Cell isolation, culture, and characterization

All protocols involving human tissue were performed under the approval of Institutional Review Board of Ajou University School of Medicine (AJIRB-MED-SMP-10-268). Human fetal cartilage tissues were harvested from four abortus within 24 hours following elective termination (n = 4, M GA11 wks, M GA12 wks, M GA12 wks, F GA12 wks) after informed consent from respective guardians. Cells were isolated from the femoral head cartilage and expanded using a previously published protocol9. Cartilage tissues were minced into small pieces and treated with 0.1% collagenase type 2 (Worthington Biochemical Corp., Freehold, NJ, USA) in high-glucose DMEM containing 1% FBS at 37 °C under 5% CO 2 . After 12 h, the released cells were centrifuged at 1700 rpm for 10 min, washed twice, and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin at a density of 8 × 103 cells/cm2. When cells reached 80% confluence, the 0.5% Trypsin-EDTA of 10X to 1 × (Gibco, NY, USA) were used to detach the cells, which replated by the same way as above. Cells were expanded for two passages with culture medium changed every 3 days. All donor cells showed no significant difference in terms of morphology, proliferation, surface marker, senescence, and chondrogenesis as previously published9. Passage 2 cells were used for this study from all groups during in vitro experiments, and M GA11 wks cells were used for in vivo investigations.

Cells at passage 2 were analyzed for expression of stem cell related surface markers by flow cytometry. Briefly, cells in the suspension were incubated with anti-CD29-PE (Catalog No.

555443), anti-CD34-FITC (Catalog No. 555821), anti-CD45-PE (Catalog No. 555483), anti-CD73-FITC (Catalog No. 561254), anti-CD90-FITC (Catalog No. 555595), anti-CD105-FITC (Catalog No. 561443), anti-SOX2-PE (Catalog No. 560291) (BD Biosciences, CA, USA) and anti-OCT3/4-APC (Catalog No. IC1759A) (R&D Systems, MN, USA) for 40 min at 4 °C. Stained cells were determined by BD FACSCanto II flow cytometer (BD Biosciences, CA, USA) and analyzed using Flowing software (http://flowingsoftware.btk.fi).

For osteogenic and adipogenic differentiation, cells at passage 2 were plated in 6-well plates at densities of 2 × 103 cells/cm2 and 2 × 104 cells/ cm2, respectively. After 24 h, the cells were incubated in the differentiation medium for each lineage. The osteogenic medium consisted of α-MEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 μg/ml ascorbate-2 phosphate (Sigma-Aldrich, MO, USA). The adipogenic medium consisted of α-MEM supplemented 10% FBS, 1 µM dexamethasone, 10 μg/ml insulin, 0.5 mM isobutyl-methylxanthine, and 0.1 mM indomethacin (Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of differentiation, cells were stained with Alizarin red S and Oil red O (Sigma-Aldrich, MO, USA) to observe the degree of mineralization and the lipid droplets, respectively. For chondrogenic differentiation, 3 × 105 cells at passage 2 were centrifuged at 500 × g for 5 min, and the cell pellet was cultured in a chondrogenic medium. The chondrogenic medium consisted of DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement, 40 μg/ml proline, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate (Sigma-Aldrich, MO, USA). After 3 weeks of induction, the samples were fixed with 4% formaldehyde and embedded in paraffin wax. Sections with a thickness of 4 μm were prepared and stained with Safranin O (Sigma-Aldrich, MO, USA) to observe the sulfated glycosaminoglycans.

We also examined the proliferation ability and phenotype of the FCPCs according to passage time. The doubling time of FCPCs was determined from passage 1 to passage 15 (n = 4). Cells were subcultured at 80% confluence. The doubling time was calculted using the following formula: DT = (T1 − T0)log2/(logN1 − logN0), where T1 − T0 = the culture period in days, N0 = the plating cell number, and N1 = the harvesting cell number. Accumulated cells numbers were calculated with passages or days.

Fabrication of cartilage gels

Cartilage gels were fabricated using a previously published protocol10. Briefly, FCPCs were cultured in a high-density monolayer (at 2 × 105 cells/cm2) with DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement, 40 μg/ml proline, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate. They were cultured until full confluency two dimensionally when cells spontaneously formed a thin membrane. Subsequently, the medium was removed and 1X Trypsin-EDTA was added and incubated for less than 5 min at 37 °C. When the membrane was peeled off from the plate, the enzyme was immediately removed and the membrane was carefully isolated using a wide-bore pipette and moved individually to a 50 ml tube filled with 10 ml of defined medium without exogenous growth factors (high glucose Dulbecco’s modified eagle’s medium with an insulin–transferrin–selenium mixture, 50 mg/ml of ascorbate 2-phosphate, 100 nM of dexamethasone, 40 mg/ml of proline, and 1.25 mg/ml of BSA). Each tube was centrifuged at 100 × g for 20 min to consolidate the membrane into a pellet-type construct. The constructs were incubated for 16 h at 37 °C and then transferred to a 6-well culture plate for extended culture for 1, 2, or 3 weeks in a 37 °C humidified atmosphere of 95% air and 5% CO2. The culture medium (5 ml) was changed every 3 days.

In vitro characterization of cartilage gels

After macroscopic examination, volume of the cartilage gels was measured using micro-CT (SkyScan, Kontich, Belgium). Scanning protocols are outlined in the Supporting Information Text43.

For histological analysis, cartilage gels were fixed in 4% formalin for 3 days and processed for tissue sectioning. Tissue sections of 4 µm in thickness were made and stained with hematoxylin-eosin (H&E), Safranin-O, and immuno-histochemical (IHC) analysis for type I collagen (1/100; Abcam, Cambridge, UK; catalog No. ab34710), type II collagen (1/100; Abcam, Cambridge, UK; catalog No. ab34712), type X collagen (1/100; Abcam, Cambridge, UK; catalog No. ab49945) using the DAB method.

For water content analysis, tissue samples were weighed (wet weight), freeze dried, and weighed again (dry weight). For glycosaminoglycans (GAG) content and DNA content analysis, samples were freeze-dried and digested with papain-digestion solution (5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, and 125 mg/ml papain type III: Sigma-Aldrich, MO, USA). For papain digestion, the sample in 1 ml papain-digestion solution was incubated at 60 °C for overnight. The total GAG content was spectrophotometrically measured by using the 1, 9-dimethylmethylene blue colorimetric assay. Chondroitin sulfate sodium salt from shark cartilage (Sigma-Aldrich, MO, USA; catalog No. C4384) was also used as a standard44. Briefly, the assay was prepared via two steps: (i) The colour reagent was prepared by dissolving 16 mg dimethylmethylene blue in 1 l water containing 3.04 g glycine, 2.37 g NaCl and 95 ml 0.1 M HCl, to give solution at pH 3.0 (Sigma-Aldrich, MO, USA). (ii) The papain-digested GAG sample prepared as above was taken, and measured by reference to a standard calibration curve. The result was analyzed by Magellan v.6.4. software and Tecan microplate reader (Tecan Group Ltd, Männedorf, Switzerland). For total collagen content analysis, samples were measured by chloramine-T hydroxyproline assay45. Souluble peptides and proteins in each standard and tissue sample were hydrolyzed to individual amino acids by adding 500 μl of 4 N sodium hydroxide (NaOH) and incubating at 121 °C and 15 psi above atmospheric pressure for 20 min (using an autoclave). Samples were allowed to cool to room temperature and then neutralized with 500 μl of 4 N HCl. Hydroxyproline amino acids were converted to pyrolle-2-carboxylate by oxidation via addition of 0.625 mL of 0.05 M chloramine-T in 74% v/v H 2 O, 26% v/v 2-propanol, 0.629 M NaOH, 0.140 M citric acid (monohydrate), 0.453 M sodium acetate (anhydrous), and 0.112 M acetic acid (glacial), followed by incubation at room temperature for 20 min. Finally, 0.625 mL of 15% w/v DMAB (1 M) in 2-propanol plus concentrated acid (a.k.a. Ehrlich’s solution) was added to each sample and vortexed immediately to facilitate mixing. Samples were incubated at 65 °C for 20 min and then rapidly cooled by immersion in room temperature water to stop chromophore development.

Genetic markers were analyzed with real-time polymerase chain reaction for 1, 2, and 3 wk cultured cartilage gels, with FCPCs and mature cartilage tissue used as controls. All adult human cartilage and osteochondral tissue were harvested from morphologically normal (ICRS grade 0) lateral condyle osteochondral tissue obtained during joint replacement surgery. Protocols and primer information are outlined in Supporting Information and Supplemental Table S1.

Biomechanical analysis of cartilage gels was done by measuring the aggregate modulus, spreadability, and adhesive strength. For aggregate modulus, tissues were subjected to an unconfined compression test using Universal Testing Machine (Model H5K-T; H.T.E, Sanford, England). Each sample was placed on a bottom plate of the machine, and compressed at a speed of 1 mm/min. The machine was stopped automatically after moving a programmed length between the top and bottom plate46,47. The spreadability of cartilage gel was determined by pressing the tissue construct between top and bottom plates covered by paper, then 500 g standardized weigth was put on the upper plate and left for about 5 minutes, as previously described48,49. Diameters of spread circles were measured in mm and were taken as comparative value for spreadability. Adhesive strength of cartilage gels after transplantation into the human cartilage defect osteochondral blocks was measured using a push-out test as in previously described50,51,52. Briefly, The adhesive strength was evaluated as the forces at ultimate failure per unit of the interfacial area and determined using 5 mm in diameter of cylindrical-shaped indenter in an Universal Testing Machine fitted with a 5N maximum load cell. Alginate gels (2%) were fabricated using previously published methods and used for controls during adhesion strength testing46,47. All in vitro characterization analyses were done 5 times each.

Remodeling process analysis using a nude mouse model

Full thickness cartilage defects of 3 mm diameter were made on 5 mm diameter human osteochondral blocks obtained from joint replacement surgery. Lateral femoral condyles blocks without gross cartilage wear were used. NCs were used as normal controls, obtained from lateral femoral condyles of fresh frozen cadavers without knee joint pathology. The 2-WCG gels were transplanted on the defects using a syringe without additional fixation. OATS was performed on osteochondral blocks as controls. Briefly, 3 mm diameter osteochondral defects were made with a matching size biopsy punch. The osteochondral block was gently reinserted into the defect similar to the OATS procedure using a plastic impactor (Arthrex, Naples, FA, USA). Osteochondral blocks were then transplanted subcutaneously into the back of nude mice (Athymic NCr-nu/nu, Koatech, Korea, n = 7 for each group of analysis), after institutional approval of Institutional Animal Care and Use Committee of Ajou University (IACUC No. 2014-0072)53. Osteochondral blocks were sampled at 2, 4, 8, and 12 weeks after transplantation and analyzed as described above. Histological images were scored for cartilage repair using O’Driscoll cartilage repair scoring system by two separate pathologists not involved in this study54. For cartilage-to-cartilage integration analysis, a human cartilage cylinder construct model was used as previously described55. Briefly, cartilage gels were implanted in the cylinder constructs and OATS was used as controls. Cylinder constructs were then subcutaneously transplanted into the back of nude mice (n = 7 for each group). The cartilage-to-cartilage integration was evaluated by safranin-O stain and mechanical test for integration strength using using a push-out test as in previously described55. A custom 2 mm in diameter of cylindrical-shaped indenter affixed to a Universal Testing Machine (fitted with a 5N, 100N, 500N maximum load cell) pushed the cartilage repair out of the cartilage annulus (1 mm/min) while recording load. Failure stress (integration strength) was calculated as the quotient of the load at failure and the interface area.

Nonhuman primate cartilage defect model investigation

All protocols involving nonhuman primates (NHP) use were approved by Institutional Animal Care and Use Committee of Seoul National University (SNU-13-0251). All experiments were performed in accordance with relevant guidelines and regulations. A total of five skeletally mature male NHPs (Macaca fascicularis) aged 54 to 69 months weighting 4–5.5 kg were used for this study. Procedures for anesthesia and surgery for femoral chondral defects on both knees followed a previously published protocol and outlined in the Supporting Information Text56. Postoperative knee circumference was measured preoperatively, and at postoperative wk 1, 2, 4, 8, 16, and 24. Intravenous blood was drawn for WBC count and C-reactive protein quantification preoperatively, and during postoperative wk 1, 4, 16, and 24. MRI was performed on each knee at postoperative wk 8, 16, and 24. MRI protocols are outlined in the Supporting Information Text. At 24 wks, all animals were euthanized for further analysis. Vital organs (heart, lung, thyroid, liver kidneys, spleen, adrenal glands, testis) and relevant knee tissue (1 mm3 of repaired cartilage of left knee defects and adjacent tibial cartilage, synovium) were harvested and prepared for cell distribution RT-PCR. Chondral defects were prepared for further histological analysis and resultant histological results were further analyzed using O-Driscoll cartilage repair scores by 2 separate pathologists twice that did not participate in this study11. Synovial tissue of each knee harvested from anterior fat pads were also prepared for histological analysis and subsequent chronic synovitis grading in the same fashion12. For cell distribution analysis, PCR for human alu sequences was performed to confirm the presence of transplanted cartilage gels in recipient NHP organs. Total DNA of organ samples was extracted with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The primers used were Human Alu F: 5′-GTAAGAGTTCCGTAACAGAGCT-3′, Human Alu R: 5′-CCCCACCCTAGGAGAACTTCTCTTT-3′, NHP gapdh F: 5′-CGGATTTGGTCGTATTGGG-3′ and NHP gapdh R: 5′-GGGATCTCGCTCCTGGAAG-3′. Samples were incubated at 94 °C for 2 min and then amplified for 25 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and extension for 59 s at 72 °C. The PCR products were analyzed by resolution on a 1.5% (w/v)57. Cell tracking within the chondral defects were also performed using human anti-nuclei antibody MAB1281, clone 235-1 (Merck KGaA, Darmstadt, Germany) immunohistochemistry. Human anti-nuclei antibody (+) cells were counted and compared in 5 histological sections 20 μm apart for each defect. The transplantation site (cartilage defects) was also evaluated for CD45 expression, with IHC using anti-CD 45 antibody (Abcam, Cambridge, UK; catalog No. ab10558).

Statistical analysis

Statistical analysis was performed using a software program (SPSS Version 18, Chicago, IL, USA). Non-parametrical tests were used throughout this study. The test is used as a non-parametric alternative of the independent two-sample t-test (Mann-Whitney) or multiple comparison (Kruskal-Wallis). Data are expressed as a mean ± standard deviation. P-values less than 0.05 were considered statistically significant58.