Klapperich Lab:Collagen-GAG Meshes
Protocol for making collagen-GAG meshes (150 ML SAMPLES).
- 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
- 250 ml jacketed beaker
- Cold water bath
- Magnetic stir plate
- Vacuum oven
- Metal mold
Making 0.05 M solution of acetic acid
Note: This solution has a shelf life of one week.
- Fill sterilized bottle with 250 mL of Millipore water.
- Add 715 µL acetic acid.
0.5% w/v Dispersion of Collagen in Acetic Acid
Note: Keep collagen mixture at below 20°C.
- 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)
- Add 150 ml of 0.05 M acetic acid to the beaker.
- 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)
- Connect the beaker inlet and outlet to the bath (Fisher Scientific), and set bath temperature at 4°C. Turn on bath and allow the bath to reach at least 10°C. Make sure bath is filled to second line. Add a few drops of Algicide each time the bath is used.
- 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.
- 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
- Measure 0.0795 g C6S into a 50 ml polypropylene centrifuge tube.
- Add 15 ml 0.05 M acetic acid to the tube. This should be 10% of the volume of the collagen mixture.
- Mix the C6S solution by shaking with cap on.
- Transfer collagen solution to a sterilized non-jacketed beaker. Add clean stir bar and place on stir plate at setting 5.
- Add C6S solution dropwise to stirring collagen solution. Roughly 1 ml/min addition.
- Remove stir bar and transfer collagen-GAG solution back to jacketed beaker.
- Continue sonicating at the same settings for 90 more minutes to ensure an even dispersion.
- Degas mixture in a vacuum flask for 30 minutes until bubbles are no longer present.
Setting the Scaffold (for any initial volume)
- Pour 4 mL collagen-C6S mixture into each well in a 25-well metal mold.
- Freeze at -20°C for 15 minutes, and then transfer to -80°C for one day.
- Transfer to sterilized lyophilizer for 2 days.
- Heat in vacuum oven at 105°C, at a vacuum reading of ~22 inHg, for 2 days in order for the meshes to be sturdier.
- Keep meshes on dessicant to keep dry.
- The mixture may be stored in a capped bottle at 4°C for about 4 months.
- Re-blend at 4°C at an amplitude of 60 with the pulser set to 5 sec for 15 minutes to use.
Relevant papers and books