Osteosoft solution

For histological analysis, osteochondral plugs were submerged in 4% buffered formaldehyde solution (VWR, Radnor, PA) for ≤7 days, and decalcified for 4–6 weeks under constant agitation using the Osteosoft solution (Merck, Burlington, MA). After decalcification, the samples were embedded in Tissue‐Tek O.C.T. and stored at −80°C. Cryosectioning was performed using the CryoStar NX70 Cryostat (Thermo Fischer Scientific, Waltham, MA) to obtain 6‐µm sections. Subsequently, the samples were prepared for Safranin O staining and Fast‐green counterstaining. Histological images were captured using a Leica microscope DM‐1000 and processed using the Leica Manager software (Leica, Wetzlar, Germany).

Spinal cord and lumbar disc 5–6 tissues were harvested, fixed and decalcified [71 (link)]. In [73 (link)] particular, the tissues were fixed for 24 h in a formaldehyde solution (10% in PBS) at room temperature and decalcified in Osteosoft solution (Merck Millipore). Three non-sequential sections were chosen from each sample for examination. Next, the samples were dehydrated through a graded series of ethanol and xylene and embedded in BioPlast Plus (Bio Optica, Milan, Italy). Sections (5 μm in thickness) were prepared from the tissues [74 (link),75 (link)]. The sections were then incubated overnight with KRT18 (CSB-MA000256, Cusabio Technology, Houston, TX, USA) or KRT19 (CSB-MA000207, Cusabio Technology). The samples were washed with PBS and incubated with secondary antibodies. Specific labeling was identified with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA) [76 (link)]. The stained sections were observed using a Leica DM6 microscope (Leica Microsystems S.p.A., Milan, Italy), following a typical procedure [77 (link)].

Organs were fixed in 4% PFA/PBS overnight and hind limb bones were decalcified in Osteosoft solution (Merck Millipore) for 4–5 days. Samples were dehydrated and paraffin-infiltrated by the STP 420 tissue processor (Thermo Fisher Scientific). 1 μm sections of paraffin-embedded tissue were stained with hematoxylin and eosin (H&E) according to standard protocols. Immunohistochemistry was performed as previously described (Bledau et al., 2014 (link)) using the following primary antibodies: rabbit polyclonal anti-myeloperoxidase (1:100; ab9535 Abcam, Cambridge, UK), rabbit polyclonal anti-von Willebrand factor (1:500; ab9378 Abcam). After counterstaining with hematoxylin, images were obtained at an Olympus BX61 upright microscope (Olympus Europa, Hamburg, Germany).

The MVs were fluorescently labeled using PKH26 (Sigma–Aldrich) as previously described [5 (link)]. The red fluorescence-labeled MVs were incubated with RAW264.7 cells at a final concentration of 10 μg/ml and incubated for 1 h. Following incubation, the cells were washed with PBS and fixed with 4% PFA. To detect locally injected MVs in mice, maxillae were fixed in 4% PFA for 24 h, demineralized using Osteosoft solution (Merck, Darmstadt, Germany) for 72 h, and embedded in paraffin. The slides were covered with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific, Waltham, MA), and the images were captured using a fluorescence microscope (Keyence Co., Osaka, Japan).

For histological analysis, osteochondral grafts were fixed in 4% buffered
formaldehyde solution (VWR, Radnor, PA, USA) for up to 1 week and decalcified
under constant agitation using Osteosoft solution (Merck, Burlington, MA, USA).
After decalcification (duration of 4-6 weeks), the osteochondral grafts were
embedded in Tissue-Tek OCT (optimal cutting temperature, VWR, Radnor, PA, USA)
and stored at −80°C. Sectioning was done using the CryoStar NX70 Cryostat
(Thermo Fischer Scientific, Waltham, MA, USA), with −25°C for the knife
temperature and −20°C for the chamber temperature. Six-micrometer sections were
obtained and processed for safranin O/light green staining. Images were taken
with a Leica DM-1000 microscope and processed using the Leica Manager software
(Leica, Wetzlar, Germany). To quantify changes within the cartilage sections
stained with safranin O/light green, a modified Mankin scoring system was used
(Table 3).^{27 (link)}
The assessment was done by 5 independent observers with a maximum score
of 15.

Fixed murine feet were decalcified using the EDTA-based Osteosoft solution (Merck) and then embedded in paraffin for histological analysis.by Ziehl-Neelsen stain, Alcian blue-periodic acid Schiff stain, and immunohistochemistry (IHC) for fibrin(ogen). For IHC staining, 5-μm tissue sections on polylysine-coated slides were deparaffinised, endogenous peroxidase quenched, epitope unmasked with heated IHC citrate buffer (pH 6.0) (Merck) and blocked with 5% bovine serum albumin. The tissue sections were incubated with anti-fibrinogen antibody (A0080, DAKO) or matched isotype control overnight at 4°C. Staining was then performed with biotinylated horse anti-rabbit IgG (Vector Laboratories) and VECTASTAIN Elite ABC kit and ImmPACT NovaRED peroxidase substrate and further counterstained with Harris Haematoxylin (ThermoFisher Scientific). Whole slide images were captured using the NanoZoomer slide scanner (Hamamatsu Photonics) and analysed using ImageScope software (Leica Biosystems) and ndp2.view software (Hamamatsu). Some photographs were taken with Micropix microscope camera (acquisition software Cytocam) attached to a Yenway CX40 laboratory microscope (Micropix).