Pepsin

The enzymes (pepsin, Ph.Eur. grade, Carl Roth GmbH, and neutrophil elastase, 20–22 U/mg, Abcam, respectively) were diluted to 5.5 µg/mL in TFA 10 mM (pepsin) or Tris buffer 0.1 M + NaCl 0.5 M pH 7.8 (neutrophil elastase) and pre-incubated for 15 min at 37 °C. Peptides were added at 100 µM and incubated at 37 °C. A sample of 40 µL was taken after 60 min of incubation and the reaction was stopped by adding 5 µL sodium carbonate solution (1 M, pepsin) and 5 µL TFA (neutrophil elastase), respectively. After 10 min, 60 µL acetonitrile/water 50:50 containing 1% TFA were added, and the resulting solution was analyzed by LC-MS (conditions: Phenomenex Kinetex 2.6 μm C18 100 Å column, 50 × 2.1 mm, flow rate 0.55 mL/min, gradient of acetonitrile in H₂O (both containing 0.1% TFA, 20–60% over 20 min)).

After examination by micro-CT, the samples were fixed in 4% PFA and then decalcified in 10% (w/v) EDTA (pH = 7.0) for 2 months at room temperature. Next, they were dehydrated and embedded in paraffin wax, sectioned into 6-μm slices and stained with H&E, toluidine blue, safranin O/fast green, and Sirius red according to the manufacturer’s protocols. Collagen II immunohistochemical staining was performed by immersing the sections into 0.25% pepsin (Abcam, United States) at 37°C for 20 min and blocking them in 10% goat serum for 1 h. After antigen retrieval, the slices were incubated with primary antibodies against collagen II (1:200; Developmental Studies Hybridoma Bank, United States) at 4°C overnight. After washing with PBS, they were incubated with goat anti-mouse IgG (1:200; Cat# NB7539; Novus) for 1 h. Finally, the sections were stained with Tris-HCl buffer containing 0.05% DAB and 0.005% hydrogen peroxide, and the nuclei were stained with hematoxylin. Photomicrographs were acquired using a Nikon microscope (Japan).
To evaluate the progress of subchondral bone reconstruction and cartilage repair, sections from three knees at 3 and 6 months per group (each sample represented three tissue sections) were blindly scored by three independent observers according to an established scoring system.