Biomedical Engineering Reference
In-Depth Information
Nerve Regeneration Template
1. Prepare vented PVC jackets by heating 12-cm sections of flexible PVC tubing
at 105 °C for 2 h, to straighten. Puncture each tube with a 25 gage needle at
90-degree intervals around the tube, spaced 1 cm apart for the length of the tube.
2. Flush silicone processing tubes (15 cm in length) with dH
2
0, and let dry.
3. Seal one end of each silicone processing tube with silicone adhesive. Inject a
cylindrical plug of adhesive, approx. 5 mm in length, into the end of silicone
tube and allow the excess to stay on the outside of the tube. The excess is
important for adhesion and can be cut off later. Let cure for 24 h at room tem-
perature to a tack-free, elastomeric state.
4. Prepare for use a 160-L liquid nitrogen tank for the bath cooling system.
5. Remove the air from the collagen-GAG suspension by placing into a 1500-ml
Erlenmeyer flask under vacuum for 10 min with agitation, or until bubbles are
no longer visible.
6. Turn on the cooling system of the axial freezing bath and set the bath tempera-
ture to − 80 °C.
5
It takes approx. 45 min of liquid nitrogen cooling for the bath
to reach this temperature.
7. Insert each plugged silicone processing tube into a prepared PVC jacket.
8. Draw collagen-GAG suspension into a 10-cc syringe (Becton Dickinson model
5604, Becton Dickinson, Rutherford, NJ) and expel all the air bubbles. Attach
a 25-gage needle (Becton Dickinson model 25G5/8, Becton Dickinson) to the
syringe and insert the needle carefully into the plugged end of the silicone
tube. The needle should be inserted far enough so that a needle length of about
3-5 mm extends beyond the Silastic plug into the tube.
9. Inject collagen-GAG suspension until the tube is full and no air remains in the
tube. Pinch the free end of the silicone processing tube against the wall of the
PVC jacket using a conical, plastic plug (the end of a pipet tip works well).
Insert the plug far enough so that the silicone processing tube is sealed, and no
suspension can leak out. Insert another conical, plastic plug into the needle end
of the tube. The plug at the needle end should not block the flow of the suspen-
sion into the silicone tube via the needle.
10. Inject additional suspension until the silicone processing tube becomes pres-
surized and expands to fill the entire PVC jacket. The silicone tube will inflate
because of pressure from the injection of additional suspension. The end of the
needle should be inside the PVC jacket to help prevent pressure build up at the
needle tip. When the silicone tube has completely filled the PVC jacket, care-
fully remove the needle; simultaneously, press the conical plug into the end of
the tube until the silicone processing tube is pinched against the PVC jacket
5
The temperature of the axial freezing bath controls the average pore diameter. In the case of
NRTs, pores with an average diameter of 5-10 µm were determined to be optimal for the regenera-
tion of axons (Chang et al. 1990; Chang and Yannas 1992; Loree et al. 1989). Freezing at − 80 °C
results in pore channels that average 5-10 µm in diameter (Fig. 3b). Higher freezing temperatures
result in larger pore diameters (Loree 1988).
