Extensive soft-tissue defects and lack of donor tissue availability may require reconstruction
with composite tissue allotransplantatiion (CTA). Currently, the main obstacles for
CTA are immunogenicity, lack of a donor population, and organ storage limitations.
Crypreservation of CTA could resolve these difficulties and broaden the application
of CTA.
Twenty-two vascularized groin flap transplantations were performed between Lewis rats.
Different cryoprotectants were used: DMSO in 18 flaps (Group 1) and glycerol in 4
flaps (Group 2). In 12 animals from Group 1, a streptokinase (STK) protocol was introduced
which included perfusion with STK solution, 5000 IU before freezing and after thawing.
Additional 1000 IU doses were administered systematically in the recipient rats after
vessel anastomosis. Femoral arteries were cannulated and attached to a perfusion pump
with the pressure limit set on 200 mmHG and flow rate at 80 ml/h. Cryoprotectant solutions
were delivered via the femoral artery. Flaps were frozen up to − 196° C using a stepwise
technique. After 3 days average, flaps were thawed. Group 1 flaps were perfused with
sucrose solutions in decreasing concentrations. Group 2 flaps were perfused with Ringer's
lactate. Flaps were transplanted as isografts to the groin region of the recipients
and the femoral vessels were anastomosed in an end-to-end manner. Flap survival was
evaluated by the patency of vascular anastomoses, flap skin color, and capillary refill.
Biopsies of the flaps were taken for histologic evaluation.
During post thawing perfusion, flap edema was observed in all 18 DMSO cryopreserved
flaps. In all cases where STK was not introduced (10 flaps), early arterial thrombosis
occurred and capillary refill was not observed, with subsequent flap failure. Administration
of streptokinase (12 flaps) extended arterial patency and allowed flap perfusion and
capillary refill. In one flap, 2-week survival was achieved; in 9 flaps, necrosis
occurred within 3 days post transplantation; 2 animals died within 16 hr post STK
administration.
Cryopreservation in a vascularized groin flap model is a challenging task. Use of
new cryoprotective solutions and fine-tuning of the thawing process may be keys to
success and longer flap survival. An anticoagulation treatment was essential in prevention
of early pedicle thrombosis. Further studies on alterations of perfusion, freezing
and thawing protocols are in progress, in order to achieve dependable cryopreservation
protocols for storage of vascularized skin allotransplants.
