of BMSCs-exos in tendon regeneration might be owed to their impact on the endogenous TSPCs in local tendon tissue. TSPCs have been utilized in tendon regeneration in various studies as they are stem/progenitor cells originated from tendon [13, 38, 39], however, culture-expanded TSPCs are prone to lose their phenotype and have an inferior regenerative capability in tendon injury[20]. Because the approaches to inhibit phenotypic drift during cell expansion in vitro are still being explored, another possible strategy is to make use of endogenous TSPCs, hence, cell-free strategies have drawn increasing attention in tendon tissue engineering field recently[40, 41]. Several strategies promoting the proliferation and tenogenic programs of endogenous TSPCs have led to better healing results, indicating that basal activation of TSPCs might not be sufficient enough for tendon regeneration [22, 42]. For example, Lee, C. H., et al. utilized connective tissue growth factor (CTGF) for patellar tendon repair and found that it promoted proliferation and tenogenic programs of TSPCs and accelerated healing in the injured tendon compared to the group without CTGF treatment [22]. In this study, we explored whether utilizing BMSCs-exos was also an effective way to activate the regenerative potential of TSPCs in the injured region of tendon. The expression of surface marker CD146 was used to identify TSPCs as previously reported [16, 22]. CD146+ TSPCs were found to be distributed in peritenon and tendon mid-substance, they stay in quiescent state in intact tendon, and switch to activated state with enhanced proliferation, migration and tenogenesis ability during tendon repair process [43]. Here we observed that BMSCs-exos delivery induced a transient increase of CD146+ TSPCs at the defect region in early period post-surgery (Fig. 5), similar with the effect of CTGF delivery in the previous study [22]. Thus, the application of BMSCs-exos might be a promising approach to activate the regenerative potential of endogenous TSPCs in tendon injured region. Compared with the application of single bio-factor, the exosome-based therapy might be a better choice because exosomes contain a cargo of multiple factors which can function synergistically[5]. On the other hand, exosomes possess great potential to be engineered to exert the required function and eliminate the side effects[44, 45]. Exosomes have also been studied as a choice of drug carrier, which makes it a safe carrier for various factors including protein, RNA and lipids[46, 47]. While the strategy of utilizing the local stem cells are under growing enthusiasm in recent years, a huge controversy gradually emerges that whether the adult stem cells 18

exist indeed[48, 49]. Previous studies have demonstrated that CD146+ cells exist in the original and neo-tissue of tendon [10]. In the present study, by utilizing the CD146+ cells isolated from rat patellar tendon tissue, we demonstrated that they indeed had the potential to undergo multipotent differentiation, and expressed the stem cell markers of CD90 and CD44 in vitro. Therefore, based on previous studies and our present study, it could be concluded that the TSPCs do exist in the tendon tissue. In general, the healing process of the tendons undergoes three main phases: initial inflammatory stage, proliferation stage and remodeling stage. Evidences have shown that in the beginning of tendon healing, blood vessels are formed in the injured site, which contributes to cell infiltration and the survival of the newly forming fibrous tissue[1]. Moreover, the angiogenesis function of exosomes has been reported in several studies[2, 3]; thus, whether fibrin-exos can enhance the forming of blood vessels in the healing tendon, and the mechanism of angiogenesis during the early stage of tendon regeneration need to be further investigated. The preferred application of exosomes was injection of exosomes aqueous solution into the circulation system or body cavity, however, it might not be suitable to inject exosomes directly into the injury area because of the difficulty to retain them locally. Only a few papers have reported about embedding exosomes inside hydrogel, for example, MSC exosomes embedded into chitosan hydrogel for skin wound healing[19, 50], Pluronic F-127 hydrogel for digestive filtula management[51], and fibrin hydrogel for incisional hernia[14], respectively. Fibrin gel is a biologically derived and FDA-approved hydrogel, and has been extensively used in clinical and scientific research for its properties of biocompatible, biodegradable and favorable for cell adhesion and infiltration[52]. Here we utilized fibrin gel as the carrier of BMSCs-exos and observed that BMSCs-exos could be well retained in the defect area, released into tissue and internalized by local CD146+ TSPCs. The dose of exosomes employed in vivo ranges from 50 µg to 250 µg in previous studies based on rat model[6]; in the present study, we utilized 20 µg BMSCs-exos embedded in fibrin glue and observed significantly improved tendon regeneration compared to control group. The reason for the relative low yet effective dose might be explained by several aspects: First, the sensibility to exosomes might be tissue specific; secondly, the exosomes here was applied in the gel and controlled-released to the target site, so that the concentration of exosomes in the injury area was likely to be higher than those applied systematically. 19

The tendon repair effect in this study was still not excellent enough since the structure of neo-tissue in the injury site was not exactly the same as the normal tendon. However, the strategy based on MSCs-exos is still a promising way with the potential and convenience to be optimized compared to cell therapy[46, 47], the biological properties of exosomes rely on the bio-factors inside the exosomes including protein, RNA and lipids, which are able to be manipulated via multiple approaches. Thus, BMSCs-exos could be engineered to further improve their regenerative efficiency and reduce possible side effects. 5. Conclusion Overall, this is the first study to investigate the paracrine effect of BMSC-derived exosomes on TSPCs, and the results showed that BMSCs-exos significantly promoted the proliferation, migration and tenogenic differentiation ability of TSPCs. Moreover, by embedding BMSCs-exos into the fibrin gel, the control-released exosomes retained the ability of being internalized by TSPCs in vivo and promoted the regeneration of patellar tendon tissue in the defect area in rats. Our findings have provided the rationale for the development of a ready-to-use, cell-free, and MSC-based therapeutic approach that is highly effective for the treatment of tendon injury.

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Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments This project was supported by the National Natural Science Foundation of China (No. 81672212, 81772417 and 81972101), the Beijing Natural Science Foundation (No. 7171014, 7174361 and 7182175), the Fundamental Research Funds for the Central Universities, and Peking University Medicine Seed Fund for Interdisciplinary Research (Nos. BMU2018MX013 and BMU2018MI006). References [1] D. Gaspar, K. Spanoudes, C. Holladay, A. Pandit, D. Zeugolis, Progress in cell-based therapies for tendon repair, Advanced drug delivery reviews 84 (2015) 240-56. [2] A.J. Lomas, C.N. Ryan, A. Sorushanova, N. Shologu, A.I. Sideri, V. Tsioli, G.C. Fthenakis, A. Tzora, I. Skoufos, L.R. Quinlan, G. O'Laighin, A.M. Mullen, J.L. Kelly, S. Kearns, M. Biggs, A. Pandit, D.I. Zeugolis, The past, present and future in scaffold-based tendon treatments, Advanced drug delivery reviews 84 (2015) 257-77. [3] J. He, Y. Wang, S. Sun, M. Yu, C. Wang, X. Pei, B. Zhu, J. Wu, W. Zhao, Bone marrow stem cells-derived microvesicles protect against renal injury in the mouse remnant kidney model, Nephrology (Carlton, Vic.) 17(5) (2012) 493-500. [4] S. Bruno, M. Tapparo, F. Collino, G. Chiabotto, M.C. Deregibus, R. Soares Lindoso, F. Neri, S. Kholia, S. Giunti, S. Wen, P. Quesenberry, G. Camussi, Renal Regenerative Potential of Different Extracellular Vesicle Populations

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Figure legends:

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Fig. 1. Paracrine effect of BMSCs on TSPCs. (A) Proliferation of TSPCs co-cultured with different amount of BMSCs assessed by EdU staining. n=4. (B) Cell viability of TSPCs co-cultured with different amount of BMSCs assessed by CCK-8 at 12 h, 24 h and 48 h. n=5. (C) PCNA mRNA level of TSPCs co-cultured with different amounts of BMSCs assessed by real-time PCR at 12 h, 24 h and 48 h. n=3. (D) Migration of TSPCs co-cultured with different amount of BMSCs in 12 and 24 h assessed by crystal violet staining. n=3. (For A-D, Group 0, 1, 2 indicated the ratio of BMSCs/TSPCs was 0, 1 and 2 respectively.) (E) Proliferation of TSPCs co-cultured with 2-fold of BMSCs pretreated with GW4869 assessed by EdU staining. n=4. (F) Cell viability of TSPCs co-cultured with 2-fold of BMSCs pretreated with GW4869 assessed by CCK-8. n=5. (G) PCNA mRNA level of TSPCs co-cultured with 2-fold of BMSCs pretreated with GW4869 assessed by real-time PCR. n=3. (H) Migration of TSPCs co-cultured with 2-fold of BMSCs pretreated with GW4869 in 24 h. n=3. Scale bar =400 µm. * p<0.05, ** p<0.01.

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Fig. 2. BMSCs-exos promote the proliferation, migration and tenogenic differentiation of TSPCs. (A) Identification of BMSCs-exos by TEM, NTA and western blotting for ALIX, CD9 and TSG101. Scale bar =100 nm. (B) Internalization

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of different concentrations of BMSCs–exos in medium by TSPCs in 12 hours assessed by fluorescence. Green represents the RNA cargo inside the BMSCs-exos, red represents cytoskeleton, blue represents nuclear. The white arrow indicates the internalized exosomes. Scale bar =10 µm. (C-G) Phenotype of TSPCs treated with 0.1, 1, and 10 µg/ml BMSCs-exos: (C) Proliferation assessed by EdU staining, n=4. Scale bar = 400µm; (D) Cell viability assessed by CCK-8, n=5; (E) PCNA mRNA level assessed by real-time PCR, n=3; (F) Migration, n=3. Scale bar = 400µm; (G) Tnmd, Mkx and Col 1a1 mRNA level assessed by real-time PCR. n=3. * p<0.05, ** p<0.01, *** p<0.001.

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Fig. 3. Release and internalization in vitro and retention in vivo of the BMSCs-exos embedded in fibrin-exo. (A) Schematic diagram of the fabrication process and characterization of fibrin-exo. (B) Standard curve of fluorescent signal and exosome concentration (R2 = 0.99537). (C) Released BMSCs-exos from fibrin-exos in PBS in 24 hours, calculated according to the standard curve. n=5. (D) Internalization of BMSCs–exos released from fibrin-exos in medium by TSPCs in 24 h assessed by fluorescence. Green represents the RNA cargo inside the BMSCs-exos, red represents cytoskeleton, blue represents nuclear. The white arrow indicates the internalized exosomes. Scale bar =10 µm. (E) Process of the rat patellar tendon

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window defect model. The black arrow indicates the patellar window defect; the white arrow indicates the implanted fibrin-exo/fibrin-vesicle. (F) Retention of BMSCs-exos embedded in fibrin-exos in the tendon defect focal at 3 days and 2 weeks post-surgery.

Fig. 4. BMSCs-exos enhance tendon regeneration in a rat patellar tendon window defect model. Fibrin-exos and fibrin-vesicle treated patellar tendons were collected and analyzed at 1, 2 and 4 weeks post-surgery. (A) Representative macroscopic images. n=4. The white arrow indicates the defect of the patellar tendon. (B) Representative HE images. n=4. Scale bar = 250 µm. (C) Representative polarized light images showing the deposited collagen at the repaired tissue site. n=4. Scale bar =250 µm. (D) Histological scores of fibrin-exos treated tendons at 4 weeks. n=4. (E) Representative immunohistochemistry images and semi-quantitative analysis of immunohistochemical staining of col I. n=4. Scale bar = 100 µm. (F)

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Representative immunohistochemistry images and semi-quantitative analysis of immunohistochemical staining of Tnmd. n=4. Scale bar = 100 µm. (G) Representative stress-strain curve of of repaired tendons at 4 weeks after implantation and calculated mechanical properties (modulus and stress at failure). n=6. Curves were shown from representative sample and data were represented as mean ± SD. *p < 0.05, ** p<0.01. Abbreviation: HE, hematoxylin and eosin; N, normal tendon; DS, defect section.

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Fig. 5. BMSCs-exos promote the accumulation of CD146+ TSPCs in the early phrase of tendon regeneration. Representative immunohistochemistry images of CD146 and statistical analysis data. The black arrow indicates the CD146+ TSPCs with residual fibrin gel surrounding. n=4. Scale bar =50 µm.

Fig. 6. BMSCs-exos internalization by CD146+ TSPCs in the defect focal. Fibrin-exos and fibrin-vesicle treated patellar tendon defect under confocal microscope 3 days post-surgery. Green represents CD146+ TSPCs, red represents DiR-labelled BMSCs-exos, blue represents nuclear. (A) Overview of the whole patellar tendon. Scale bar =400 µm. (B) Representative images in the defect region. The white arrow represents the internalization of BMSCs-exos by CD146+ TSPCs. Abbreviation: N, normal tendon; DS, defect section. Scale bar =25 µm.

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Fig. 7. BMSC-exos promotes the proliferation of CD146+ TSPCs in the defect focal. Patellar tendon defect was treated with fibrin-exos or fibrin-vesicle, and proliferated cells were labeled by EdU for 1-3d, 4-7d and 8-14d post-surgery. Green represents CD146+ TSPCs, red represents EdU+ cells, blue represents nuclear. n=5. Scale bar = 25 µm.

graphical abstract

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Declaration of interests

☒ The 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.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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