2.5 Histology and
Immunohistochemistry Analysis
1. Paraformaldehyde (PFA): 4% in PBS.
2. Hematoxylin.
3. Safranin O-fast green.
4. Primary antibody: rabbit anti-human polyclonal antibodies
against collagen type II and aggrecan.
5. Secondary antibody: goat anti-rabbit antibody.
6. Vectastain ABC kit.
7. DAB Peroxidase Substrate Kit.
2.6 DMMB Assay for
sGAG Content
Evaluation
1. 1,9-dimethylmethylene blue (DMMB) solution at a concentration of 16 μg/mL.
2. Quant-iT PicoGreen dsDNA Assay Kit.
2.7 Total RNA
Extraction, Reverse
Transcription, and
Real-Time PCR
Analysis
1. TRIzol reagent.
2. RNeasy Mini Kit.
3. TaqMan Micro RNA reverse transcription kits.
4. SYBR Green Real-Time PCR kit.
3 Methods
3.1 Preparation of
Alginate Capsule
1. Load 4% sodium alginate into a 5 mL syringe, and connect the
syringe to the feed tube of the coaxial nozzle.
2. Load 4% CaCl 2 into another 5 mL syringe, and connect the
syringe to the inner tube of the coaxial nozzle.
3. Print the alginate at the air pressure of 82.7 kPa, and the CaCl 2
at a feed rate of 16 mL/min (Fig. 1b) (see Note 3).
4. Collect the pre-crosslinked alginate capsules in a CaCl 2 pool
(see Note 4).
5. Leave the alginate capsules in CaCl 2 solution overnight for
complete crosslinking (Fig. 1c).
3.2 Cell Preparation
1. Rinse the articular cartilage in HBSS buffer with 100 U/μL
penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL
fungizone.
2. Mince the cartilage into fine pieces.
3. Digest the cartilage pieces overnight in digestion buffer.
4. Culture the isolated cells in growth medium at 37
C, 5% CO 2 .
5. Fabrication of tissue strands.
6. Expand the chondrocytes to approximately 100 million cells.
7. Suspend the cells in culture medium in a 50 mL conical tube.
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Immunohistochemistry Analysis
1. Paraformaldehyde (PFA): 4% in PBS.
2. Hematoxylin.
3. Safranin O-fast green.
4. Primary antibody: rabbit anti-human polyclonal antibodies
against collagen type II and aggrecan.
5. Secondary antibody: goat anti-rabbit antibody.
6. Vectastain ABC kit.
7. DAB Peroxidase Substrate Kit.
2.6 DMMB Assay for
sGAG Content
Evaluation
1. 1,9-dimethylmethylene blue (DMMB) solution at a concentration of 16 μg/mL.
2. Quant-iT PicoGreen dsDNA Assay Kit.
2.7 Total RNA
Extraction, Reverse
Transcription, and
Real-Time PCR
Analysis
1. TRIzol reagent.
2. RNeasy Mini Kit.
3. TaqMan Micro RNA reverse transcription kits.
4. SYBR Green Real-Time PCR kit.
3 Methods
3.1 Preparation of
Alginate Capsule
1. Load 4% sodium alginate into a 5 mL syringe, and connect the
syringe to the feed tube of the coaxial nozzle.
2. Load 4% CaCl 2 into another 5 mL syringe, and connect the
syringe to the inner tube of the coaxial nozzle.
3. Print the alginate at the air pressure of 82.7 kPa, and the CaCl 2
at a feed rate of 16 mL/min (Fig. 1b) (see Note 3).
4. Collect the pre-crosslinked alginate capsules in a CaCl 2 pool
(see Note 4).
5. Leave the alginate capsules in CaCl 2 solution overnight for
complete crosslinking (Fig. 1c).
3.2 Cell Preparation
1. Rinse the articular cartilage in HBSS buffer with 100 U/μL
penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL
fungizone.
2. Mince the cartilage into fine pieces.
3. Digest the cartilage pieces overnight in digestion buffer.
4. Culture the isolated cells in growth medium at 37
C, 5% CO 2 .
5. Fabrication of tissue strands.
6. Expand the chondrocytes to approximately 100 million cells.
7. Suspend the cells in culture medium in a 50 mL conical tube.
178
Pallab Datta et al.