Animals and treatments
The procedures of animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji University. Young (2 months, weight 20–25 g) and old (22 months, weight 32–40 g) C57BL/6 J mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). At least one week of acclimation was allowed before advanced experiments. The mice were raised under specific pathogen-free (SPF) conditions with a 12 h light/dark cycle at a temperature of 18–22 °C and humidity of 50–60%. Both male and female mice were used in the ADMSC isolation, while only male mice were used in tendinopathy related animal experiments. A total of four groups of mice, including the control, tendinopathy, tendinopathy with ADMSCyoung-EVs and tendinopathy with ADMSCold-EVs treatment groups, were involved in this study and 12 mice were included in each group. To induce a tendinopathy phenotype, 20 μL of 1% type I collagenase (Sigma‒Aldrich, USA) was injected around the Achilles tendon. For EV treatments, EVs at a concentration of 109 particles/20 μL local injection were administered once after tendinopathy induction.
ADMSC isolation and EVs production
ADMSC isolation and primary culture were conducted from both young and old C57BL/6 mice as described previously [9, 43]. Briefly, both young and old mice were sacrificed, and the adipose tissues on the inguinal were collected for ASMSC isolation. The adipose tissues were digested with 1 mg/mL collagenase I at 37 °C for 30 min. The cell suspensions were filtrated and then centrifuged at 1200 r/min for 7 min. The pellets were resuspended, and ADMSCs were cultured with DMEM/F12 1:1 medium (Gibco, Thermo Fisher Scientific, USA) containing 15% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA). ADMSCs from young or old mice were recorded as ADMSCyoung or ADMSCold, respectively, and ADMSC of the passage four was used in advanced in vitro experiments. The cultured ADMSCs were collected, digested with trypsin and then washed 3 times with PBS. After adjusting the cell suspensions to approximately 1 × 106 cells/ml, the cell suspensions were centrifuged at 1500 r/min for 5 min and then washed with PBS. A total of 100 μl of PBS was added to generate a cellular sample and 10 μl of CD29 (Cat.No. 11-0291-82, Thermo Fisher Scientific, USA), CD90 (Cat.No. 11-0900-81, Thermo Fisher Scientific, USA) and CD45 (Cat.No. 11-0451-82, Thermo Fisher Scientific, USA) primary antibodies were added. After being incubated at room temperature for 20 min and washed with PBS to remove the the unconjugated antibodies, 500 μl of PBS was used to resuspend the suspensions for flow cytometry identification in an LSRII flow cytometer (BD Biosciences) and analyses with FlowJo software. EVs production and characterization were conducted following our previous protocol [44]. The culture supernatants of the ADMSCs were collected and gently centrifuged to remove cells and cellular debris. The supernatants were centrifuged at 100,000 × g at 4 °C for 2 h, and the pellets were then resuspended in PBS. After isolation, ADMSC-derived EVs were obtained and stored at − 80 °C until use. A total of 10 μL of EVs was dropped on copper grids, and the morphology of EVs was observed with a transmission electron microscope (Tecnai Spirit, USA). EVs were diluted into 200 μL, and the diameter distributions and zeta potentials were detected using a dynamic light scattering (DLS, Malvern Instruments, UK). The amounts of EVs were measured with nanoparticle tracking analysis (NTA, Particle Metrix, Meerbusch, Germany). Exosomal markers, including Alix (Cat.No. 12422-1-AP, Proteintech, USA) and CD9 (Cat.No. 14025-1-AP, Proteintech, USA), were imaged by immunofluorescence in ADMSCyoung-EVs and ADMSCold-EVs. The EVs were fluorescently labelled by incubation with CD9 and Alix antibodies respectively. Fluorescence images of EVs were obtained from Nikon A1 plus multiphoton laser scanning confocal microscopy. Protein was extracted from EVs using RIPA buffer and sonicated at 4 °C. The expression levels of EV markers, including CD9, CD63 (Cat.No. PA5-100713, Thermo Fisher Scientific, USA) and Alix, as well as NAMPT (Cat.No. 11776-1-AP, Proteintech, USA) were analyzed by western blotting. The detailed operations of western blot were shown below.
Safety assessments
Because both EVs were exogenous and demonstrated potential toxicity, safety assessments were conducted by both observing histological imaging and assessing the physiological function of the liver and kidney. Hematoxylin and eosin (HE) staining was used for histological observation, and biochemistry examinations were used to examine liver and kidney functions. At least 0.75 mL of blood was collected from all mice, and serum samples were obtained for advanced analyses. Blood biochemical analyses were performed at the Shanghai Biological Model Organism Research Center.
Histological examination and TUNEL staining
The Achilles tendon along with part of the tibia and calcaneus were taken and quickly placed in 4% paraformaldehyde for fixation. The fixed specimens were routinely sectioned and stained. Masson staining was used to image the collagen fiber using a commercial kit (Masson's Trichrome Stain Kit, Solarbio, Beijing, China) according to the manufacturer's instructions. Paraffin-embedded tissue sections were then deparaffinized in xylene and dehydrated in ethanol gradients. The staining was performed according to the instructions of the TUNEL staining kit (Sigma‒Aldrich, USA). Apoptotic cells were positive cells, which were brownish yellow or tan under the light microscope, and non-apoptotic cells were negative cells, which appeared blue.
Biomechanical testing
For the biomechanical test, freshly isolated tendons were maintained in PBS and stored at − 20 ℃ until use. Both ends of the tendon of the experimental specimen are directly fixed on the instrument to ensure that when the maximum tensile force is reached, the tendon tear site is the tendon or tendon-bone junction rather than the tendon side or the humeral head side falling off the instrument. During the biomechanical experiment, the tensile test was performed on a universal material testing machine (AG-10KNX, Shimadzu, Japan), and the load rate was 0.4 mm/s with a preload of 1 N. The loading load when the tendon was pulled off was observed and recorded in detail and used as the maximum load (N). The stiffness of tendon tissues (force required per mm displacement) was analyzed based on the force/elongation curve.
ELISA
The concentrations of Col I (Cat. No. KL-ColI-Mu, Kanglang Bioengineering, Shanghai, China), Col III (Cat. No. 02666, Yansheng Bioengineering, Shanghai, China), MMP3 (Cat. No. MMP300, R&D, USA), MMP9 (Cat. No. MMPT90, R&D, USA) and TIMP-1 (Cat. No. MTM100, R&D, USA) in the supernatant from tendon tissue homogenates were measured using commercial ELISA kits (Cat. No. DAB142, DAB140B; R&D, USA). The procedures were conducted following the instructions of the manufacturer. The OD value at 450 nm was detected with a Multiskan Microplate reader (Thermo Fisher Scientific, USA) used for analyses.
SA-β-gal staining
A senescence β-galactosidase staining kit (Beyotime Biotech, Hangzhou, China) was used to detect cellular senescence in cultured cells. Staining was conducted according to the manufacturer’s instructions. The senescent cells were stained blue, and the percentage of positive.
NAD+/NADH assay
NAD+ content and the NAD+/NADH ratio were analyzed with an NAD/NADH Assay Kit (Cat. No. ab65348, Abcam; UK). Tissue lysates were used to quantify NAD+ contents and the.
Cell culture
Primary cultures of tenocytes and macrophages were used for advanced experiments. Briefly, tendon tissue was digested with 1 U/ml dispase (STEMCELL Technologies, USA) and 2 mg/ml collagenase (Worthington Biochemical, USA). Isolated tenocytes were kept in α-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and type I collagen (Sigma‒Aldrich, USA). After sacrificing the mice, the peritoneal macrophages were isolated and extracted. After 48 h of culture, the morphology of the cells was observed under a microscope and identified by flow cytometry.
A cellular crosstalk system was generated to mimic inflammatory conditions in the tendon microenvironment. Macrophages were treated with IL-1β or EVs, and the culture media were collected to treat the tenocytes. IL-1β pretreatment in macrophages was conducted to induce tendinopathy-related inflammation, and tenocytes were collected for advanced analyses.
In vitro experiments
IL-1β treatments in tenocytes and macrophages were used to induce an inflammatory response, and TGF-β1 treatments in tenocytes were adopted to analyze the fibrotic pathological process. EVs labeled with DII iodide were used in the EV uptake assay. The cell death status was analyzed with a Cytotoxicity Assay Kit (Beyotime, Hangzhou, China), and propidium iodide (PI) was used to stain dead cells with red fluorescence. The relative ratio of dead cells was used in the data analyses. The cellular senescence status was detected by two independent methods, including biological markers, including p21Cip1 and p16INK4a, and SA-β-gal staining. The cellular migration assay was conducted with a transwell test, and the migrated cells were stained with crystal violet. For the pathological fibrosis assay, α-SMA and phalloidin immunofluorescent staining of tenocytes in different groups was detected. Total collagen contents in the supernatants were analyzed using a mouse total collagen assay kit (DASF, Nanjing, China). The relative ratios of collagen contents compared with the control group were recorded and analyzed. The mRNA expression levels of fibrotic genes (COL I, COL III, Den and Cx43) and extracellular matrix remodeling-related genes (MMP1, MMP3, MMP9, TIMP1 and TIMP2) were evaluated using RT‒PCR. Mitochondrial membrane potential detection was conducted using a JC-1 kit (Cat. No. C2003S, Beytime, Hangzhou, China). The formation of aggregates by JC-1 produced red fluorescence, which indicated that the membrane potential was high, while JC-1 monomers indicated that the mitochondrial membrane potential was low and thus produced green fluorescence. Both the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were used in cellular energy metabolism analyses. Tenocytes in different groups were seeded on Seahorse XF24 plates (Agilent Technologies, Santa Clara, CA, USA) at 1 × 104 cells/well. For OCR detection, 1 μM oligomycin, 2 μM FCCP, and 2 μM Rot/AA were added to the activated probe. Another plate of cells was placed into the prewarmed Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA, USA) for ECAR detection. The detection solution was heated to 37 °C, the pH was adjusted to 7.4, the cells were washed twice with 1 mL of detection solution, and then 10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG were added to the activated probe. The results were calculated and analyzed according to the obtained real-time detection values of ECAR and OCR. ATP production and LDH release in cultured cells were detected using an ATP Assay Kit (Beyotime, Hangzhou, China) and LDH Release Assay Kit (Beyotime, Hangzhou, China), respectively. A phagocytosis assay was conducted to analyze the phagocytic functions in macrophages with different treatments.
Macrophage M2 polarization was identified using both flow cytometry with CD206 antibody (Thermo Fisher Scientific, USA) and the expression of M1/M2 markers (CD86, iNOS, IL-6, TLR4, Arg-1, Fizz-1, Ym-1 and CD206). The senescence-associated secretory phenotype (SASP) was identified by detecting the mRNA expression of related key factors including IL-6, IL-8, TGF-β1, MCP-1, MMP3, TNF-α and CXCL1. The Nile red fluorescent beads at a concentration of 5 × 105 beads/ml (Invitrogen, USA) were incubated with macrophages for 2 h. After two washes with PBS, the extracellular beads were removed. Then, the cellular samples were fixed with 4% PFA for 15 min, stained with DAPI, and observed under a fluorescence microscope.
Signaling experiments
To detect the signaling pathways of the protective effects of EVs in tenocytes and macrophages, 100 ng/mL recombinant IL-1β (Sino Biological Inc., Beijing, China) was used in this study. The potential roles of the NAMPT/SIRT1/PPARγ/PGC-1α pathway in tenocytes and the NAMPT/SIRT1/Nf-κb p65/NLRP3 pathway in macrophages were analyzed. NAMPT inhibitor FK566 (10 nM) or SIRT1 inhibitor EX-527 (100 nM) pretreatments were conducted to study the roles of NAMPT and SIRt in the signaling pathway.
Immunofluorescence.
Tenocytes and macrophages in logarithmic phase were digested into single-cell suspensions, and the plated cells were used in advanced experiments when grown to 70–80% confluence. The cells in different groups were treated for 24 or 48 h and then used in immunofluorescence assays. After washing with PBS and fixing with 4% paraformaldehyde, the p16INK4A (1:200, ab211542, Abcam, UK), p21CIP1 (1:200, 10355-1-AP, Proteintech, USA) and α-SMA (1:200, 19245, CST, USA) primary antibodies were added and incubated overnight at 4 ℃. After incubating with the fluore scently labeled secondary antibody (Thermo Fisher Scientific, USA) at room temperature for 2 h, the cell plates were washed with PBS. DAPI incubation was conducted for nuclear staining at room temperature for 2 min, and finally, the cells were observed with a fluorescence microscope (IX53, Olympus, Tokyo, Japan). Cytoskeleton staining was conducted with a phalloidin product (A12379, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.
RT‒PCR
RT‒PCR was adopted to detect the expression of key genes involved in NAD+ metabolism, macrophage polarization, SASP status and extracellular matrix remodeling. Total RNA samples from cultured ADMSCs, tenocytes and macrophages in each group were extracted with TRIzol reagent (Invitrogen, USA). After removing the mixed DNA and DNase, quantification of extracted RNA was conducted by spectrophotometry (Nanodrop 2000, Invitrogen, USA). First-strand cDNA was synthesized from total RNA with a Fast King RTPCR synthesis kit (Tiangen, China). RT‒PCR was performed with the FastQuant RT Kit (Tiangen, China), and the reaction program was as follows: predenaturation at 95 °C for 15 min; denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s, 40 cycles. β-actin was adopted as an internal reference. The relative expression of mRNA was calculated by the 2−ΔΔCt method. The primers are listed in Additional file 1: Table S1.
Western blot
ADMSCs, tenocytes and macrophages in different groups were used in protein content assays. RIPA lysis buffer was used for total protein extraction, and the protein concentration was determined by a BCA kit (Beyotime. Hangzhou, China). After adding loading buffer, the extracted protein samples were boiled and used for advanced experiments. Then, the proteins were separated by SDS‒PAGE and primary antibodies, including NMAPT (1:1000, ab236874, Abcam, UK), SIRT1 (1:500, 2028S, CST, USA), PPARγ (1:1000, ab272718, Abcam, UK), PGC-1α (1:500, ab106814, Abcam, UK), p65 K310Ac (1:1000, ab19870, Abcam, UK), NLRP3 (1:1000, 19771-1-AP, Proteintech, USA), ASC (1:1000, 10,500-1-AP, Proteintech, USA) and β-actin (1:1000, Cat. No. 3700, CST, USA) at 4 ℃ overnight. The membranes were incubated with secondary antibody for 2 h at room temperature. ECL chemiluminescence was used to detect the signal. β-actin was adopted as an internal reference, and the bands were quantitatively analyzed by ImageJ software.
Statistical analysis
The experimental data were analyzed using GraphPad 8.4.3 software (GraphPad Software Inc., CA, USA). Continuous data are expressed as the mean ± standard deviation. Student’s t test was used to analyze the difference between two groups, and one-way analysis of variance (ANOVA) was used to compare the means among multiple groups. The SNK test was used to compare the means of each group pairwise if there were significant differences. A P < 0.05 was considered statistically significant.
