深冷冻保存对离体牙牙周膜细胞活性影响的研究

目的：探讨应用不同降温方法和不同冷冻保护液的深冷冻保存技术，对人离体牙牙周膜细胞活性的影响。方法：收集新鲜拔除的人第一或第二前磨牙25颗随机分为5组；其中4组使用含海藻糖或不含海藻糖的冷冻保护液，分别应用程序降温或快速降温至－196℃，冷冻保存一周；一组新鲜拔除牙齿为对照组。分别刮取牙根面中1／3的牙周膜组织，消化法收集细胞，台盼蓝染色，高倍镜下计数活细胞数，并计算细胞存活率。结果：不同降温方法不同冷冻保护液深冷冻保存与对照组相比，牙周膜细胞存活率无明显差异。其中，使用含海藻糖的冷冻保护液程序降温的方法牙周膜细胞存活率最高。结论：应用深冷冻技术保存牙齿，牙周膜细胞的活性无明显变化，其中使用含海藻糖的冷冻保护液程序降温的方法对牙周膜细胞的活性影响最小。

Studies on the effect of cryopreservation on the activity of periodontal ligament cells

Objective:The purpose of this study was to explore the influences of different cooling methods and cryoprotectants on the viability of periodontal ligament cells in vitro. Methods:A total of 25 fresh teeth ( the first premolar or second premolar) were col?lected and randomly divided into five groups. Four experimental groups were cooled to -196℃ by programmed freezer or rapid freezer in cryoprotectants with or without trehalose for 1 week. In the control group, the fresh teeth were not given any treatment. The PDL atta?ched to the middle third of roots was shaved from the root surface using scalpel knife. Filtered fluid was collected after digestion using trypsin for 5 mins and repeated for 5 times. After centrifugation, the supernatant was discarded. After resuspending the cells with phos?phate buffered saline ( PBS) , the living PDLCs were stained by Trypan blue and counted under high magnification. The cell survival rates of different groups were calculated and statistically analysed. Results:There were no statistically significant difference in survival rates of PDLCs between all groups. But in the group cryopreserved by programmed freezer with trehalose, the survival rates was the highest compared to that of the others. Conclusions: The cryopreservation do not cause obvious damage to the periodontal ligament cells. In our opinion, this technique could be applied in teeth preservation. On the other hand, using the programmed freezer with tre?halose in cryoprotectants have minimal effect on the viability of periodontal ligament cells.