Tissue Engineering of Cartilage - Methods in Molecular Biology: Extracellular Matrix Protocols - page 287

woven, fiber networks (felt). The selection of biomaterials can influence the
growth of the construct (8 , 9) . The extent and rate of tissue that is formed is
dependent on the culture time and method, as well as the quality and quantity
of cells applied to the scaffold. The methods described here will result in the
formation of a uniformly distributed tissue containing proteoglycan, collagen,
and water comprising up to 5%, 1% and 85-95%, respectively, of the total
construct weight. These levels are equivalent to the lower levels of the corre-
sponding matrix components of normal articular cartilage. The constructs
grown in this manner consist of a smooth, hyaline-like cartilage that surrounds
chondrocytes in lacunae, with a fibrous capsule localized at the periphery of
the construct. While this tissue does not have the compressive and tensile prop-
erties of normal articular cartilage, tissue-engineered constructs have been suc-
cessful in repairing cartilage defects in vivo (10) .
The method below describes how a cartilage construct can be generated in
vitro, using simple tissue-culture techniques. The tissue generated using this
method will have type II collagen and the proteoglycan aggrecan as major com-
ponents of its extracellular matrix. Metabolically active chondrocytes will
superficially be distributed relatively homogeneously throughout the tissue.
The tissue will also have a fibrocartilaginous layer surrounding the cartilage.
These constructs can be used for cartilage transplantation studies and in vitro
experimental studies.

2. Materials
2.1. Tissue Harvest and Chondrocyte Isolation (see Note 1)
All of the following materials must be cell culture grade and sterile.
1. Femurs (skinned): rabbits (</= 1 yr), sheep (</= 3 wk), or cows (</= 3 wk).
2. Plastic culture dishes: (10 cm diameter) and tubes (50 mL): Obtain tissue-culture
grade vessels.
3. Isopropanol (70%, diluted in DI - H 2 O).
4. Phosphate-buffered saline (PBS + Ca 2+ and Mg 2+ ).
5.

Culture medium A (gentamicin solution): Dilute gentamicin in PBS to 25 µ g/mL.

6. Culture medium B (cell-culture medium): To Dulbecco's Modified Eagle's

Medium (DMEM), add fetal bovine serum; FBS (10%), L -glutamine (2 m M ),
nonessential amino acids (0.1 m M ), L - proline (50 µ g/mL), sodium pyruvate

(1 m M ), and gentamicin (25 µ g/mL).

7. Culture medium C (tissue digestion solution): Dissolve bacterial collagenase

type II to a final concentration of 506.0 U/mL in culture medium B.

8. Orbital shaker: Obtain a unit that can be placed on an incubator shelf.

9. Cell filter (70- µ m filters that fit atop 50-mL conical cell culture tubes).

10. Pipeter/pipet tips (with accuracy for pipetting 5 µ L, 200 µ L, and 1000 µ L).

11. Trypan blue (0.04%).