온혈허혈시간과 냉동보존온도와 보존액 조성에 따른 기관의 생육성 비교 = The Effects of the Warm Ischemic Time, the Preserving Temperature and the Cryopreservation Solution on the Viability of Tracheas

다국어 초록 (Multilingual Abstract)

Background: Tracheal reconstruction after extended tracheal resection still remains as a major surgical challenge because good clinical outcomes are usually correlated with limited tracheal resection. Recent investigations with a using cryopreserved trachea for the reconstruction of a trachea have been carried out to overcome this problem. In this study, we analyzed viability of tracheas, which is an important determining factor for the success of transplanting a cryopreserved trachea and the development of post-transplantation tracheal stenosis, according to three different experimental factors: 1) the warm-ischemic time, 2) the cryopreservation solution and 3) the preserving temperature, to determine a better cryopreservation protocol and a better composition of the cryopreservation solution. Material and Method: Rats tracheas were harvested for different warm-ischemic times (0 hr, 12 hrs, 24 hrs). The tracheas were treated with recombinant insulin growth factor-1 (IGF-1) and they were stored at three different temperatures (4℃, −80℃, −196℃) for two weeks. After two weeks, we thawed the stored trachea and isolated the cells of the tracheas with using type II collagenase. We cultured the cells for seven days and then we compared the cellular viability by the MTT reduction assay. Result: Though cryopreservation is required to preserve a trachea for a longer time period, the viability of the tracheas stored at −80℃ and −196℃ was significantly reduced compared to that of the tracheas stored at 4℃. The viability of the tracheas with warm-ischemic times of 12 hrs and 24 hrs was also reduced in comparison to the tracheas with a warm-ischemic time of 0 hrs. Our data showed that the warm ischemic time and the parameters of cryopreservation negatively affect on trachea viability. However, a cryopresrvation solution containing IGF-1 improved the cellular viability better than the existing cryopreservation solution. For the warm ischemic time group of 0 hr, the addition of IGF-1 improved the viability of trachea at all the preserving temperatures. Conclusion: These experiments demonstrate that the viability of a cryopreserved trachea can be improved by modifying the components of the cryopreservation solution with the addition of IGF-1 and reducing the warm-ischemic time.

국문 초록 (Abstract)

배경: 기관재건술은 제한된 경우에서만 뚜렷한 효과를 얻을 수 있어 광범위한 기관절제술 후의 기관재건술은 아직 의학적으로 해결되지 못하고 있는 난제들 중의 하나로 남아 있다. 이 어려운 문제를 해결하기 위한 방법으로 냉동 보존된 기관을 이용하여 기관을 재건하려는 노력이 이루어지고 있다. 냉동 보존된 기관을 이용한 재건에서는 수술의 성공 여부에 가장 중요한 결정인자가 바로 기관의 생육성이다. 이에 저자들은 기관의 냉동 보존 시 냉동 보존액의 조성과 온혈허혈시간의 정도에 따른 차이, 그리고 보존온도의 정도에 따른 차이에 따른 기관연골의 생육성의 차이를 비교 검토하여, 보다 나은 냉동 보존방법을 알아보고자 하였다. 대상 및 방법: 정해진 온혈허혈시간(0시간, 12시간, 24시간)의 경과 후 쥐의 기관을 채취하여, recombinant insulin growth factor-1 (IGF-1)을 처치하고 3가지의 보존 온도(4℃, −80℃, −196℃)에서 2주간 보존하였다. 보존 후 해동하여 type II collagenase효소를 이용하여 기관의 세포를 채취하였다. 채취한 세포를 7일간 배양한 뒤 MTT reduction assay를 이용하여 각 군의 기관 세포의 생육성을 비교하였다. 결과: 기관을 오랜 기간 보존하기 위해서는 냉동보존은 필요하지만, −80℃와 −196℃에서의 냉동 보존은 대조군과 4℃ 보존군에 비해 통계적으로 유의할 정도로 기관의 생육성을 감소시키는 것으로 관찰되었고, 12시간과 24시간의 온혈허혈시간도 온혈허혈시간 0시간 군에 비해 기관의 생육성을 감소시키는 것으로 관찰되었다. IGF-1을 첨가한 냉동 보존액은 기존의 냉동 보존액보다 기관의 생육성을 향상시키는 것을 확인할 수 있었고, IGF-1로 인한 생육성 향상은 4℃ 보존군에서는 모든 온혈허혈시간에서, 온혈허혈시간 0시간 군에서는 모든 보존온도에서 관찰되었다. 결론: 온혈허혈시간을 최대한 줄이며 냉동 보존액에 IGF-1을 첨가하여 보존액의 조성을 조정함으로써, 냉동 보존 시 보다 나은 기관의 생육성을 유지할 수 있을 것으로 판단되었다.

참고문헌 (Reference)

• 1 deLorimier AA, "Tracheobronchial obstruction in infants and children. Experience with 45 cases" 212 : 277-289, 1990
• 2 Tojo T, "Tracheal replacement with cryopreserved tracheal allograft: Experiment in dogs" 66 : 209-213, 1998
• 3 Sakata J, "Tracheal composites tissue engineered from chondrocytes, tracheal epithelial cells, and synthetic degradable scaffolding" 26 : 3309-3310, 1994
• 4 Lenot B, "Tracheal allograft replacement. An unsuccessful method" 7 : 648-652, 1993
• 5 Tojo T, "Tracheal allogenic immunoresponse is reduced by cryopreservation: Canine experiment" 28 : 1814-1815, 1996
• 6 Fuchs JR, "Tissue engineering: A 21st century solution to surgical reconstruction" 72 : 577-591, 2001
• 7 Herman DB, "The fate of meniscus cartilage after transplantation of cryopreserved non-tissue-antigen-matched allograft: a case report" 266 : 145-151, 1991
• 8 Ingham E, "The effects of variation of cryopreservation protocols on the immunogenicity of allogeneic skin grafts" 30 : 443-458, 1993
• 9 Aoki T, "Successful tracheal transplantation using cryopreserved allografts in a rat model" 16 : 169-173, 1999
• 10 Moriyama H, "Revascularization of canine cryopreserved tracheal allografts" 69 : 1701-1706, 2000
• 11 Yokomise H, "Reliable cryopreservation of trachea for one month in a new trehalose solution" 110 : 382-385, 1995
• 12 Hartog JM, "Reconstruction of the temporomandibular joint with cryopreserved cartilage and freeze-dried dura: a preliminary report" 48 : 919-925, 1990
• 13 Mabrut JY, "Mechanical and histological characteristics of human trachea before and after cryopreservation: An opportunity for tracheal tissue banking" 33 : 609-611, 2001
• 14 Nakanishi R, "Maximal period of cryopreservation with the bicell biofreezing vessel for rat tracheal isografts" 117 : 1070-1076, 1999
• 15 Kirklin JK, "Long-term function of cryopreserved aortic homografts: a ten-year study" 106 : 154-166, 1993
• 16 Yokomise H, "Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability" 111 : 930-934, 1996
• 17 Lang FJ, "Long segment congenital tracheal stenosis: treatment by slide tracheoplasty" 34 : 1216-1222, 1999
• 18 Negoescu A, "In situ apoptotic cell labeling by the TUNEL method: Improvement and evaluation on cell preparations" 44 : 959-968, 1996
• 19 Yokomise H, "High-dose irradiation prevents rejection of canine tracheal allografts" 107 : 1391-1397, 1994
• 20 Lim CY, "Flow cytometric analysis of endothelial cell viability in arterial allograft" 30 : 553-558, 1997
• 21 Hashimoto M, "Feasibility of cryopreserved tracheal xenotransplants with the use of short-course immunosuppression" 121 : 241-248, 2001
• 22 Vacanti CA, "Experimental tracheal replacement using tissue-engineered cartilage" 29 : 201-205, 1994
• 23 Mukaida T, "Experimental study of tracheal allotransplantation with cryopreserved grafts" 116 : 262-266, 1998
• 24 Teng MS, "Enhancing osteochondral allograft viability: effects of storage media composition" 466 : 1804-1809, 2008
• 25 Sotres-Vega A, "Cryopreserved tracheal grafts: a review of the literature" 19 : 125-135, 2006
• 26 Carlos AA, "Cell viability and protein composition in cryopreserved cartilage" 460 : 234-239, 2007
• 27 Kushibe K, "Assessment of cartilage viability in the cryopreserved tracheal allograft by measurement of Na235SO4 incorporation" 32 : 1655-1656, 2000
• 28 Kushibe K, "Assessment of cartilage viability in long-term cryopreserved tracheal allografts" 33 : 625-626, 2001
• 29 Aziz DM, "Assessment of bovine sperm viability by MTT reduction assay" 92 : 1-8, 2006
• 30 Bando K, "Anterior pericardial tracheoplasty for congenital tracheal stenosis: intermediate to long-term outcomes" 62 : 981-989, 1996