All experiments were conducted with the approval of the Tokai University Ethics Committee and with either informed consent from the patient or parental permission.
Fabrication of PD sheets
Cartilage tissue was obtained from eight patients (average age 13.4 months, range 8–17 months, four boys and four girls) who underwent polydactyly surgery at Tokai University Hospital. A summary of the PD sheet fabrication process is shown in Fig. 1a. Cartilage tissue was minced with scissors and subsequently incubated in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS; AusGeneX, Molendinar, Australia), 1% antibiotic–antimycotic solution (AB; Gibco), and 5 mg/mL collagenase type 1 (CLS1; Worthington, Mannheim, Germany) for 1.5 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cell suspension was washed and passed through a 100-μm strainer (BD Falcon, Franklin Lakes, NJ, USA).
The collected cells were seeded at a density of 1 × 104 cells/cm2 on six-well culture plates (Corning, Corning, NY, USA) in DMEM/F12 supplemented with 20% FBS and 1% AB and incubated at 37 °C. After 4 days, 100 μg/mL ascorbic acid (Nissin Pharmaceutical, Yamagata, Japan) was added to the medium and the medium was replaced every 3 or 4 days. Cells were passaged once or twice when they reached confluence and then cryopreserved. To fabricate PD sheets, cells were thawed and passaged once and then seeded on temperature-responsive culture inserts (CellSeed Inc., Tokyo, Japan) at 1 × 104 cells/cm2. After 2 weeks, the culture plates were kept at 25 °C for 30 min to promote detachment of PD sheets from the inserts and the sheets were collected onto a polyvinylidene difluoride (PVDF) membrane. PD sheets were manipulated and visually confirmed to check for strength and any tearing.
Fabrication of adult chondrocyte sheets
Adult knee articular cartilage and synovium were obtained from 11 patients (average age 74 years, range 67–79 years, five men and six women) who underwent TKA surgery at Tokai University Hospital. TKA sheets were fabricated according to previously reported methods [24, 25], which are similar to those used to create autologous chondrocyte sheets in our clinical study. A summary of the TKA sheet fabrication process is shown in Fig. 1b.
Briefly, cartilage and synovium were minced and subsequently incubated in DMEM/F12 supplemented with 20% FBS, 1% AB, and 5 mg/mL CLS1 for 4 and 2 h, respectively, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cell suspensions were washed and passed through 100-μm strainers. Chondrocytes were cryopreserved, and synovial cells were seeded at 1 × 104 cells/cm2 and cryopreserved after confluence. To fabricate TKA sheets, chondrocytes were seeded on temperature-responsive culture inserts and cocultured with synovial cells for 2 weeks and three chondrocyte sheets were layered onto a PVDF membrane and cultured further for 1 week. TKA sheets were then manipulated and visually confirmed to check for strength and any tearing.
Cell count and viability
PD sheets and TKA sheets were washed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco). The sheets were then incubated in TripLE Express® (Gibco) at 37 °C for 15 min and centrifuged at 1500 rpm for 5 min. The cell sheets were resuspended in 0.25 mg/mL Collagenase P (Roche, Basel, Switzerland) at 37 °C for up to 30 min and then centrifuged at 1500 rpm for 5 min. The isolated cells were finally resuspended in DMEM/F12, and cell count and viability were determined using the trypan blue exclusion assay.
Flow cytometric analysis
After obtaining the cell count, isolated cells were washed with DPBS containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid (EDTA; Gibco). About 1.5 × 105 cells were mixed in each tube with the following antibodies: hCD31–fluorescein isothiocyanate (FITC) (clone: 5.6E, Beckman & Coulter, Brea, CA, USA), hCD44–FITC (clone: G44-26), hCD45–FITC (clone: J.33, Beckman & Coulter), hCD81–allophycocyanin (APC) (clone: JS-81, BD Bioscience, Franklin, NJ, USA), and hCD90–APC (clone: 5E10, BD Bioscience). The cells were incubated for 90 min at 4 °C and then washed with DPBS containing 0.2% BSA and 1 mM EDTA. Fluoroprobe-labeled mouse IgG1 antibody (clone: 679.1Mc7, Beckman & Coulter) and mouse IgG2b antibody (clone: MG2b-57, Beckman & Coulter) were used as negative controls. Stained cells were analyzed using a FACSVerse™ cell sorter (BD Bioscience).
Histological and immunohistochemical staining
PD sheets and TKA sheets were harvested after culture and then embedded and frozen in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan). Then, sections 10 μm thick were stained for proteoglycans with Safranin O or toluidine blue using standard methods. Sections 20 μm thick were immunostained with anti-human type I collagen (COL1; 1:200; Southern Biotech, Birmingham, AL, USA), type II collagen (COL2; 1:200; Kyowa Pharma Chemical, Toyama, Japan), fibronectin (FN; 1:500; Merck, Darmstadt, Germany), and aggrecan (ACAN; 1:10; R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. The sections were washed and incubated at room temperature for 1 h with the secondary antibody Alexa Fluor 488-conjugated goat anti-mouse Ig (Thermo Fisher Scientific, MA, USA) for COL2 and FN or Alexa Fluor 546-conjugated donkey anti-goat Ig (Thermo Fisher Scientific) for COL1 and ACAN. After immunostaining, the sections were washed and mounted with VECTASHIELD Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Microscopic images were captured under a BZ-8000 microscope (Keyence, Osaka, Japan).
Measurement of humoral factors
A random selection of fabricated PD sheets and TKA sheets were cultured for 72 h in 3 mL of DMEM/F12 supplemented with 1% FBS and 1% AB. Supernatants were collected and centrifuged at 15,000g for 10 min to remove cell debris. The concentrations of transforming growth factor beta-1 (TGF-β1; R&D Systems), melanoma inhibitory activity (MIA; Roche), tissue inhibitor of metalloproteinases (TIMP1; R&D Systems), matrix metalloproteinase-3 (MMP3; Sigma-Aldrich), stanniocalcin-1 (STC1; Cusabio, College Park, MD, USA), and hyaluronan and proteoglycan link protein 1 (HAPLN1; US Biological, Salem, MA, USA) were measured using enzyme-linked immunosorbent assay (ELISA) kits. The signal detected for blank medium containing 1% FBS was subtracted to adjust for proteins contained in FBS. Measurements were repeated at least twice for each donor, and averages were used.
Statistical analysis
Numerical results are expressed as mean and standard deviation. Statistical analysis was performed using SPSS 23.0 software (IBM Corp., Armonk, NY, USA). Differences between the two groups were identified using Student’s t test. The level of significance was set at P < 0.05.