Cd105

ADSCs and WJ-MSCs were detached using 0.25% trypsin EDTA (Gibco), and the collected cells were washed with PBS containing 1% BSA. The cells were divided into 1 × 10⁶ cells and incubated for 1 h at 4 °C with primary antibodies against CD34 (Bioss, Beijing, China), CD45 (Proteintech, Rosemont, IL, USA, clone number. 4E9B2), CD73 (R&D Systems, Minneapolis, MN, USA), CD90 (R&D Systems, Minneapolis, MN, USA), and CD105 (Novus Biologicals, Centennial, CO, USA, clone number. MEM-229). The cells were then washed with PBS containing 1% BSA and incubated for 1 h at 4 °C with secondary antibodies against PE-conjugated anti-rabbit (R&D Systems, MN, USA), PE-conjugated anti-sheep (R&D Systems, MN, USA), and APC-conjugated anti-mouse (R&D Systems, MN, USA) antibodies. Appropriate secondary antibodies were used according to the primary antibodies. Unlabeled cells and those labelled only with the secondary antibody were used as negative controls. Subsequently, the cells were washed with PBS containing 1% BSA. The fluorescence of the stained samples was analyzed using a FACSCalibur flow cytometer (BD Biosciences) and BD CellQuest Pro software v9.

To investigate the ability of the various scaffolds on recruiting SMSCs in vivo, the PCL, PCL/MECM, and PCL/MECM-KGN μS scaffolds were implanted in the meniscus of the Sprague–Dawley rats. CD73 and CD105, which were defined as MSC markers and assessed by immunofluorescence staining to determine the endogenous MSC recruitment capacity of the three scaffolds. Briefly, after the samples were harvested, we used 4% paraformaldehyde to fix them at room temperature for 30 min, and then, the target antigens were retrieved by 2% sodium citrate for 20 min and blocked by 10% goat serum. Subsequently, the samples were incubated with primary antibodies against CD73 (1:150, Novus Biologicals) and CD105 (1:150, Novus Biologicals) at 4°C overnight, followed by incubation with secondary antibodies, which were conjugated with Alexa Fluor488 and Fluor594 (1:100, Abcam, Cambridge, United Kingdom) for 1 h and DAPI (1:1000, Life Technologies) for 10 min. The samples were imaged by a TCS-SP8 confocal microscope (Leica, Germany) to determine the total cell numbers and CD29 and CD90 double-labeled cell numbers.

Eight healthy, 18‐week‐old male Sprague Dawley rats weighing 200–220 g were employed for the in vivo ESPC recruitment study. This study was designed with four groups as follows: the (A) control group, (B) GE hydrogel group, (C) Apt‐hydrogel group, and (D) Apt/GF‐hydrogel group (n = 4 knees in each group). More information about animal surgery is given in Data S1 1.5.
To evaluate the cell lineage recruited to the defect site, CD73 and CD105, which were defined as MSC markers, were assessed by immunofluorescence staining. Briefly, samples (n = 4 knees) were first fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature. The target antigens were retrieved by immersing the specimens in 2% sodium citrate for 20 min and blocking them with 10% goat serum. Subsequently, the specimens were incubated overnight at 4°C with CD73 (1:200, Novus Biologicals) and CD105 (1:100, Novus Biologicals). The next day, the samples were washed and incubated with secondary antibodies conjugated with Alexa Fluor488 and Fluor594 (Abcam, Cambridge, UK) for 1 h at room temperature. Next, the samples were counterstained with DAPI (1:1000, Life Technologies) to label the nuclei and observed with a Leica TCS‐SP8 confocal microscope (Leica, Germany). The number of total cells and CD73/CD105 double‐positive cells were analyzed with ImageJ software (USA).