| Objectives: Bisphosphonates had made remarkable curative effects in the treatment of osteoporosis and bone metastasis of malignant tumors. With the wide application of these drugs, Bisphosphonate-associated osteonecrosis of the jaws as one of the adverse drug reaction had caused widespread concern in the whole of world, but up to now the mechanism was still unclear. This study observed histopathological changes of mandible after using zoledronate injection, and explored basic pathological change. It helped to provide experimental data and basic research for clinical prevention and treatment.Methods:Thirty-six healthy female SD rats with age of three month and weights from 200 to 240 gram were selected for the study. All the rats were free access to food and water for one week. All the rats were divided into three groups, with each group which included 12 samples. Group A(Simple zoledronate group): peritoneal injection was done by zoledronate with 0.2mg/kg(concentration of 0.1 mg/ml) twice a day for 8 weeks and physiological saline 0.5ml per 100 g in weihgt was given by gavage once a day. Group B(zoledronate combined calcium): peritoneal injection was done by zoledronate with 0.2mg/kg(concentration of 0.1 mg/ml) twice a day for 8 weeks. Concentration of 0.84% of Calcuim carbonate suspension liquid 0.5ml per 100 g in weihgt was given by gavage once a day. Group C(control group): peritoneal injection was done by zoledronate with physiological saline 2ml/kg for 8 weeks, and physiological saline 0.5ml per 100 g in weihgt was given by gavage once a day for 8 weeks. In eight weeks after injection, all of each group(12 SD rats) was randomly divided into extraction group(3 rats) and non-extraction group(9 rats). Extraction group: the first molar on the mandibular left side was extracted under 10% chloral hydrate general anesthesia. The animals were sacrificed and their mandibular were collected in 4 weeks after extraction. The non-extraction group: 3 SD rats were randomly sacrificed on the 8, 10, 12 weeks after injection and mandibule specimens were collected. All specimens were fixated in the 10% formaldehyde solution for 48 hours, and rinsed for 24 hours by water, and rinsed for 3 min by 0.01 M phosphate buffer, and decalcified in 10% EDTA solution for one and half months. All specimens were cleaned under running water for 24 hours, and fixated in the 10% formaldehyde solution for 30 min, and dehydrated in ethanol, and cleared in xylene, and finally infiltrated. Then 4 microns thick tissue was cut off along the sagittal plane and stained with hematoxylin-eosin(HE) for histological observation and histomorphometry examination. Bone tissue morphology metrology was detected by Computer medical digital pathological image analysis system Med6.0. After immunohistochemical staining, average optical density of maxillofacial Vascular endothelial growth factor(VEGF) was measured by the professional Image analysis software Image- pro plus 6.0, and then statistical analysis was performed.Statistical analysis:Statistical analysis was performed by SPSS 21.0. All data were presented as Mean±Sd and tested normality and Homogeneity of variance. Factorial design analysis of variance was used when when normality(and homogeneity of variance) assumptions were satisfied, SNK(Student-Newman- Keuls) test was used between two groups, P<0.05 was considered as the level of statistical siginificance.Results:1 General observationNon-extraction group: the gum was not red and swollen, without bleeding, exudation, fistula and sequestrum in 8, 10, 12 weeks after injection.Extraction group: in 4 weeks after extraction, group A: the gum was red, swollen and ulcerated. Group B: the gum was red, but sequestrum was not exposured. Group C:extraction sockets healled well, without red, swollen and bleeding.2 Histological observationsIn 8 weeks after injection, group A:inflammatory cellular infiltration, trabeculae disordered arrangement. Inflammatory cells mainly included multinucleated giant cells, lymphocytes, plasma cells and so on. Capillaries and damaged trabeculae was observed, but without sequestrum, mainly limited around apical alveolar. Group B: inflammatory cellular infiltration was also observed around apical alveolar with smaller scale. Lymphocytes, plasma cells, and neutrophils were widely distributed around trabeculae with some damaged trabeculae. Group C: finer trabeculae arranged regularly and connected into a net, and inflammatory cellular infiltration was not obvious. Scattered inflammatory cells and intact trabeculae without absorption could be seen occasionally without inflammatory cellular infiltration around the periodontal membrance.In 10 weeks after injection, group A and B: bigger scale with large amount of inflammatory cellular infiltration. Lymphocytes, plasma cells, and neutrophils were more intensive. Trabeculae was disorderly and absorpted more obviously, without sequestrum. There was no inflammatory cellular infiltration around the periodontal membrane. Group C: scattered inflammatory cells, disordered arrangemen of trabecula and intact rabecular without absorption could be seen around the periodontal membrane.In 12 weeks after injection, group A and B: large amount of inflammatory cellular infiltration could be seen around the periodontal membrane. Trabeculae was disorderly, which absorpted more obviously, inflammatory cell distributed around the trabeculae, but sequestrum was not detected. Trabeculae, was thick and connected, arranged regularly in normal area. Group C: Inflammatory infiltrates was not obvious. Trabeculae arranged regularly without absorption and with inflammatory cell distributed around the periodontal membrane.In 4 weeks after extraction: inflammatory infiltrates increased more widely and seriously in extraction group A and B than 12 weeks. New born trabeculae was not found in dental socket. The scope of inflammatory cells was more extensive and intensive, trabeculae absorpted more obviously than non-extraction group but without sequestrum. Group C: Tooth extraction sockets filled with many immature trabeculae, which was slender, loose arrangement, and connected into a net. A few inflammatory cells and regularly arranged of trabeculae without absorption could be seen surround in dental socket. There was no significant difference between extraction and no-extraction group in 4 weeks after extraction.3 Bone histomorphometry results:3.1 Cortical bone of mandibleComparison of cortical bone thickness among three groups: there was no interaction between group and time. There was statistical difference in cortical bone thickness among the three groups(group C<group A<group B)(P<0.05). Compared from three time points, there was no statistical difference in cortical bone thickness among the three groups(P>0.05).3.2 Cancellous bone in mandible apical regionIn 8, 10, 12 weeks after injection, comparison of trabecular area, circumference and width, inflammatory infiltrates among three groups. There was significant difference in trabecular area and circumference among the three groups(group A<group B<group C)(P<0.05). There was no significant difference in trabecular width between group A and group B(P>0.05), but there was significant difference between group A and group C, group B and group C(P<0.05). There was no significant difference in inflammatory infiltrates between group A and group B(P>0.05), but there was significant difference between group A and group C, group B and group C(P<0.05). There was no significant difference in trabecular area, trabecular circumference and width, inflammatory infiltrates among three time points(P>0.05).In 12 weeks after injection, Factorial analysis was used to be, there was no interaction between extraction and different groups. Comparison of trabecular area, circumference, width and inflammatory infiltrates among three groups. There was no significant difference in trabecular area, circumference and width between group A and group B(P>0.05), but there was significant difference between group A and group C, group B and group C(P<0.05). There was no significant difference in inflammatory infiltrates between group A and group B(P>0.05), but there was significant difference between group A and group C, group B and group C(P<0.05). Comparison of trabecular area, circumference, width between extraction and non-extraction group. There was significant difference in trabecular area, circumference and width between two groups(extraction group <non-extraction group, P<0.05). There was no significant difference in inflammatory infiltrates between two groups(P>0.05).3.3 Cancellous bone in mandible non- apical regionIn 8, 10, 12 weeks after injection, Factorial analysis was used, there was no interaction between different groups and time. Comparison of trabecular area, circumference and width among three groups. There was significant difference in trabecular area and circumference among three groups(group C<group A<group B)(P<0.05).There was no significant difference in trabecular width among the three groups(P>0.05). Comparison of trabecular area, circumference and width in three time points. There was no significant difference in bone trabecular area between 10 weeks and 12 weeks(P>0.05), but there was significant difference between 8 weeks and 10 weeks, 8 weeks and 12 weeks(P<0.05). There was no significant difference in trabecular circumference between 8 weeks and 10 weeks(P>0.05), but there was significant difference between 8 weeks and 12 weeks, 10 weeks and 12 weeks(P<0.05). There was no significant difference in trabecular width between 8 weeks and 10 weeks, 10 weeks and 12 weeks(P>0.05), there was significant difference between 8 weeks and 12 weeks(P<0.05).4 The average optical density value of VEGF in blood vessels4.1 Comparison the average optical density value of VEG in blood vessels in 8, 10, 12 weeks after injection. There was interaction between time and groups(P<0.05). There was significant difference in average optical density value of VEGF among three groups(group A<group B<group C)(P<0.05). There was significant difference in three time points(IOD8 < IOD 10< IOD 12)(P<0.05).4.2 Comparison the average optical density value of VEGF in blood vessels between extraction and non-extraction group in 12 weeks after injection. There was interaction between extraction and group(P<0.05). There was significant difference in average optical density value of VEGF between two groups(non-extraction group<extraction group, P<0.05). There was significant difference in average optical density value of VEGF among three groups(group A<group B<group C)(P<0.05).Conclusions:1 There was histomorphological variation on rat mandible after using Zoledronate. This drug increased the thickness of the cortical bone of mandible and bone mineral density of cancellous bone in non-apical region. But in cancellous bone of apical region, trabecular was arranged disorderly and surrounded by a mixed inflammatory infiltration.2 Vascularization on mandible was inhibited after using Zoledronate. So there was less blood suppy on mandible.3 After long-term application of zoledronate, there was variation on organization structure of mandibular before necrosis occurred on mandible. It may be pathological basis of mandible necrosis after extraction and mandible surgery. |
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