PU-MC composites were fixed in 70% methanol at 37 °C to prevent hydrogel solid-gel phase transition and incubated in 5% D(+) sucrose (Sigma Aldrich, Milan, Italy) solution in PBS for 12 h at 37 °C before embedding them in Jung tissue freezing compound and cryosectioning at 10 μm (Microm HM560 CryoStar, Thermo Scientific, Waltham, USA)10 (link). The presence of GAGs was investigated using safranin-O (Sigma Aldrich, Milan, Italy). The deposition of collagen type I, type II and type X was determined by an immunofluorescence staining. After enzyme pre-treatment (0.5 U/mL Hyaluronidase), sections were incubated with antibodies against collagen I (COL 1, 1:150, rabbit polyclonal, ab34710 from Abcam, Cambridge, UK), collagen II (COL 2, 1:50, rabbit polyclonal, ab34712 from Abcam, Cambridge, UK) and collagen X (COL 10, 1:50, rabbit polyclonal, ab58632 from
Abcam, Cambridge, UK). Sections were incubated overnight at 4 °C, washed 3 times with PBS and co-stained using an appropriate secondary antibody (Alexa-Fluor conjugated anti-rabbit, 1:500 in PBS, AP132JA4 from Sigma Aldrich, Milan, Italy). Samples were visually investigated by a fluorescence microscope (Leica DM5500 B, Leica Microsystems, Basel, Switzerland).
Abcam, Cambridge, UK). Sections were incubated overnight at 4 °C, washed 3 times with PBS and co-stained using an appropriate secondary antibody (Alexa-Fluor conjugated anti-rabbit, 1:500 in PBS, AP132JA4 from Sigma Aldrich, Milan, Italy). Samples were visually investigated by a fluorescence microscope (Leica DM5500 B, Leica Microsystems, Basel, Switzerland).