Gels containing cells were first fixed with 4% paraformaldehyde in serum-free DMEM at 37°C for 30 to 45 min. The gels were then washed in phosphate-buffered saline (PBS) containing calcium (cPBS; GE Healthcare), incubated in 30% sucrose (Thermo Fisher Scientific) at 4°C overnight, and placed in a mixture of 50% of a 30% sucrose in cPBS solution and 50% O.C.T. (Tissue-Tek) for 4 hours. The mix solution was removed, and the gels were then embedded in O.C.T., frozen, and sectioned with a thickness of 30 to 80 μm using a cryostat (CM1950, Leica). The sectioned samples were stained using standard immunohistochemistry protocols. The samples were permeabilized with Dulbecco’s PBS (DPBS) containing 0.5% Triton X-100 (Sigma-Aldrich), blocked with DPBS containing 1% bovine serum albumin (Sigma-Aldrich), 10% goat serum (Invitrogen), 0.3 M glycine (Thermo Fisher Scientific), and 0.1% Triton X-100. The following antibodies and reagents were used for immunohistochemistry: p27Kip1 antibody (catalog no. ab32034, Abcam), Ki-67 antibody (catalog no. RM-9106S, Thermo Fisher Scientific), collagen antibody (catalog no. ab34710, Abcam), fibronectin (catalog no. ab2413, Abcam), laminin-5 (catalog no. ab78286, Abcam), α-tubulin (catalog no. 3873, Cell Signaling Technology), and Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and Alexa Fluor 488 phalloidin to stain actin (Invitrogen). The Click-IT EdU cell proliferation assay (Invitrogen) and TUNEL assay (Thermo Fisher Scientific) were used to assess the proliferation and apoptosis of cells, respectively.

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