Effects of Cryopreservation and Replantation on Muscles: Application Scope of Limb Cryopreservation

Wang successfully replanted the severed fingers of 2 patients after cryopreservation in 2002 and 2003, which has enabled us to share our own experience for the knowledge interests of our colleagues and to further develop this technology.

Fifteen healthy adult male Sprague-Dawley rats were selected and divided into 5 groups (group 1: normal control, group 2: cryopreservation with protectant, group 3: cryopreservation without protectant, group 4: 6-hour postoperative, and group 5: 72-hour postoperative). After harvesting the hind limbs, cryoprotectant was applied to 20 limbs, and the rest were cryopreserved without cryoprotectant for 15 days. After being thawed, the amputated limb was replanted in situ. Nerves, skins and gastrocnemius muscles were collected for hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and transmission electron microscopy observation.

Muscle and skin tissues treated with cryoprotectant restored a better outline after being frozen than those not treated, whereas nerves were not significantly different between the 2 groups. After replantation, some of the myofibrils of the muscle were in disarray, but the sarcomere structure remained intact at approximately 6 hours postoperatively. At 72 hours, a transmission electron microscopy scan showed that the myofibrillar arrangement was disorderly, with segmental myofilament breakage, and the sarcomere structure was destroyed in some cases. In addition, the scan revealed increased apoptotic cells and collapse of basic structures in the skin and nerves.

Relative to that of skin and neuronal tissue, the replantation of muscle tissues through the cryopreservation method is more difficult.