8. Centrifuge the cell suspension at 2000 rpm for 10 min to
obtain the cell pellet. Discard supernatant (see Note 5).
9. Aspirate the cell pellet into the syringe unit (see Notes 6 and 7).
10. Inject the cell pellet into the fabricated alginate tubular constructs (see Note 8).
11. Incubate the pellets for 7 days (Fig. 1d).
12. Dissolve the alginate tube in sodium citrate solution for 5 min
to obtain the pure cartilage strands (Fig. 1e).
13. Culture tissue strands in medium as Subheading 3.3, step 4.
3.3 Cell
Viability Assay
1. Rinse the strand with HBSS twice, 15 min each time.
2. Place the strand into calcein/ethidium solution.
3. Incubate the strand for 30 min at 37
C with 5% CO 2 .
4. Remove the calcein/ethidium solution.
5. Rinse the strand with HBSS twice, 15 min each time.
6. Image the strand using a laser scanning confocal microscope
(LSCM), with a depth of 1000 μm at 20 μm intervals for Z-axis
projections.
7. Apply ImageJ software for intensity quantification of the redand green-stained tissue strands.
3.4 Histology
Analysis
1. Fix cells in the strand with PFA.
2. Freeze the strand, and cut it into 10 μm sections.
3. Perform hematoxylin and Safranin O-fast green staining
according to standard protocols [11].
3.5 Immunohistochemistry Analysis
1. Prepare the tissue sections as Subheadings 3.6, steps 1 and 2.
2. Incubate the section for 30 min in blocking solution to prevent
nonspecific binding.
3. Incubate the section with rabbit anti-human polyclonal antibodies against collagen type II and aggrecan overnight at room
temperature.
4. Use the goat anti-rabbit secondary antibody for detection.
5. Visualize the reaction products by the Vectastain ABC kit and
the DAB peroxidase substrate kit according to the manufacturer’s instructions.
6. Omit the primary antibodies to generate negative controls.
7. Observe the section under a microscope.
3.6 DMMB Assay for
sGAG Content
Evaluation
1. Dilute the cell lysates to contain 4 Â 10
3 cells/μL in 50 μL, and
add 150 μL 1,9-dimethylmethylene blue (DMMB) dye solution (see Note 9).
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obtain the cell pellet. Discard supernatant (see Note 5).
9. Aspirate the cell pellet into the syringe unit (see Notes 6 and 7).
10. Inject the cell pellet into the fabricated alginate tubular constructs (see Note 8).
11. Incubate the pellets for 7 days (Fig. 1d).
12. Dissolve the alginate tube in sodium citrate solution for 5 min
to obtain the pure cartilage strands (Fig. 1e).
13. Culture tissue strands in medium as Subheading 3.3, step 4.
3.3 Cell
Viability Assay
1. Rinse the strand with HBSS twice, 15 min each time.
2. Place the strand into calcein/ethidium solution.
3. Incubate the strand for 30 min at 37
C with 5% CO 2 .
4. Remove the calcein/ethidium solution.
5. Rinse the strand with HBSS twice, 15 min each time.
6. Image the strand using a laser scanning confocal microscope
(LSCM), with a depth of 1000 μm at 20 μm intervals for Z-axis
projections.
7. Apply ImageJ software for intensity quantification of the redand green-stained tissue strands.
3.4 Histology
Analysis
1. Fix cells in the strand with PFA.
2. Freeze the strand, and cut it into 10 μm sections.
3. Perform hematoxylin and Safranin O-fast green staining
according to standard protocols [11].
3.5 Immunohistochemistry Analysis
1. Prepare the tissue sections as Subheadings 3.6, steps 1 and 2.
2. Incubate the section for 30 min in blocking solution to prevent
nonspecific binding.
3. Incubate the section with rabbit anti-human polyclonal antibodies against collagen type II and aggrecan overnight at room
temperature.
4. Use the goat anti-rabbit secondary antibody for detection.
5. Visualize the reaction products by the Vectastain ABC kit and
the DAB peroxidase substrate kit according to the manufacturer’s instructions.
6. Omit the primary antibodies to generate negative controls.
7. Observe the section under a microscope.
3.6 DMMB Assay for
sGAG Content
Evaluation
1. Dilute the cell lysates to contain 4 Â 10
3 cells/μL in 50 μL, and
add 150 μL 1,9-dimethylmethylene blue (DMMB) dye solution (see Note 9).
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