骨质疏松SD大鼠药物相关性颌骨骨坏死模型的建立

目的：建立骨质疏松SD大鼠药物相关性颌骨骨坏死（medication-related osteonecrosis of the jaw,MRONJ）动物模型。方法：6月龄雌性SD大鼠去势法建立骨质疏松模型,术后12周皮下注射阿仑膦酸钠1.0 mg/（kg·d）,60 d后随机分为两组：第1组（对照组）给药后不予干预;第2组（实验组）连续给药后予以拔除双侧上下颌第一磨牙。术后2、4、8周每组随机处死5只大鼠并进行临床、血清学、组织病理学及组织形态学检测。采用SPSS 19.0软件包进行统计学分析。结果： 临床观察实验组术后2周开始出现MRONJ改变,发病率显著高于对照组（P<0.01）;下颌骨发生率（56.7%）高于上颌骨（26.7%）。血清RANKL和OPG浓度随时间发生显著改变,但与MRONJ发病率无显著相关性（P>0.05）。H-E染色观察MRONJ病变区骨小梁结构紊乱,骨细胞溶解导致大量空虚骨陷窝形成（P<0.05）。根据骨坏死病变和软组织损害程度对所有MRONJ动物进行分级分期,对照组发病率和病变程度显著低于实验组（P<0.05）,MRONJ病变程度随着时间变化逐渐加重。Micro-CT显示MRONJ病变区域有骨微裂发生,骨质溶解破坏; MRONJ大鼠BMD、Tb.N在MRONJ 2期与其他分组有显著差异（P<0.05）,MRONJ 0期时Tb.Sp与正常组差异显著（P<0.05）。结论：骨质疏松SD大鼠动物模型上使用高剂量阿仑膦酸钠皮下注射60 d后予以配合拔牙,术后2周开始出现MRONJ,8周可以建立较为稳定的MRONJ动物模型;拔牙是MRONJ的好发因素;MRONJ病变程度与时间有相关性;血清RANKL/OPG比值变化可能与MRONJ有相关性;Tb.Sp可能是病变早期骨形态学标志,病变区可观察到骨微裂。

The establishment of a bisphosphonate-related osteonecrosis of the jaw animal model in osteoporosis rats

PURPOSE: To establish medication-related osteonecrosis of jaw-liked animal model in osteoporosis rats. METHODS: Thirty six-month-old female osteoporosis Sprague-Dawley rats were created by bilateral ovariectomy. After 12 weeks, all rats were administrated by subcutaneous injections of alendronate(1.0 mg/kg/day dosage) for 60 days then randomized into two groups: Group 1 (control group) no intervention after ALN administration; group 2 (experimental group) bilateral maxillary and mandible first molars were extracted after ALN administration. Five rats were randomly euthanized at 2 weeks, 4 weeks and 8 weeks after interventions from each group, and underwent clinical, serological, histopathologic and histomorphometric evaluations. SPSS 19.0 software package and R program were used for statistical analysis. RESULT: MRONJ lesions were observed in group 2 rats 2 weeks after operation, the occurrence rate was significantly greater than group 1 (P<0.01); Mandible had higher incidence of 56.7% than maxillar which had a probability of BRONJ of 26.7%. The time-varying serum concentration of RANKL and OPG were unrelated with the occurrence. Hematoxylin-Eosin staining demonstrated that trabecular bone structure of MRONJ lesion areas was disorganized, large number of empty bone sinkholes were generated by the nuclear of osteocyte karyolysis (P<0.05). According to the degrees of osteonecrosis lesion and soft tissue damage, the morbidity and pathological degree of group 1 were significantly lower than that of group 2(P<0.05). The degree of MRONJ lesions was escalated over time. Micro-CT revealed that the MRONJ lesion area had microcracks and osteolysis. The value of Tb.N and BMD had significant changes in Stage 2 MRONJ rats compared to other stages(P<0.05). The difference of Tb.Sp between stage 0 and non-MRONJ rats was statistically significant(P<0.05). CONCLUSIONS: The MRONJ animal model had been successful established by subcutaneous injection of high dose ALN for 60 days associated with teeth extraction in osteoporosis rats, the typical lesions had been observed 2 weeks after intervention; while the animal model stabled 8 weeks after operation. The lesion degree of BRONJ had close relation to time. With regard to local risk factors of BRONJ, this study confirmed that teeth extraction was a remarkable risk factor of BRONJ. The variation of serum RANKL/OPG ratio might be related to BRONJ, Tb.Sp might be effective bone morphogenetic marker associated with BRONJ in the early stage, and microcracks had been observed in the lesion area.