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Cryofracture Technique

For Jeffrey Dunn and Douglas Goodwin, Radiology department, DMS

1. Primary Fixation:

Kneecaps were fixed by immersion with 2% Glutaraldehyde(GTA) in 0.1 M NaCacodylate buffer, pH 7.4 (NaCac) for 3 hours at RT or. Overnight at 4oC; or 2% GTA in 0.1 M HEPES buffer, pH 7.4 for long term storage. Or, sometimes samples were fixed in Formalin.

2. Samples were trimmed to a size for cryo-fracture after fixation, but before de- mineralization. Retain 5mm of subchondral bone, to support the cartilage during processing. 1.5cm by 1.5 cm is a good starting point, including 5mm of subchondral bone, to support the cartilage.

3. PRE-CUTS for cryo-fracture: see figure on last page for a good image of this process.

After fixation and prior to dehydration, make pre scores in the subchondral bone(using a saw) to direct fracture plane. Jeffery, etal 1991 states "two saw cuts were made thorough the bone to within 0.2 cm(2mm) of the subchondral bone plate to facilitate fracturing specimen after freezing. Split lines were made in the cartilage surface with fine, round- bodied needle." All stated the importance of sample orientation relative to split lines. Clark ( 1991) states that a fracture must be parallel to the collagen fibers for their orientation to be recognized. Where fiber curve significantly, they are seen only when they curve in the plane of section.

4. Demineralize:

After fixation, rinse in HEPES buffer or NaCac buffer and then follow standard procedure for demineralizing in Nitical. Rinse in HEPES buffer or NaCac buffer and return to 2% GTA) in 0.1 M NaCac buffer or 2% GTA in 0.1 M HEPES buffer, pH 7.4 for long term storage.

5. Dehydration:

Samples are washed in buffer for at least 4 hours.

Dehydrated to 100% Ethanol: rinse in dH2O

50%ETOH 1 hour

70%ETOH 1 hour

100% ETOH 1 hour

100%ETOH overnight

2 1 hr. changes in 100% ETOH

6. Cryofracture

Immersion freeze in LN2.

Use a pre-cooled Stanley disposable cold chisel blade to fracture. Sample should be able to stand by itself in a pan of LN2. Hold pre-cooled blade with vise grips just at the pre-cut site along the bone surface. Fracture open with a solid tap from a large rubber mallet. return piece to 100% ETOH.

7. Drying process:

two methods, either works fine:

a. t-butyl alcohol, following the technique of Inoue and Osatake(1988):

100%ETOH/TBA 1:1, 2 changes for 1 hour each.

100%ETOH/TBA 1:2 overnight

TBA: 4-5 hours, 3 changes each

Freeze at 4oC (takes @ 5 minutes

pump with house vacuum(w solid CO2 trap to collect TBA vapors) until sublimated. Pump for 1 hour past sight of last TBA. (takes sevral hours)

b. HMDS:

100%ETOH/HMDS1:1, 2 changes for 1 hour each.

100%ETOH/HMDS1:2 overnight

HMDS: 4-5 hours, 3 changes each; remove as much fluid as possible and pump with house vacuum for 24 hours.

8. MOUNT on Al stubs and sputter coat with Au or AuPd.

References

Clark,J.M. (1985) "Organization of collagen in cryofractured articular collagen: a SEM study", J. of Orthopaedic Research 3: 17-29

Clark,J.M. (1991) "Visualization of collagen fiber alignment in a joint surface: a SEM study of tibial plateau in dog, rabbit and man", J. of Orthopaedic Research 9: 246-257

Jeffrey, A.K., etal, (1991) "3-d Collagen Architecture on Bovine Articular Cartilage", The Journal of Joint and Bone Surgery 73-B, 99795-801

Humpheys, W.J. etal, (1975) "TEM of tissue prepared for SEM by ethanol cryofracturing". Stain technology 50:119-125

Inoue, T. and Osatake, H., (1988) "A new drying method for SEM; the t-butyl alcohol freeze- drying method", Arch. Histol. Cytol. 51:53-59

Notzli, H. and Clark,J.M. (1997) "Deformation of loaded articular cartilage prepared for SEM with rapid freezing and freeze-substitution fixation", J. of Orthopaedic Research 15: 76-86

Last Updated: 10/2/08