Biomedical Engineering Reference
In-Depth Information
Fig. 4. MTT testing on engineered tissues (chondrocytes and collagen-based membranes)
indicated as Case 1, 2 and 3. Samples were analysed after 0, 7 and 14 days in culture. Data
are expressed as optical density at 570 nm.
3.2.1 Materials and methods
Tree engineered tissues -i.e. chondrocytes and collagen-based membranes (see section 2.4.1)-
were embedded in OCT, snap-frozen in liquid nitrogen, cut into 25 m sections, air-dried
and stored at -20 °C until use. These slides were transferred at room temperature, air-dried
for 20 minutes and fixed in 4% PFA at room temperature for 20 minutes. The following
primary antibodies were used: mouse anti-human collagen type II monoclonal antibody and
mouse anti-human proteoglycans (Chemicon International Temecula, CA, USA). Air-dried
fixed samples were rehydrated. Slides for collagen type II determinations were treated with
0.1 % hyaluronidase (Sigma) in Phosphate Buffered Saline (PBS) at 37°C for 5 minutes for
epitope unmasking; those for proteoglycans with chondroitinase ABC (Sigma) in Tris-HCl
pH=8 for 30 minutes at room temperature. The slides were then incubated with the primary
antibodies diluted 1:40 (collagen type II) or 1:50 (proteoglycans) in 0.04M Trizma Base Saline
(TBS) pH 7.6 containing 1% BSA and 0.1 % Triton X-100 for 1 hour at room temperature.
After washes in PBS with the addition of 1% BSA, the slides were incubated with
biotinylated immunoglobulins specific for various animal species (BioGenex, San Ramon,
CA, USA) for 20 minutes at room temperature. Then samples were incubated with a
phosphatase-labeled streptavidin (BioGenex) for 20 minutes at room temperature, and after
washes the reactions were developed using fast red substrate (BioGenex). Negative controls
were performed by omitting the primary antibody. Slides were counterstained with
hematoxylin and mounted in glycerol gel. All the samples were analysed using a Zeiss
Axioscope Microscope (Carl Zeiss).
