Effect of 3 Preservation Methods (Freezing, Cryopreservation, and Freezing plus Irradiation) on Human Menisci Ultrastructure An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard

• Jacquet Christophe
• Erivan Roger
• Argenson Jean-Noël
• Parratte Sebastien
• Ollivier Matthieu

Background: Three main meniscus preservation methods have been advocated: freezing (-80 degrees C), freezing with gamma irradiation (-80 degrees C + 25 kGy), and cryopreservation (-140 degrees C). Hypothesis: All preservation methods will result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. Study Design: Controlled laboratory study. Methods: Five human intact menisci were collected from 5 patients undergoing total knee arthroplasty. The inclusion criteria were patients <70 years old with primary unilateral (medial) femorotibial knee osteoarthritis and without surgical or traumatic history on the operated knee. Four cubes (9 mm(3)) were cut inside of the white, or avascular, area of each specimen's middle horn and divided into 4 groups: fresh control, frozen (-80 degrees C), cryopreserved (-140 degrees C), and frozen + irradiated (-80 degrees C + 25 kGy). Specimens of the control group were evaluated at day 1, and specimens from the frozen, cryopreserved, and frozen + irradiated groups were evaluated after 1 month of storage. Evaluation was performed with electron microscopy according a validated protocol to analyze (1) mean diameters of the collagen fibers in longitudinal and transverse sections in 5 points per section and (2) validated architectural scores. Results: No significant difference was found between the control and cryopreserved groups regarding mean transverse and longitudinal diameters (transverse: 95.39 15.87 nm vs 99.62 19.23 nm, P = .1; longitudinal: 96.31 +/- 13.96 nm vs 94.57 +/- 16.42 nm, P = .1). Significant differences were found between the control and frozen groups (transverse: 95.39 +/- 15.87 nm vs 70.20 +/- 13.94 nm, P < .001; longitudinal: 96.31 +/- 13.96 nm vs 71.28 +/- 10.64 nm, P < .001) and the control and frozen + irradiated groups (transverse: 95.39 +/- 15.87 nm vs 63.1 +/- 15.57 nm, P < .001; longitudinal: 96.31 +/- 13.96 nm vs 60.9 +/- 14.8 nm, P < .001). Regarding architectural score calculation, there were significant differences between the control and frozen groups (4.5 +/- 1.3 vs 2.3 +/- 1.4, P = .02) and the control and frozen + irradiated groups (4.5 +/- 1.3 vs 1.4 +/- 0.9, P = .02). Conclusion: Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing + irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and the organization of collagen fibers, and this method should be used with caution to preserve and sterilize meniscus tissue.