Phosphotungstic acid

TR-701 was purchased from Shanghai Macklin Biotechnology Co., Ltd. OH–PLGA–COOH 50/50 (PLGA, Mw = 20,000) was purchased from Jinan Daigang Biomaterial Co., Ltd. Poloxamer188 (P188) was purchased from BASF. DiO (green fluorescent probe), Cell counting kit-8 (CCK-8), 4% paraformaldehyde, antifade mounting medium, PBS, 10 $\times$ PBS were purchased from Biyotime Biotechnology Co., Ltd. Saline and agar powder were purchased from Shanghai Yuanye Biotechnology Co., Ltd. 2% Phosphotungstic acid was purchased from Beijing Solarbio Science and Technology Co., Ltd. Carbon support film (230 mesh) was purchased from Beijing Zhongjingkeyi Technology Co., Ltd. Dialysis bag (36 mm, MD44, MW:3500) was purchased from Shanghai labeen Biotechnology Co., Ltd. 0.25% Trypsin-EDTA, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco, USA. DMEM high glucose medium was purchased from HyClone Biochemical Products (Beijing) Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Shanghai MP Biomedical Co., Ltd. Sodium chloride (>99.5%) was purchased from Shanghai Sangon Biotechnology Co., Ltd. Sodium bicarbonate (>99%) was purchased from Shanghai Saen Chemical Technology Co., Ltd. Yeast extract and tryptone were purchased from Oxoid, USA. Acetone and EDTA-2Na were purchased from Sinopharm Chemical Reagent Co., Ltd.

After 8-week treatment, the rabbits were sacrificed, and tissue specimens surrounding fracture locations were collected for further analyses. Afterwards, specimens were subsequently fixed in 10% PBS-buffered formaldehyde, followed by decalcification, embedding and sectioning. For H&E staining, the slides were incubated with hematoxylin (Solarbio, China) for 10 min and with Eosin (Solarbio, China) for 3 min, respectively. Thereafter, the slides were fixed with 70% ethanol for 20 s, 90% ethanol for 20 s, 100% ethanol for 60 s and xylene for 3 min. For Masson staining, the slides were stained with Weigert’s iron-hematoxylin (Solarbio, China) for 5 min, phosphomolybdic–phosphotungstic acid (Solarbio, China) for 45 s and a solution containing 1% orange G and 0.25% aniline blue (Solarbio, China) for 5 min. Next, these slides were rinsed with 1% acetate solution (Solarbio, China) and stained with 0.12% ponceau xylidine (Solarbio, China) for 20 min. After rinsing with 1% acetate solution, the slides were then incubated with 2.5% phosphotungstic acid (Solarbio, China) for 10 min, rinsed with 1% acetate solution, and dehydrated in ethanol and xylene. Histological images were collected through Pannoramic 250 Flash III (3DHISTECH Ltd, Budapest, Hungary), and were analyzed by CaseViewer 2.3 and Image J software (National Institutes of Health, USA) accordingly.

A total of 20 μL of isolated exosomes were loaded onto copper grids (Ted Pella Inc., Redding, CA, USA) and allowed to absorb for 2 min. The samples were further fixed with 2% phosphotungstic acid (Solarbio) for 10 min. After air drying at room temperature, the samples were observed under a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan).

SjEVs or SjEV-depleted ESPs suspension was adsorbed onto 200 mesh formvar-coated grids (Agar Scientific, UK) for 2 min at room temperature (25°C). The grids were then stained with 2% phosphotungstic acid (Solarbio, China) for 2 min and examined under a TEM (FEI, Netherlands). The size distributions of EVs were determined by NTA using the ZetaView system (Particle Metrix, Germany).

A 10-μl aliquot of EVs suspended in PBS was pipetted on Formvar copper grid (Znongjingkeyi, Beijing, China) for 3 min and then excess liquid was removed by a dry paper towel. The copper grid was washed with sterile PBS and immediately stained with 3% phosphotungstic acid (Solarbio, China) for 30 s and allowed to air-dry. Grids were imaged by transmission electron microscopy (TEM) using a Hitachi HT7700 (Hitachi Ltd., Tokyo, Japan).