Support Protocol One

Type II collagen (CII) is present only in hyaline cartilage and the vitreous humor of the eye. When large quantities of CII are desired, fetal bovine or porcine articular cartilages are convenient sources and can be purchased from slaughterhouses. Sternal cartilage from young chickens is also a good source of CII and can be obtained from wholesale meat markets that process chicken breasts. In general, only negligible quantities of native CII can be solubilized from normal cartilage using nondenaturing solvents.

This protocol describes the purification of CII from chick and fetal bovine cartilage. CII can be purified from the cartilage of other species (e.g., rat, mouse, or pig; Table 15.5.1) using the same procedure.

Table 15.5.1 Sources of Type II Collagen

Species	Suggested tissue
Chicken	Sternum
Bovine	Knee joint
Porcine	Knee joint
Rat	Knee, hip, shoulder points
	Xiphoid process and costal cartilage
Mouse	Sternochondral cartilage

Regardless of the source, however, care must be taken to remove all sources of type I collagen--e.g., the perichondrium--prior to extraction of the CII. Ideally, 100 to 400 g of cartilage should be processed at one time, given the time required for the entire procedure. Smaller quantities can be processed (5 to 10 g) when the quantity of cartilage is limited--e.g., when processing from rat or mouse sternums. It is critical to minimize contamination of CII with type I collagen.

Materials

• Fetal bovine joints or chick sternums
• 70% (v/v) isopropyl alcohol
• Tris-buffered guanidine (see recipe)
• 10 mM, 100 mM, and 500 mM acetic acid
• 70% (v/v) formic acid
• Pepsin (3x crystallized; Sigma)
• 5 M NaCl (APPENDIX 2)
• 10 mM Na2HPO4
• Tris-buffered saline (TBS): 50 mM Tris_Cl, pH 7.4 (APPENDIX 2)/200 mM NaCl
• DE-52 anion-exchange resin (Whatman)
• Scalpel or single-edged razor blade
• Food processor or blender with sharp blades
• Sorvall RC-5B or equivalent centrifuge with 250-ml centrifuge tubes and 1-liter centrifuge bottles
• Dialysis tubing (MWCO 10,000)
• 5 x 20-cm chromatography column
• 5% SDS-PAGE gel (UNIT 8.4)
• Additional reagents and equipment for dialysis (APPENDIX 3H), DE-52 ion-exchange chromatography (as for IgG purification; UNIT 2.7), and SDS-PAGE (UNIT 8.4)

NOTE: All steps in this procedure must be performed at at 4°C unless otherwise indicated. Although it is nearly impossible to perform this procedure under totally sterile conditions, maintaining the collagen preparation at such a low temperature will not only help to maintain its native form, but will also help reduce bacterial or mycobacterial growth.

Excise and Process cartilage

1. Rinse tissue liberally with 70% isopropyl alcohol to disinfect. Carefully dissect the articular cartilage from bovine joints, meticulously freeing it from bone and other noncartilaginous tissues.

   A scalpel or a single-edged razor blade is recommended for gross dissection and excision. The cartilage is identifiable as a firm, elastic, translucent tissue that is easily sliced. Noncartilaginous tissues are tough, white, nonelastic, and more difficult to cut. Care should be taken to avoid collecting fibrocartilage, as it contains potentially contaminating type I collagen.

   When working with chicken sternums, removal of the perichondrium (which is rich in potentially contaminating type I collagen) can be facilitated by overnight incubation at 4°C in 50 mM Tris_Cl (APPENDIX 2) containing 1 M NaCl. The remaining cartilage is easily removed from the sternum by hand.

2. Cut the cartilage into ~5-mm pieces using a scalpel. Using a food processor or blender, mince the cartilage pieces as finely as possible.

   This process is facilitated by the use of just enough cold distilled water and chips of ice to cover the cartilage pieces. Ice chips are required to keep the temperature of the cartilage pieces below 4°C. Although the cartilage will not homogenize, the smaller the size of the pieces obtained, the greater the yield of CII.

3. Remove the water by centrifuging in 1-liter centrifuge bottles, 45 to 60 min at 4600 x g, at 4°C, using a Sorvall RC-5B or equivalent centrifuge. Stir the minced cartilage overnight at 4°C with 10 ml of Tris-buffered guanidine per gram of minced cartilage.

   This step removes much of the proteoglycan.

4. Collect the minced cartilage by centrifuging in 1-liter centrifuge bottles, 30 min at 4600 x g, at 4°C, then remove the supernatant. Wash three times to remove the guanidine, each time by adding 10 to 15 ml cold distilled water per gram of minced cartilage, centrifuging 30 min at 4600 x g, at 4°C, then discarding the supernatant.

   Solubilize and purify CII.

5. Resuspend the cartilage pellet in 20 vol of 500 mM acetic acid and adjust the pH of the suspension to 2.8 using 70% formic acid.
6. Add 1 g pepsin for every 20 g (wet weight) of cartilage and stir the suspension gently for 36 to 48 hr in the cold.

   The pepsin can be added as dry weight or first dissolved in cold distilled water or 0.5 M acetic acid. The optimal pepsin:cartilage ratio is 1:20 (weight/wet weight). The pepsin will digest the cartilage and release soluble CII, causing the solution to become viscous.

7. Separate the solubilized CII from the cartilage residue by centrifuging in 1-liter bottles, 30 min at 5000 x g, at 4°C. Discard the pellet.

   If separation is poor because of high viscosity, dilute with additional 500 mM acetic acid and repeat centrifugation.

8. Add 5 M NaCl to the supernatant slowly (over a 30 to 45 min period) to a final concentration of 0.8 M. Allow collagen to precipitate at 4°C for 12 to 24 hr.
9. Recover the collagen precipitate by centrifuging in 1-liter bottles, 60 min at 4600 x g, 4°C. Discard supernatant. Dissolve precipitate in 400 ml of 100 mM acetic acid by stirring overnight at 4°C.
10. Centrifuge the collagen solution in 250-ml centrifuge bottles, 1 hr at 5000 x g to remove any insoluble material.

    If the solution is highly viscous, dilute with additional 100 mM acetic acid before centrifugation.

11. Repeat the salt precipitation of the CII (steps 8 to 10) three additional times and dissolve the last collagen precipitate in 400 ml of 100 mM acetic acid.

    These steps are necessary to obtain highly purified CII. The volume of acetic acid depends on the initial amount of cartilage, assumed to be 200 g in this protocol.

12. Precipitate the collagen by dialysis against several changes of 10 mM Na2HPO4 at 4°C using MWCO 10,000 dialysis tubing (see APPENDIX 3H). Collect the collagen precipitate by centrifuging in 250-ml bottles, 40 min at 9000 x g, 4°C, then wash the precipitate twice each time by centrifuging 40 min at 9000 x g, removing the supernatant, adding 200 ml of 10 mM Na2HPO4 to the pellet, then centrifuging again and removing the supernatant.

    This step inactivates and helps remove any residual pepsin.

13. Solubilize the collagen precipitate in 400 ml TBS, then dialyze overnight against TBS at 4°C using MWCO 10,000 dialysis tubing (APPENDIX 3H).
14. Pass the dialyzed solution over a preswollen 5 x 20-cm DE-52 column which has been equilibrated with TBS (see UNIT 2.7; use TBS in place of Tris_Cl).

    CII elutes from the DE-52 column unretarded; residual proteoglycans and pepsin are retained by the resin.

15. Precipitate the collagen from the column effluent by adding 5 M NaCl slowly (over a 30- to 45-min period) to a final concentration of 2.5 M. Allow collagen to precipitate at 4°C for 12 to 24 hr.
16. Collect the collagen precipitate by centrifugation in 250-ml centrifuge bottles for 60 min at 4000 x g, 4°C and remove the supernatant. Dissolve the pellet by stirring in 400 ml of 100 mM acetic acid, then dialyze exhaustively at 4°C against 10 mM acetic acid using MWCO 10,000 dialysis tubing to remove the salt. Lyophilize desalted CII and store at -20°C (stable _3 years if handled properly).

    The volume of acetic acid used at this step depends on the initial amount of cartilage; see step 11, annotation.

    See Critical Parameters for additional guidelines on how to store the CII.

17. Determine purity of CII by SDS-PAGE using a 5% gel (see UNIT 8.4).