摘要：

目的：探讨在制备种植窝过程中不同温度的冷却液对周围骨组织损伤情况的影响。方法：将48只大鼠随机分为3组，每组使用不同温度（4 ℃、20 ℃、40 ℃）的0.9%氯化钠冷却液建立双侧上颌磨牙区种植窝模型，术中使用K型热电偶和有限元热传导模型记录和模拟备孔区骨组织温度变化。术后1、4、7、14 d处死取材，用mirco-CT检测备孔区新骨形态学参数，组织脱钙固定后制作石蜡切片，使用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal-deoxynucleotidyl transferase mediated nick end labeling, TUNEL）检测凋亡细胞，抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色检测破骨细胞，免疫荧光检测Runt相关转录因子2（Runt-related transcription factor 2，Runx2）表达水平，实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测热休克蛋白70（heat shock protein 70，Hsp70）基因的转录水平。结果：成功建立大鼠上颌骨种植窝制备模型，建立的有限元热传导模型模拟的备孔区温度与实验测量值相似，4 ℃组冷却效果最佳，40 ℃组备孔区温度超过组织损伤的阈值（42 ℃）。术后1 d，TUNEL染色结果显示，40 ℃组备孔区凋亡细胞最多（P<0.05），4 ℃与20 ℃组凋亡细胞数量差异无统计学意义（P>0.05）；术后1 d，3组Hsp70的转录水平未见明显差异（P>0.05）；术后4 d，40 ℃组TRAP阳性细胞数量最多（P<0.05）；术后7 d，Runx2免疫荧光染色结果显示，4 ℃组和20 ℃组的Runx2表达水平高于40℃组（P<0.05）；术后14 d，micro-CT结果显示，40 ℃组种植窝新生骨的骨密度最低（P<0.05）。结论：冷却液对术区的冷却有显著效果，使用合适温度的冷却液有助于减少制备过程中的骨组织损伤。在充足流量的冷却液灌流下，4 ℃与20 ℃ 0.9%氯化钠溶液的冷却效果无差异。

关键词: 牙种植, 冷却, 骨生成, 细胞凋亡

Abstract:

Objective: The aim of this study was to explore the effect of normal saline at different temperatures on the damage of surrounding bone tissue during the preparation of implant sockets. Methods: A total of 48 rats were randomly divided into three groups. Each group used different temperatures (4 ℃, 20 ℃, 40 ℃) of 0.9% sodium chloride cooling solution to establish the implant socket preparation model at bilateral maxillary molar region. K-type thermocouple and finite element heat conduction model were used to record and simulate the temperature changes of bone tissue in the socket preparation area during operation respectively. At 1, 4, 7 and 14 days after operation, all rats were sacrificed for samples, and morphology parameters of new bone were detected by mirco-CT. After fixation and decalcification, paraffin sections were made. Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) was used to detect apoptotic cells. Tartrate resistant acid phosphatase(TRAP) staining was used to detect the osteoclasts. Runt-related transcription factor 2 (Runx2) expression was detected by immunofluorescence, and heat shock protein 70(Hsp70) transcriptional level was detected by real-time quantative polymerase chain reaction(RT-qPCR). Results: The model of rat maxillary implant socket preparation was successfully established, and the established finite element heat conduction model simulated the temperature of the hole preparation area, which was similar to the experimental value. The cooling effect of 4 ℃ group was the best, and the temperature of 40 ℃ group exceeded the tissue damage threshold of 42 ℃. TUNEL staining showed that the counts of apoptotic cells in 40 ℃ group was the most(P<0.05), but there was no significant difference in the counts of apoptotic cells in 4 ℃ and 20 ℃ groups at the 1st day after operation (P>0.05); Hsp70 transcriptional level among the three groups did not show significant differences at the 1st day after operation (P>0.05); The number of TRAP positive cells in 40 ℃ group was the most at the 4th day after operation (P<0.05). Runx2 immunofluorescence staining showed that Runx2 expression level in both 4 ℃ group and 20 ℃ group was higher than that in 40 ℃ group at the 7th day after operation(P<0.05); micro-CT results showed that the bone marrow density (BMD) in 40 ℃ group was the lowest at the 14th day after operation(P<0.05). Conclusion: The coolant had a significant cooling effect on the operative area, and the use of appropriate temperature coolant was beneficial to reduce bone tissue damage during the preparation process. There was no difference in cooling effect between 4 ℃ and 20 ℃ 0.9% sodium chloride solution under sufficient flow of coolant irrigation.

Key words: dental implant, cooling, osteogenesis, apoptosis