Experimental Study of Cryopreserved Allogenic Ear Cartilage

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 09671259
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General surgery
• Research Institution: JIKEI UNIVERSITY SCHOOL OF MEDICINE
• Principal Investigator: MIYAWAKI Takeshi JIKEI UNIVERSITY SCHOOL OF MEDICINE, ASSISTANT, 医学部, 助手 (70246445)
• Co-Investigator(Kenkyū-buntansha): TAKEISHI Meisei JIKEI UNIVERSITY SCHOOL OF MEDICINE,LECTURER, 医学部, 講師 (00256405) ; HIRASE Yuichi JIKEI UNIVERSITY SCHOOL OF MEDICINE,LECTURER, 医学部, 講師 (00181157)
• Project Period (FY): 1997 – 1999
• Keywords: cryopreservation / allograft / ear cartilage graft / cartilage preservation

Research Abstract

The authors have established a cryopreservation method of long-term preservation nd published several reports on allogenic transfers of skin, nerves and vessels in rats, pigs and monkeys. Herein is reported a study of long-term storage and allogenic transplantation of the ear cartilage of rabbits which was based on previous ezperimental data.
Materials and Methods : Experiment 1 ; Male 8-week-old Japanese white rabbits(2.0-2.5kg) were used. Under anesthesia by intravinous administration of pentobalbital sodium, a piece of ear cartilage with midauricular vessels was harvested at the base of an ear. Glycerol(1.4 mol)was injected through the midauricular artery for cryoprotection. Twelve rabbits were divided into three(cryopreservation, direct freezing and control)groups. In the cryopreservation group, 3×1 cm ear cartilage specimens were harvested, frozen stepwise to -70℃, and stored at -196℃ in a liquid nitrogen tank for more than weeks. The thawing process consisted of a rapid resolution to 20℃, followed by soaking in physiological saline at 4℃ for one hour. In the direct freezing group, each cartilage specimen was frozen very rapidly without any cryoprotection and then stored at -196℃ for three weeks. Histrogical study was conducted utilizing light and electron microscopy. Viable cells were counted in three fields consisted of perichondral cells and premature and cature chondrocytes and this was compared with fresh ear cartilages serving as a control group. Experiment 2 ; Eighty cryopreserved ear cartilage specimens were transplanted above the thigh fascia of different rabbits as allogenic transfers. At 1, 2, 4, 6, 8, 12, 16, 20, 21, 28, 31, 32, 46, 52, and 55 weeks after surgery, the grafted cartilage were harvested for histological examinations performed by light and electron microscopy. Viable cells were counted in the manner described in experiment 1 and compared with fresh auto and allografts.
Results : Experiment 1 ; Chondrocytes in the cryopreserved group revealed a well-preserved cellular level structure. In constant, the freezing group demonstrated collapse and destruction of the cellular matrix.
Experiment 2 ; Histological findings indicated cellular infiltration was minimal in the cryoallograft group, similar to the fresh autograft group, and marked in the fresh allograft group. Chondrocytegenesis was observed in three groups of different periods. There was a very slow decrease in the number of viable cells in the cryopreserved allograft group, and a relatively rapid decrease in the fresh allograft.
Conclusion : The authors' cryopreservation technique proved its versatility for long-term storage of viable chondrocytes, and a significantly slow decrease in viable cell count was observed without active cellular infiltration despite alligenic transfers. It has great potential for allogenic transfer of ear catilage at clinics.