Temporal expression of SOX9 and type II collagen in spheno-occipital synchondrosis of mice after mechanical tension stimuli

OBJECTIVE: To associate the expressions of SOX9 and type II collagen during growth in the synchondrosis with and without tensile stress in order to understand the role of these factors in the growth of cartilage in spheno-occipital synchondrosis. MATERIALS AND METHODS: Sixty 1-day-old male BALB/c mice were randomly divided into experimental and control groups. Each group was subdivided again into five different time points which were 6, 24, 48, 72, and 168 hours. Each subgroup consisted of five mice. Each mouse was sacrificed using an overdose of pentobarbitone sodium. The synchondroses were aseptically removed and incubated in a 24-well plate with or without tensile stress in tissue culture. Tissue sections were stained immunohistochemically to quantitatively analyze the expression of SOX9 and type II collagen. RESULTS: There was a statistically significant increase of 57% (P < .001) in the expression of SOX9 between the experimental and control groups at 24 hours, followed by a significant increase of 44.4% (P < .001) in the expression of type II collagen at 72 hours. CONCLUSIONS: SOX9 may play an important role for early differentiation of chondrocytes and increase the expression of type II collagen, a major component of the extracellular matrix, during the growth of cartilage in the spheno-occipital synchondrosis.     

Effect of tensile force on expression of PTHrP and thickness of hypertrophic zone in organ-cultured mouse spheno-occipital synchondroses

OBJECTIVE: Responses of spheno-occipital synchondroses to direct tensile stress have not been identified before. This study was, therefore designed to evaluate expression of PTHrP, and thickness of hypertrophic zone in spheno-occipital synchondroses in response to such stress, using mouse in vitro model. METHODS: Spheno-occipital synchondroses together with adjacent structures were excised from fifty-five 2-day-old mice that were randomly assigned to 6 control and 5 experimental groups for 5 experimental periods (n=5). In the experimental groups, tensile force of 0.2g was applied across the synchondroses, using helical springs. In 5 control groups, the springs were made inactive. Both groups were then cultured for 6, 24, 48, 72 h and 7 days. Another control group was cultured without any springs for 7 days to compare with natural growth of the synchondroses from a group of five 9-day-old mice. Alcian blue-PAS staining was used to study growth of the synchondroses; immunohistochemical staining to identify PTHrP and type X collagen expression. The area of PTHrP expression and thickness of hypertrophic zone, demarcated by type X collagen expression, were measured. RESULTS: Quantitative analysis showed that PTHrP expression increased significantly at hour 24 of the force application in the experimental group (p<0.05), then reduced from hour 24 to 72 with a significant drop from hour 24 to 48 (p<0.01); and the thickness of hypertrophic zone significantly increased at hour 48 (p<0.01). CONCLUSIONS: Our findings suggested that the growth of spheno-occipital synchondroses could be modified by tensile stress; and a light continuous force could enhance its growth, as evidenced by an increase in PTHrP expression and thickness of hypertrophic zone.     

张应力对小鼠蝶枕软骨联合细胞增殖及低氧诱导因子-1α表达的影响

目的:了解循环张应力对小鼠颅底蝶枕软骨联合细胞(SOSCs)增殖及低氧诱导因子-1α表达的影响.方法:采集1 d龄小鼠的SOSCs进行体外培养,对第三代细胞加载牵张形变率分别为3％、6％、9％,频率为1 Hz,持续时间为1 h的循环张应力;用流式细胞术测算细胞增殖指数,用蛋白免疫印迹技术分析Hif-1α的表达水平.用按...     

机械张力作用下新生大鼠颅骨缝组织中NOS三种同形异构体的表达

目的：观察体外培养的新生SD大鼠颅顶骨骨缝在牵张力作用下,一氧化氮合酶（Nitric Oxide Synthase,NOS）三种同形异构体（eNOS,nNOS,iNOS）的表达及分布;探讨一氧化氮（nitric oxide,NO）在机械应力调节骨缝组织改建过程中所起的作用。方法：取一周龄SD大鼠颅顶骨正中矢状缝组织块进行体外器官加力培养。实验组施加0.2g张力,对照组将弹簧固定不施力,两组分别于0、1、6、24、48h收获标本。标本用常规免疫组化的方法,分别检测NOS三种同形异构体在颅骨缝组织中的分布和表达变化情况。结果：正常新生SD大鼠颅骨缝组织中内皮型一氧化氮合酶（eNOS）及神经元型一氧化氮合酶（nNOS）有轻微表达,而诱导型一氧化氮合酶（iNOS）基本不表达;施加机械张力后,三种NOS在颅骨缝组织中的表达强度及位置随受力时间长短均有改变。eNOS在最初就有少量表达,6h表达开始增加,24h呈强阳性高表达,48h各种细胞表达强度均有所减弱。nNOS在不同时间点均有较弱表达,其结果无统计学意义（P〉0.05）。iNOS在对照组和加力0h基本未见表达;加力1h后偶有成骨细胞、成纤维细胞弱表达;加力6h组可见散在轻微表达;加力24h组各种骨缝细胞均显著表达;加力48h,骨缝组织各种细胞表达呈强阳性。结论：机械牵张力刺激可以导致内源性eNOS及iNOS生成的增加,NO可能在机械张力调节骨缝组织改建过程中发挥重要作用。     

多囊蛋白-1在生长期小鼠颅底蝶枕软骨联合的表达

目的:了解多囊蛋白-1(PC1)在不同生长阶段小鼠蝶枕软骨联合表达的时空特征。方法:采集1、4、8周龄小鼠的蝶枕软骨联合标本,制备组织切片后进行PC1免疫组织化学染色,观察PC1的表达位置并测算单位面积组织内PC1阳性面积的百分比。结果:PC1在1周龄小鼠蝶枕软骨联合表达于储备层、前肥大层和早期肥大层;4周龄时主要表达于储备层、增殖层和前肥大层;8周龄时主要表达于前肥大细胞和早期肥大细胞。1、4、8周龄组PC1阳性面积百分比的均值分别为4.51%、5.7%、4.59%;4周龄组与1周龄及8周龄组间的差异都具有显著性(P<0.05)。结论:生长期小鼠蝶枕软骨联合表达PC1,但不同生长阶段的表达区域和水平存在差异。     

不同加载时间周期性张应力对大鼠颅底软骨细胞的影响

目的: 构建颅底软骨细胞体外培养力学刺激模型,研究周期性张应力对颅底软骨细胞主要细胞外基质（Ⅱ型胶原）和Sox9表达的影响。方法: 采用FX-5000T应力加载系统对体外培养的第2代大鼠颅底软骨细胞分别施加3、6、12、24 h的周期性张应力,加力值均为10%形变率,频率均为1 Hz。加力后即刻收集细胞,提取总RNA,实时荧光定量 PCR技术检测颅底软骨细胞Ⅱ型胶原（type-Ⅱcollagen,Col-Ⅱ）和Sox9 mRNA的表达。采用SPSS 17.0软件包对数据进行统计学分析。结果: 与对照组（加力0 h）相比,加力3 h组Col-Ⅱ和Sox9表达下降,且后者有显著差异（P<0.05）;加力6 h组Col-Ⅱ和Sox9表达显著下降（Col-Ⅱ,P<0.01;Sox9,P<0.05）;加力12 h组Col-Ⅱ和Sox9表达较6 h组有所上升,与对照组相比差异无统计学意义;加力24 h组中,两者表达与对照组相比显著升高（P<0.05）。结论: 周期性张应力可影响颅底软骨细胞增殖及细胞外基质合成,加力短时间基质合成抑制,增加加力时间则明显促进基质合成,且成软骨分化标志物Sox9 mRNA表达水平差异先于Col-Ⅱ出现。     

Rapid Maxillary Expansion Affects the Spheno-occipital Synchondrosis in Youngsters: A Study with Low-Dose Computed Tomography

Objective:To test the null hypothesis that the spheno-occipital synchondrosis does not show bony displacement in response to rapid maxillary expansion (RME) therapy in youngsters.Materials and Methods:A total of 16 computed tomography (CT) records were taken from 8 growing patients (2 males and 6 females), before (T0) and after (T1) treatment with RME. All patients had been diagnosed originally with transverse maxillary deficiency. The mean chronological age of the patients was 9.8 ± 1.8 years (range, 8 to 11.4 years). High-resolution multislice multidetector CT was used to study quantitatively the extent of the opening of the spheno-occipital synchondrosis following RME. A low-dose CT scan protocol was used (80 kV, 10 mA) and the data file of each patient was transferred to a workstation where the anteroposterior width of the spheno-occipital synchondrosis was measured on axial images.Results:Before treatment with RME (T0), the anteroposterior mean width of the spheno-occipital synchondrosis was 1.73 ± 0.46 mm immediately after the active phase of expansion (T1), and the width of the synchondrosis increased to 2.30 ± 0.47. This difference was statistically significant according to the Wilcoxon signed rank test (P < .05).Conclusion:Rapid maxillary expansion leads to a small immediate widening of the spheno-occipital synchondrosis in youngsters.     

静态张应力作用下人牙周膜干细胞核心结合因子Cbfa1表达的变化

目的检测静态张应力作用对人牙周膜干细胞核心结合因子Cbfa1表达变化的影响。方法有限稀释法克隆化培养纯化人牙周膜干细胞(hPDLSCs),应用内外双层套筒式硅胶膜细胞加力装置对人牙周膜干细胞加力,加力时间点为0、3、6、12、24小时。采用原位杂交和实时定量PCR检测各加力时间点Cbfa1 mRNA的表达;蛋白免疫印迹法(Western blot)检测人牙周膜干细胞受力后Cbfa1蛋白的表达。结果人牙周膜干细胞受到静态张应力作用后3小时、6小时、12小时Cbfa1 mRNA表达均升高(P0.05),且以加力3小时最为明显,6小时后开始下降,24小时后基本恢复到不加力时的表达水平。加力3小时Cbfa1蛋白表达未见明显升高,加力6小时后Cbfa1蛋白逐渐升高,一直持续到加力24小时(P0.05)。结论静态张应力作用下,人牙周膜干细胞核心结合因子Cbfa1在RNA水平和蛋白水平的表达均发生变化,说明Cbfa1在机械力促使人牙周膜干细胞向成骨细胞方向分化的过程中起到了重要作用。     

不同加载频率及力值的循环张应力对大鼠颅底软骨细胞的影响

目的 构建颅底软骨细胞体外培养力学模型,研究不同条件循环张应力（cyclic tensile strain, CTS）对颅底软骨细胞的影响。方法 采用FX-5000T应力加载系统对体外培养的第2代大鼠颅底软骨细胞施加不同条件的循环张应力,加力大小分别为5%、10%延伸率,加力频率分别为0.3、0.5、1.0 Hz,加力时间为24 h。加力后即刻收集细胞,提取总RNA,实时荧光定量PCR检测颅底软骨细胞Ⅱ型胶原(type-Ⅱcollagen,Col-Ⅱ)和Sox9 mRNA的表达。数据采用SPSS 17.0软件包进行统计学分析。结果 循环张应力大小为10%延伸率、频率为0.5 Hz或1.0 Hz时可显著促进大鼠颅底软骨细胞Col-Ⅱ及Sox9表达（P<0.05）。结论 适宜条件的循环张应力可促进大鼠颅底软骨细胞基质合成。     

机械牵张后大鼠颅缝成骨样细胞Etsl和Cbfal基因表达的研究

目的：探讨大鼠颅骨缝成骨细胞在张应力作用下Etsl和Cbfal基因表达的变化，以揭示骨缝牵张成骨的分子机制。方法：分离培养新生大鼠颅缝成骨样细胞，应用四点弯曲细胞加力装置对其施加单一周期的机械张力，并通过SYBR Green实时定量RT-PCR技术，对Etsl和Cbfal的基因表达进行检测。结果：Etsl和Cbfal的mRNA水平分别在机械张应力作用后6h及12h内显著增高，随后，mRNA水平逐渐恢复到对照组水平，Etsl先于Cbfal表达，在加力后表达立即升高，并在0．5h达到最高水平，而Cbfal则首先经历一个短暂的潜伏期，后表达逐渐升高，在6h达到最高水平，Cbfal的最高表达水平约为Etsl的2．58倍。结论：Etsl和Cbfal在骨缝牵张中对成骨细胞合成分泌骨基质蛋白可能起着不同的调节作用，而它们的功能具有时序性。高频率的牵张（〉2次／24h）更有利于Etsl和Cbfal的最佳表达。     

Magnetic resonance images and histology of the spheno-occipital synchondrosis in young monkeys (Macaca fuscata)

Magnetic resonance images and the histology of spheno-occipital synchondrosis were examined in young monkeys in order to compare the magnetic resonance images with their histologic observations. In serial magnetics resonance images of posterior cranial base, the spheno-occipital synchondrosis showed a low signal zone with unclear boundaries, running through the posterior cranial base perpendicularly to the clivus. The zone was always interposed between nonsignal zones. These observations were the same as those in young juvenile human beings. The histologic sections also revealed that the low signal zone was really the spheno-occipital synchondrosis, which consisted of hyaline cartilage and that the nonsignal zones were bone tissues. The chondroblasts in the spheno-occipital synchondrosis were arranged bipolarly. Intense alkaline phosphatase activity was located in the areas along the bone. Tetracycline labeling was also noticed in the bone formed in the endochondral ossification. These results suggest that magnetic resonance imaging enables us to observe the spheno-occipital synchondrosis in the posterior cranial base and also to elucidate its influences on the growth of maxilla and mandible in the future. (Am J Orthod Dentofacial Orthop 1999;115:138-42)     

Correlation of spheno-occipital synchondrosis fusion stages with a hand-wrist skeletal maturity index: A cone beam computed tomography study

ObjectivesTo examine the correlation between spheno-occipital synchondrosis fusion stages and the hand-wrist skeletal maturity index.Materials and MethodsDigital records of 164 individuals (77 males, 87 females) aged 10 to 18 years old were examined. Three-dimensional CBCT scans and hand-wrist two-dimensional radiographs were scored for the spheno-occipital synchondrosis fusion stages and hand-wrist skeletal maturity index, respectively. Statistical analyses were performed for associations using R software with a significance threshold of P< .01.ResultsA significant positive relationship was demonstrated between spheno-occipital synchondrosis fusion stages and hand-wrist skeletal maturity in both sexes. The Kendall''s rank correlation τ between hand-wrist skeletal maturity index and spheno-occipital synchondrosis fusion percentage were high and positive in males and females (r = .74 and r = .71, respectively).ConclusionsThe significant, positive relationship between the hand-wrist skeletal maturity index and spheno-occipital synchondrosis fusion stages support the idea of using spheno-occipital synchondrosis fusion as a biological indicator for craniofacial and mandibular growth spurt prediction.     

不同加载频率拉应力对髁突软骨细胞增殖分化的影响

目的:探讨不同加载频率的拉应力对髁突软骨细胞增殖、分化的影响。方法:体外分离2周龄新西兰兔髁突软骨细胞,对细胞施加2 h/d,为期3 d的不同频率(0 Hz、0.1 Hz、0.5 Hz、1 Hz)的拉应力。采用CCK8检测细胞增殖水平,实时荧光定量RT-PCR检测Sox9、Runx2、VEGF的表达。结果:细胞增殖水平在静止性(0 Hz)组显著高于周期性加力(0.1~1 Hz)组和对照组;Sox9、Runx2、VEGF表达在0.5 Hz组和1 Hz组显著高于静止性(0 Hz)组和对照组。结论:不同加载频率拉应力对髁突软骨细胞增殖分化有差异性影响。     

Three-dimensional assessment of the spheno-occipital synchondrosis and clivus after tooth-borne and bone-borne rapid maxillary expansion:: A retrospective CBCT study using voxel-based superimposition

ObjectivesTo assess changes in spheno-occipital synchondrosis after rapid maxillary expansion (RME) performed with conventional tooth-borne (TB) and bone-borne (BB) appliances.Materials and MethodsThis study included 40 subjects with transverse maxillary deficiency who received TB RME or BB RME. Cone-beam computed tomography images (CBCT) were taken before treatment (T0), and after a 6-month retention period (T1). Three-dimensional surface models of the spheno-occipital synchondrosis and basilar part of the occipital bone were generated. The CBCTs taken at T0 and T1 were registered at the anterior cranial fossa via voxel-based superimposition. Quantitative evaluation of Basion displacement was performed with linear measurements and Euclidean distances. The volume of the synchndrosis was also calculated for each time point as well as the Nasion-Sella-Basion angle (N-S-Ba°). All data were statistically analyzed to perform inter-timing and intergroup comparisons.ResultsIn both groups, there was a small increment of the volume of the synchondrosis and of N-S-Ba° (P < .05). Basion showed a posterosuperior pattern of displacement. However, no significant differences (P > .05) were found between the two groups.ConclusionsAlthough TB and BB RME seemed to have some effects on the spheno-occipital synchondrosis, differences were very small and clinically negligible.     

Time and pattern of the fusion of the spheno-occipital synchondrosis in patients with skeletal Class I and Class III malocclusion

Objectives:To investigate the time and pattern of fusion of the spheno-occipital synchondrosis in patients with skeletal Class I and Class III malocclusion using cone-beam computed tomography (CBCT).Materials and Methods:A total of 262 CBCT images were collected: 140 skeletal Class I (0° < ANB < 4°; 71 males, 69 females) and 122 skeletal Class III (ANB ≤ 0°; 61 males and 61 females). The fusion stages were identified using CBCT images of a six-stage system defined by the appearance of osseous cores and ossifying vestige in the synchondrosis. The age distributions of each stage and the pattern of fusion were evaluated.Results:The stages of fusion progressed with increasing age (P < .05, r = .824), and the age distributions in the female groups were generally 1 to 3 years younger than those in the male groups. However, no significant differences were observed between the skeletal Class I and Class III groups regarding the time of ossification of the synchondrosis. The osseous cores appeared most frequently in the supero-center part, followed by the mid-center part of the synchondrosis.Conclusions:The time and pattern of fusion of the spheno-occipital synchondrosis are not apparently different between patients with Class I malocclusion and those with Class III malocclusion. The osseous cores appear frequently in the supero-center and mid-center of the synchondrosis with various patterns before the end of the pubertal growth spurt period.     

Dietary loading and aggrecanase-1/TIMP-3 expression in rat mandibular condylar cartilage

AIMS: To examine the expression of aggrecanase-1 and a tissue inhibitor of metalloproteinases (TIMP-3) in the condylar cartilage of young rats and to determine their relationship during altered dietary loading at different time points after weaning. METHODS: One hundred Sprague-Dawley rats were randomly assigned to 1 of 2 groups: the soft-diet group, which served as the control group (n=50), or the hard-diet group, which served as the experimental group (n=50). Ten soft- and 10 hard-diet rats were killed at 6 hours, 12 hours, 24 hours, 48 hours, and 9 days after weaning (i.e., after initiation of diet change for hard-diet rats). The right-side temporomandibular joints (TMJs) were prepared for immunohistochemical staining. The cartilage from the left-side mandibular condyles of all 10 animals in each group was combined for Western blot analysis. RESULTS: Immunohistochemical analysis revealed strong staining for aggrecanase-1 localized mainly in the chondrocytes of proliferative and upper hypertrophic cartilage zones at all time points in both groups. The immunohistological expression of aggrecanase-1 was significantly higher in the hard-diet group at 12 and 24 hours than in the soft-diet group. Strong staining for TIMP-3 was mainly localized in the chondrocytes of proliferative and upper hypertrophic zones at all time points in both groups. The expression of TIMP-3 in the hard-diet group was at a significantly lower level compared to the soft-diet group at 6 hours. Western blot analysis also showed time-related differences in aggrecanase-1 and TIMP-3, but there was no significant difference between the 2 groups. CONCLUSION: The temporary change in aggrecanase-1 and TIMP-3 expression reflects the complex interaction of these enzymes in the physiologic range and cartilage response to altered dietary loading.     

Histological and histochemical study of the spheno-occipital synchondrosis of the cranial base on BALB/c-bm/bm mouse

Murine brachymorphism (bm) has cartilage with undersulfated glycosaminoglycans. The reason for inferior growth of craniofacial structure in BALB/c bm homozygous mouse has not been clarified. The spheno-occipital synchondrosis, which is the growth site of the cranium, was histologically and histochemically investigated in this study.

BALB/c mice and BALB/c-bm/bm mice were used. Sagittal sections of the cranial bases were stained by the method of hematoxylin and eosin, alcian blue critical electrolyte concentration, or sensitized high iron diamine method after treatment with/without testicular hyaluronidase.

The results showed that chondroitin sulfate and keratan sulfate were undersulfated in the cartilage of the spheno-occipital synchondrosis in BALB/c-bm/bm mice. Sections stained using hematoxylin and eosin showed that columns of chondrocytes were irregular in arrangement, and normal endochondral ossification was not seen in the cartilage of the spheno-occipital synchondrosis in BALB/c-bm/bm mice.     

改变食物硬度对大鼠生长早期髁突软骨中VEGF、aggrecanase-1和TIMP-3mRNA表达的影响

目的:通过改变食物硬度造成大鼠生长早期颞下颌关节软骨的负荷改变,检测髁突软骨中血管内皮生长因子(vascular endothelial growth factor,VEGF)、聚集蛋白聚糖酶-1(aggrecanase-1)和金属蛋白酶抑制剂因子-3(tissue inhibitor of metalloprotei...     

静态张应力作用下人牙周膜成纤维细胞β-catenin和Wnt3a蛋白的表达

目的:研究人牙周膜成纤维细胞在不同大小机械张应力作用下Wnt/β-catenin信号通路中β-catenin和Wnt3a蛋白的表达变化。方法:运用酶解组织块法体外培养人牙周膜成纤维细胞,将细胞按加力时间的不同(2 h、4 h、6 h)分为3组,每组再按形变率不同将其分为8%、12%和16%3个亚组,并取加力时间0h、形变率0%作为对照组。用western blot技术检测细胞在不同时间点和不同形变率作用下Wnt/β-catenin信号通路中β-catenin和Wnt3a蛋白的表达变化。结果:Western blot结果显示,人牙周膜成纤维细胞在机械力加载后β-catenin 和Wnt3a蛋白表达显著减少(P<0.05)。在加力时间相同的组内(2 h、4 h、6 h),β-catenin 和Wnt3a蛋白表达随着形变率的增大而逐渐减少(P<0.05)。结论:β-catenin和 Wnt3a参与了静态张应力作用下牙周膜成纤维细胞的代谢,机械力对牙周膜成纤维细胞中Wnt/β-catenin信号通路有抑制作用。