Table 1.
Primers used for RT-PCR analysis of chondrocyte genes.
Fig 1.
Determination of the thrombin and TGF-β1-transduced chondrocytes mixing ratio.
(A) The mixture comprising 0.9 ml of TGF-β1-transduced chondrocytes and 0.1 ml of thrombin did not retain the shape of the beads and flowed down. (B) The mixture comprising 0.8 ml of TGF-β1-transduced chondrocytes and 0.2 ml of thrombin maintained the bead shape well and became firm at approximately 30 s. Mixtures comprising different ratios of TGF-β1-transduced chondrocytes and thrombin: (C) 0.7 ml of TGF-β1-transduced chondrocytes and 0.3 ml of thrombin, (D) 0.6 ml of TGF-β1-transduced chondrocytes and 0.4 ml of thrombin, and (E) 0.5 ml of TGF-β1-transduced chondrocytes and 0.5 ml of thrombin exhibited rapid bead formation. Manipulating these beads was difficult. Determination of the fibrin and atelocollagen mixing ratio: (F) mixing ratios of 0.6 ml of fibrin and 0.4 ml of atelocollagen, (G) 0.7 ml of fibrin and 0.3 ml of atelocollagen (failed to maintain the bead shape at approximately 30 s), and (H) 0.8 ml of fibrin and 0.2 ml of atelocollagen (maintained the bead shape well at around 30 s). Determination of the mixing ratio of TGF-β1-transduced chondrocytes, thrombin, atelocollagen, and fibrin mixture: (I) Two syringes were prepared as one set of injection, and four sets of injection were prepared. Microscopic findings of TGF-β1-transduced chondrocyte-atelocollagen mixture: TGF-β1-transduced chondrocytes were well distributed in the atelocollagen mixture beads, and the round shape of the beads was well maintained after 21 days of culture. Cells exhibited proliferation activity around the mixture beads. (J) × 40 (K) × 100.
Fig 2.
(A) After 21 days of culture, the size of the TGF-β1-transduced chondrocyte-atelocollagen mixture beads (upper) under non-differentiation and (lower) and chondrogenic differentiation conditions was compared. (B) The size of the TGF-β1-transduced chondrocyte-atelocollagen mixture beads under chondrogenic differentiation conditions was significantly reduced. Analysis of gene expression. Real-time quantitative polymerase chain reaction analysis of (C) type II collagen, (D) aggrecan, and (E) SOX9 expression of TGF-β1-transduced chondrocytes cultured for 21 days under non-differentiation and chondrogenic differentiation conditions, and TGF-β1-transduced chondrocytes-atelocollagen cultured for 21 days under chondrogenic differentiation conditions (* P <0.05, ** P <0.01, *** P <0.001). Histological images of the TGF-β1-transduced chondrocyte-atelocollagen mixture: (F) Hematoxylin-eosin, (G) toluidine blue, (H) Alcian blue pH 2.5, and (I) type II collagen. (F-I, × 400).
Fig 3.
Scanning electron microscopy of TGF-β1-transduced chondrocyte-atelocollagen mixture.
(A), (B) TGF-β1-transduced chondrocyte-atelocollagen mixture after 21 days of culture. A large number of cells surrounded the mixture bead and were in close contact with each other (white arrowhead). Profound fibers with striation, which is specific for collagen fiber, and formation of mesh structure for cell attachment were observed. Transmission electron microscopy of TGF-β1-transduced chondrocyte-atelocollagen mixture: (C), (D) TGF-β1-transduced chondrocyte-atelocollagen mixture after 21 days of culture. Numerous vesicles (white arrowhead) in the cytoplasm indicated the active secretory function of cells and production of proteoglycan granules and fibrillar collagens with striation (black arrowhead) for matrix formation. Biochemical staining of TGF-β1-transduced chondrocyte-atelocollagen mixture: viable cells appeared green and dead cells appeared red after staining with calcein acetoxymethyl ester and ethidium homodimer. (E) At 48 h and (F) 7 days after fluorescent dye staining. Original magnification × 100.
Fig 4.
Process of knee joint model production using 3D printing technique.
(A) Image segmentation based on the knee T2 magnetic resonance imaging data, (B) conversion of each divided part to stereolithography files, (C) implementation of functional elements with computer-aided design, and (D) output using a 3D printer. Arthroscopic application of TGF-β1-transduced chondrocyte-atelocollagen mixture. (E) Knee joint model from the 3D printing technique. Arthroscopic findings after applying the TGF-β1-transduced chondrocyte-atelocollagen mixture: (F) Medial femoral condyle defect, (G) trochlear defect, (H) application of the mixture to the medial femoral condyle defect, and (I) application to the trochlear defect.