Tissue Engineering of Cartilage - Methods in Molecular Biology: Extracellular Matrix Protocols - page 292

b. Embedding and sectioning: Embed the fixed tissue in paraffin after dehydra-
tion with ethanol, as described above. Cut sections with a rotary microtome to
4 or 5 m. and place onto microscope slide. Deparaffinize sections with

xylene, then rehydrate by 5 min sequential incubations in 100%, 90%, and
70% ethanol. Incubate in water for 5 min. If preservation of newly seeded
cartilage constructs with negligible levels of extracellular matrix is desired,
embed tissue in methacrylate).

µ

2. Histochemical staining for extracellular matrix components

a. Conventional staining: To examine cell and tissue morphology, total collagen,
and sulfated-glycosaminoglycan (S-GAG), stain with Hematoxylin and Eosin,
Trichrome, (12) and Safranin-O (13) respectively, as described .
b. Immunostaining (collagens and S-GAGs):
i. To detect collagen type I and II, obtain antibodies specific for either col-

lagen type I or II and appropriate enzyme-conjugated secondary antibod-
ies and substrate. If no signal is detected, enzymatically pretreat the
sections with chondroitinase A,B,C (14) , with or without subsequent treat-
ment with pepsin, trypsin, or a combination of these enzymes, as recom-
mended by a histochemical vendor.

ii. To detect chondroitin-4-sulfate and chondroitin-6-sulfate, use mono-

clonal antibodies 2B6 and 3B3, respectively, as described (14 , 15) .

3.3.2. Biochemical Analyses
1.

Construct sample preparation: Record the total construct weight (wet weight), as
well as the dry weight of the constructs after drying with a SpeedVac (Savant
Instruments, Holbrook, NY) or by lyophilization. Enzymatic digestion of dried
cartilage constructs with papain is recommended, because it renders the samples
amenable to the evaluations described below. Follow the procedure described by
Kim et al. (16) .

2. Cell number: The total cell number in cartilage constructs can be calculated after

performing a DNA dye binding assay on papain digested cartilage constructs (16) .

3. Total collagen: The total collagen in cartilage constructs can be calculated after

determination of the hydroxyproline content from acid hydrolyzed papain
digested cartilage constructs (17) .

4. Total S-GAG: The total S-GAG content in cartilage constructs can be determined

after performing a dye-binding assay on papain-digested constructs (18) .

4. Notes
1.

It is recommended to obtain articular cartilage from very juvenile, skeletally
immature donors. Cells isolated from tissue obtained from adult donors or juve-
nile donors close to skeletal maturity of various species may produce a more
fibrocartilaginous matrix, as compared to the hyaline-like cartilage produced by
cells derived from tissue of very juvenile donors

2. Entangled, nonwoven polyglycolic acid (PGA) felt:

Scaffolds composed of 100% PGA degrade very rapidly [4]. Relatively slower