摘要
目的:应用X射线能谱分析仪从微观上分析功能矫治前伸大鼠下颌后髁突前、中、后部钙磷元素的代谢情况及在保持过程中的改变,从而以Ca/P的动态变化评价功能矫治过程中的骨改建情况,通过检测全过程的钙磷能谱变化评价II类牙合功能矫治的远期效果,为临床提供相应的实验室依据。
方法:选取健康的5周龄雄性SD大鼠35只(清洁级),体重约90-100g,由河北医科大学实验动物中心提供。经实验室饲养7天,待大鼠适应实验室环境后随机分为:对照组(2周组,4周组,6周组);实验组(戴用矫治器2周组,戴用矫治器4周组,戴用矫治器2周后摘除并观察2周组,戴用矫治器6周组)。每组5只,自由摄食饮水。实验组大鼠配戴自制的上颌可摘式斜面导板矫治器引导下颌前伸,每日戴用时间为10-12小时;对照组不做任何处理,为自然生长组。大鼠断颈处死后取双侧髁突,其中右侧髁突于4%多聚甲醛中固定,10%EDTA脱钙,逐级酒精脱水后常规石蜡包埋,沿髁突矢状切片,厚约5μm,取中间部位的切片,进行组织切片制备,HE染色后光镜下观察其形态,并在10倍镜下应用vista病理图像分析软件采图,测量髁突前、中、后部软骨厚度;左侧髁突于2.5%戊二醛中固定24小时以上,液氮制冷条件下沿矢状方向劈开髁突,暴露观察面。样本经0.1mol/LPBS反复冲洗,再置于0.1mol/LPBS液中超声清洗5分钟,梯度酒精逐级脱水,乙醚置换酒精,干燥后使用导电胶粘于观测台,在扫描电镜定位下分别对髁突前、中、后斜面做X射线能谱分析及元素定量测定。将全部记测数值按顺序输入计算机同时用SPSS 13.0统计分析软件对样本数据进行分析处理。使用t检验比较各组特征数值,分析各组数据之间的差异。
结果:
1 HE染色后光镜下观察髁突软骨层变化:
1.1髁突软骨层形态学变化
软骨细胞根据形态可将其分为:①纤维层:由平行于髁突表面的胶原纤维构成,含有纤维细胞;②生发层:细胞体积小,呈圆形或卵圆形,排列紧密,核大,胞外基质少;③成熟层:细胞体积明显增大,卵圆形,软骨基质明显增多,细胞排列不规律;④移行层:细胞肿胀,较成熟层明显增大。胞核固缩或肿胀,细胞外软骨基质增多、均匀。软骨层移行并无确切分界,其下方为骨小梁与软骨表面垂直排列。髁突软骨由后向前逐渐变薄,以成熟层、移行层的变化最为明显。对照组大鼠在生长发育过程中可以观察到髁突软骨出现增龄性变化,整个软骨层逐渐变薄,以中后部变化最为明显。细胞体积变小,骨小梁变粗。
1.2髁突软骨层厚度变化
1.2.1前部软骨:戴用矫治器2周后其厚度与对照组相比无统计学差异(P>0.05),直到戴用6周后软骨层较对照组变薄(P<0.05),放弃保持的2+2周组大鼠髁突前部软骨较4周组变厚(P<0.05),与对照组相比无统计学差异(P>0.05)。
1.2.2中部软骨:戴用矫治器2周后较对照组变厚(P<0.05),放弃保持的2+2周组与4周组相比其变化无统计学差异(P>0.05)。
1.2.3后部软骨:后部软骨层厚度变化较大与对照组相比变厚(P<0.01),放弃保持的2+2周组其后部软骨层与4周组相比变薄(P<0.01),但与对照组相比仍然存在差异(P<0.01)。
2髁突钙磷能谱变化:
2.1前部:
戴用矫治器2周后,Ca元素峰值强度较低,而P含量相对升高,实验组Ca/P较对照组低(P<0.01);
戴用矫治器4周时,能谱分析显示Ca峰值强度略有升高,变化不明显,而P元素相对降低,实验组Ca/P有所升高但低于对照组(P<0.05);
6周时,Ca元素含量进一步升高,与P含量差距变大,实验组Ca/P继续升高,但仍然与对照组的Ca/P存在差距(P<0.05);
放弃保持的2+2周组与一直戴用矫治器的4周组相比,前部软骨P含量相对升高,Ca/P降低(P<0.05)。
2.2中部:
Ca、P元素总体变化趋势和前部相似,在戴用矫治器2周后Ca/P明显降低(P<0.05);
戴用4周后Ca/P与对照组相比无统计学差异(P>0.05);
放弃保持的2+2周组与一直戴用矫治器的4周组相比Ca/P略有降低(P<0.05)。
2.3后部:
戴用矫治器2周后,Ca元素峰值强度较低,而P含量相对升高,实验组Ca/P较对照组低(P<0.01);
戴用矫治器4周时, Ca峰值强度升高, P元素相对降低,实验组Ca/P虽有所上升,但仍然比对照组低(P<0.05);
6周时,Ca元素含量进一步升高,此时实验组虽然比对照组的Ca/P低但是已无统计学差异(P>0.05);
放弃保持的2+2周组与一直戴用矫治器的4周组相比,Ca/P降低(P<0.05)。
结论:
1功能矫治2周时大鼠髁突软骨层细胞增生活跃,中后部软骨层厚度明显增大,前部厚度于矫治6周时变薄。治疗后放弃保持则出现明显复发,前、后部变化较显著。
2对照组大鼠髁突骨矿化程度(Ca/P)在整个实验过程中呈逐渐升高的趋势,是一种发育性的改变。
3实验组大鼠髁突骨矿化程度(Ca/P)在治疗初期明显降低,随后逐渐升高接近对照组,但中部改建完成较快,前部较慢在实验结束时仍未稳定。
4放弃保持后,髁突Ca/P降低,是一种复发性表现,这一现象早于软骨厚度的变化。
Objective: Use the X-ray spectrum analyzer from the microcosmic view to analyze the anterior,the middle,and the posterior condyle of the rats. The elements of calcium and phosphorus after mandibular protrusion and between the process of maintaining should be detected. We choose the dynamic changes of Ca/P to evaluate the bone alteration and analyze the dynamic changes of calcium and phosphorus during the whole process to evaluate the long-term effects of II malocclusion after using functional appliance in order to provide the experimental basis for clinical treatment.
Methods: 35 5-week-old male SD rats were chose (clean level). They weighed about 90-100g. They were provided by the experimental animal center of Hebei Medical University. Fed the rats in the laboratory for seven days until they adopt the condition and then divided into 7 groups randomly: control groups (2 weeks group, 4 weeks group, 6 weeks group), experimental groups (the group of wearing the appliance for 2 weeks, the group of wearing the appliance for 4 weeks, the group of wearing the functional appliance for 2 weeks and casting off the appliance for 2 weeks, the group of wearing the appliance for 6 weeks.) Each group included five rats. Free drinking and timing eating. The rats of experimental groups wore movable upper jaw inclined bite plate to keep the mandible protrusion. The wearing time was 10-12h / d. The control groups received no treatment. They were natural growth groups. The rats were sacrificed and took the bilateral condyle. The right condyle was fixed in the 4% paraformaldehyde and decalcified in the 10%EDTA.The tissues were dehydrated and then embedded in paraffin. The histological section were cut into 5μm in thickness. Took the middle parts of the slice to make tissue sections. The sections were observed through optical microscopy after stained with hematoxylin and eosin. Took the image under the microscope at 10 times and measured the change of the cartilage’s thickness. The left side of condyle was fixed in 2.5% glutaraldehyde for more than 24h.After being frozen in liquid nitrogen the condyle was split into two parts and the observing surface was exposed. The samples were cleaned in 0.1mol/LPBS, and washed in ultrasonic cleaner for 5minutes. Fixed the samples on the observatory using the conductive adhesive after be dehydrated and dried .The elements concentration was detected and analyzed by x-ray energy dispersive system . All the measured values would be recorded by the computer with the SPSS 13.0 statistical analysis software. Compared the characteristic values of each group using the t test and analyzed the differences between the various sets of data.
Results:
1 Observe the changes of the condylar cartilage layer under the optical microscope after be stained with hematoxylin and eosin
1.1 The morphological changes of condylar cartilage layer
The chondrocytes can be classified according to morphology:①The fiber layer: The collagen fibers parallel the condylar surface and contain fiber cells.②The germinal layers: The cells are small round or oval and closely arranged. Nucleus is big and extracellular matrix is less;③The mature layers: The cell’s volume and the cartilage matrix increase significantly. The cells are ova. The cells arrange irregular.④The transitional layers: The cells are swelling and increase significantly than mature level. Nucleus is condensed or swelling. The cartilage matrix outside the cells is uniform and increases a lot. Transitional layers of the cartilage don’t have the exact boundaries. The trabecular bone locates under the cartilage and perpendicular to the surface. The condylar cartilage becomes thinner from posterior part to anterior part and especially the mature layer and the transitional layer.The condylar cartilage of the control group rats during the growth can be observed age-related changes.The entire cartilage layer becomes thinner gradually. The most obvious changes occur in the rear part. The volume of the cell becomes smaller and the trabecula becomes thicker.
1.2 The change of the thickness of the condylar cartilage
1.2.1 The anterior cartilage
The change after wearing the appliance for 2 weeks had no significant difference comparing with the control group (P>0.05). After wearing the appliance for 6weeks the cartilage became thinner than the control group (P<0.05). The group which gave up maintaining the treatment effect, its anterior condylar cartilage became thicker comparing with the 4W group (P<0.05) and ultimately had no significant difference with the control group (P>0.05).
1.2.2 The middle cartilage
After wearing the appliance for 2 weeks the cartilage became thicker comparing with the control group (P<0.05). The group which gave up maintaining the treatment effect changed little (P>0.05).
1.2.3 The posterior cartilage
The thickness of the posterior cartilage changed significantly comparing with the control group (P<0.01). The group which gave up maintaining the treatment effect its posterior cartilage layer became thinner comparing with the 4W group. (P<0.01) But the difference with the control group still had statistical significance (P<0.01).
2 The condylar changes in the spectroscopy of calcium and phosphorus
2.1 The anterior part
After wearing the appliance for 2 weeks, the elements of Ca had a lower peak intensity, while the content of P was relatively higher. In the control group the Ca / P was higher than the experimental group (P<0.01). After wearing the appliance for 4 weeks, the energy spectrum analyzer showed that the peak intensity of Ca was slightly elevated but did not change significantly, while the element of P was relatively lower. In the experimental group the Ca / P had increased but still be lower than the control group (P<0.05). In the 6W group, the element of Ca content increased further, and had a bigger gap with the content of P. The Ca / P still had statistical significance between the control group and experimental group (P<0.05). Comparing with the 4W group, the content of P at the front cartilage in the 2 +2 W group was higher and the Ca / P was lower (P<0.05).
2.2 The middle part
The trends of the Ca and P elements were similar to the front. After wearing the appliance for 2 weeks the Ca / P decreased significantly (P<0.05).The Ca / P of the 4W group had no significant difference with the control group (P>0.05). The Ca / P of the 2 +2 W group was slightly lower comparing with the 4W group (P<0.05).
2.3 The posterior part
After wearing the appliance for 2 weeks, the elements of Ca had a low peak intensity, while the content of P was relatively higher. In the control group the Ca / P was higher than the experimental group (P<0.01). In the 4W group the peak intensity of Ca increased and the element of P was relatively lower. In the control group the Ca / P was higher than the experimental group (P<0.05). In the 6W group the content of Ca increased further. Although the Ca / P of the experimental group was low comparing with the control group but the difference had no statistical significance. (P>0.05).Comparing the 2+2W group with the 4W group, the difference had obvious statistical significance (P<0.05).
Conclusion:
1 After wore the functional appliance for 2 weeks the condylar cells proliferated actively in the experimental group. The posterior and the middle cartilage became thicker. The anterior cartilage became thinner in the 6W group. During the puberty if we gave up maintaining the effect of the treatment would bring the apparent recurrence especially at the front and the rear condylar.
2 The bone mineralization of the control groups’condylar (Ca / P) during the entire experimental process was gradually increased and the trend was a growing change.
3 The bone mineralization of the experimental groups’condylar (Ca / P)was gradually decreased at the beginning and then increased to normal level. The middle part reconstructed faster while the front part reconstructed slower. In the end of the experiment the front condylar had not yet stabilized.
4 When give up wearing functional appliance to keep the effect, the Ca / P of the condylar became lower. This was a recurrent manifestation. The phenomenon presented earlier than the change of the cartilage thickness.
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