歯の凍結保存法に関する実験的研究

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  • Experimental Study on the Cryopreservation of Teeth

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For the purpose of establishing a method to preserve frozen extracted teeth for the transplantation of long-term cryopreserved teeth, we investigated in vitro the cell adhesion, proliferation, morphology and bioactivity of human periodontal membrane-derived fibroblasts that had been frozen in D-MEM at freezing rates (℃/min) of -0.5, -5, -10 and -15 using DMSO, glycerol and trehalose as antifreezing agents. In addition, X-ray and histological findings in beagle dogs were comparatively examined to know the condition of autotransplantation of long-term cryopreserved teeth.<br/> In the cell adhesion and proliferation tests, freezing in D-MEM＋ 10% glycerol and D-MEM＋10% DMSO showed the best cell activity, followed by D-MEM＋ 10% trehalose and D-MEM in this order. The freezing rate of -0.5℃/min was most appropriate among the four freezing rates tested. In morphological observation, the fibroblasts cryopreserved in D-MEM＋10% glycerol and D-MEM＋10% DMSO showed good growth of cell processes with cell morphology nearly identical to fresh cells.<br/> In X-ray observation of the long-term cryopreserved teeth autografted in beagle dogs, immediately replanted teeth and the teeth cryopreserved in D-MEM＋10% glycerol showed no dental root absorption, and radiolucent images suggesting probably the alveolar hard line and periodontal membrane cavity were observed. The teeth cryopreserved in D-MEM, however, showed obscure images of both the alveolar hard line and periodontal membrane cavity, and an image suggesting bony fusion was observed. In histological observation, immediately replanted teeth and the teeth cryopreserved in D-MEM＋10% glycerol were histologically similar, and showed neither dental root absorption nor fusion in comparison with healthy teeth. Fibrous connective tissue was also observed between the alveolar bone and the cementum. Teeth cryopreserved in D-MEM showed proliferated bony tissues on almost all dental root surfaces and fused to the alveolar bone without intervening fibrous connective tissue.<br/> These results suggest that the gentle freezing of teeth at a freezing rate of -0.5℃/min using glycerol as an antifreezing agent is a very effective method of cryopreservation and useful enough for clinical application.<br/>

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