Klapperich Lab:Collagen-GAG Meshes

Overview
Protocol for making collagen-GAG meshes (150 ML SAMPLES).

Reagents

 * 250 mL of 0.05M acetic acid
 * 0.75 g of collagen (Type I, Insoluble from Bovine Achilles Tendon)
 * 0.0795 g chondroitin-6-sulfate

Equipment

 * Homogenizer
 * 250 ml jacketed beaker
 * Cold water bath
 * Ultrasonicator
 * Magnetic stir plate
 * Lyophilizer
 * Vacuum oven
 * Metal mold

Making 0.05 M solution of acetic acid
Note: This solution has a shelf life of one week.
 * 1) Fill sterilized bottle with 250 mL of Millipore water.
 * 2) Add 715 µL acetic acid.

0.5% w/v Dispersion of Collagen in Acetic Acid
Note: Keep collagen mixture at below 20&deg;C.
 * 1) Add 0.75 g of collagen (Type I, Insoluble from Bovine Achilles Tendon) to a 250 ml jacketed beaker beaker. (0.5 % w/v, this type of collagen should be stronger than from calf skin)
 * 2) Add 150 ml of 0.05 M acetic acid to the beaker.
 * 3) Mix solution with homogenizer at full speed for approximately 5 minutes, or until no large fibrils or chunks are apparent. Make sure that tip has been washed thoroughly (ethanol, rinse with Millipore water)
 * 4) Connect the beaker inlet and outlet to the bath (Fisher Scientific), and set bath temperature at 4&deg;C. Turn on bath and allow the bath to reach at least 10&deg;C. Make sure bath is filled to second line.  Add a few drops of Algicide each time the bath is used.
 * 5) Set the sonicater controller to the ID03 program. (Amplitude 100% (dial), 59 min, 5 sec on, 10 sec off). Press RECALL. Type in 03 and press ENTER.
 * 6) Place sonicater horn in collar, making sure tip of sonicater does not touch the bottom of the beaker. Press START/STOP on sonicater controller.

Addition of the 0.53 % w/v Solution of Chondroitin 6-Sulfate (C6S) in Acetic Acid

 * 1) Measure 0.0795 g C6S into a 50 ml polypropylene centrifuge tube.
 * 2) Add 15 ml 0.05 M acetic acid to the tube. This should be 10% of the volume of the collagen mixture.
 * 3) Mix the C6S solution by shaking with cap on.
 * 4) Transfer collagen solution to a sterilized non-jacketed beaker. Add clean stir bar and place on stir plate at setting 5.
 * 5) Add C6S solution dropwise to stirring collagen solution. Roughly 1 ml/min addition.
 * 6) Remove stir bar and transfer collagen-GAG solution back to jacketed beaker.
 * 7) Continue sonicating at the same settings for 90 more minutes to ensure an even dispersion.
 * 8) Degas mixture in a vacuum flask for 30 minutes until bubbles are no longer present.

Setting the Scaffold (for any initial volume)

 * 1) Pour 4 mL collagen-C6S mixture into each well in a 25-well metal mold.
 * 2) Freeze at -20&deg;C for 15 minutes, and then transfer to -80&deg;C for one day.
 * 3) Transfer to sterilized lyophilizer for 2 days.
 * 4) Heat in vacuum oven at 105&deg;C, at a vacuum reading of ~22 inHg, for 2 days in order for the meshes to be sturdier.
 * 5) Keep meshes on dessicant to keep dry.

Contact

 * Cass