Factors regulating endochondral ossification in the spheno-occipital synchondrosis

OBJECTIVES: To identify the temporal pattern of core-binding factor alpha1 (Cbfa1) and vascular endothelial growth factor (VEGF) expressions in the spheno-occipital synchondrosis in vitro with and without tensile stress. MATERIALS AND METHODS: Sixty male BALB/c mice were randomly divided into an experimental group (with tensile stress) and a control group (without tensile stress) at each of five time points. Animals were sacrificed and the cranial base synchondroses were aseptically removed. In the experimental groups, mechanical stress was applied on the surgical explants with helical springs and incubated as organ culture for 6, 24, 48, 72, and 168 hours. In the control group, the springs were kept at zero stress. Tissue sections were subjected to immunohistochemical staining for quantitative analysis of Cbfa1 and VEGF expression. RESULTS: Quantitative analysis revealed that Cbfa1 and VEGF expressions reached a peak increase at 24 and 48 hours, respectively. Compared with the control groups, both Cbfa1 and VEGF were expressed consistently higher in the experimental groups at all time points. CONCLUSION: Mechanical stress applied to the spheno-occipital synchondrosis elicits Cbfa1 expression and subsequently up-regulates the expression of VEGF. Increased levels of expression of both factors could play a role in the growth of 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.     

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。     

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.     

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)     

Growth and growth pressure of mandibular condylar and some primary cartilages of the rat in vitro

To compare the in vitro development of the secondary cartilage of the mandibular condyle with that of primary cartilages, several cartilaginous explants derived from 4-day-old rats were cultured in a serum-free culture system. The following cartilages were used: the mandibular condylar cartilage, the distal epiphyseal cartilage (including the growth plate) of the third metatarsal, a fragment of costal cartilage (including the osteochondral junction) of the fourth rib, the spheno-occipital synchondrosis and the chondroepiphysis of the femoral head. In addition, with a specially designed, in vitro pressure registration system, the maximal growth pressures for each of the explants, except the femoral head, were determined. The results show an independent growth potential for the primary cartilages of the epiphyseal and costal growth plates with a maximal growth pressure of 9.5 and 7.8 g/mm2, respectively. The primary cartilage of the spheno-occipital synchondrosis, on the other hand, although it possesses an independent growth potential, could exert a maximum growth pressure of only 1.5 g/mm2. The secondary cartilage of the mandibular condyle showed a limited intrinsic growth potential, as well as a low maximal growth pressure (2.6 g/mm2). If calculated per dividing and/or matrix synthesizing cell (cells mainly responsible for the cartilage growth), the cells of the condylar cartilage showed the least growth potency (0.08 mg/cell in comparison to 1.9, 1.5 and 0.3 for epiphyseal, costal, and synchondroseal cartilages, respectively.     

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。     

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。     

生长期兔髁突软骨细胞体外培养方法及其生物学特性研究

目的 建立髁突软骨细胞体外培养模型,研究其生物学特性。 方法 分离4周龄健康新西兰大白兔双侧髁突软骨,采用胰酶和胶原酶联合消化方法收集4 h和12 h 2个时间点的髁突软骨细胞,连续体外培养并传代至P10。使用倒置显微镜观察细胞形态;采用甲苯胺蓝、阿利新蓝和免疫细胞化学染色对髁突软骨细胞进行鉴定;利用细胞计数绘制细胞生长曲线;通过Western 免疫印迹分析细胞Ⅱ型胶原、Ⅹ型胶原和SOX9在蛋白水平的表达情况。采用SPSS13.0软件包对Western 印迹条带灰度值进行统计学分析。 结果 兔髁突软骨细胞体积较大,呈纺锤形或多角形,铺路石样排列,细胞爬片染色结果为阳性。随着细胞传代“成纤维样”细胞比例逐渐加重,P2代之前的细胞形态差异较小。细胞生长曲线显示,兔髁突软骨细胞的体外培养符合经典的S形生长曲线。Western 印迹结果表明,4h和12 h所收髁突软骨细胞Ⅱ型胶原、Ⅹ型胶原和SOX9表达无显著差异。 结论 使用本方法培养兔髁突软骨细胞简单有效。髁突软骨细胞体外培养存在去分化现象,但P2代以内的髁突软骨细胞生物学特性相对稳定,适合用于后期实验研究,P3代以后的髁突软骨细胞逐渐丧失原有细胞的特性。     

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...     

The human spheno-occipital synchondrosis II. A histological and microradiographic study of its growth

The spheno-occipital synchondrosis, dorsum sellae and clivus were studied post mortem in tissue samples from 53 males and 25 females, aged 2 days to 24 years 11 months. The investigation aimed at ascertaining the growth and remodelling changes in these structures during the growth period. Light microscopic and microradiographic techniques were used. In the early ages the organization of the synchondrosis was similar to that of an epiphyseal disc, except that the morphology of the synchondrosial cartilage was bipolar. With increasing age the hyaline cartilage in the upper part of the synchondrosis was replaced by fibro-cartilage, while the central zone in the lower part underwent asbestos transformation. The synchondrosis was completely ossified by 16—17 years of age in girls, and about two years later in boys. Appositional growth and endochondral ossification continued up to 3 years of age in cartilage covering the superior and clival aspects of the dorsum sellae. The cartilage on the superior aspect of dorsum sellae persisted until after puberty and occasionally longer. Minor areas of resorption were seen i the posterior wall of the sella turcica. This resorption, together with the growth of the cartilage covering the dorsum sellae, may cause a shift of the cephalometric reference point sella in an upward direction during the growth period. The intracranial surface of the occipital bone showed resorption. On the extracranial surface there was simultaneous bone apposition.     

The Temporomandibular Joint: A Morphologic Study on A Human Autopsy Material

The spheno-occipital synchondrosis and clivus in 32 males and 21 females, aged 2 days to 24 years 11 months, were studied post mortem to ascertain the time of closure of the synchondrosis. The material studied consisted of the major part of the clivus and dorsum sellae, which were decalcified and serially sectioned in the sagittal plane. The first sign of closure of the synchondrosis was the appearance of a bony bridge in the superior part. Closure was found to occur about 2 years earlier in girls than in boys. The synchondrosis was never completely open in any of the females above 13 years 9 months, the corresponding age for the boys was 16 years. The major part of the dorsum sellae of preparations from young subjects consisted of cartilage and minor cartilaginous areas were seen in almost all preparations from subjects below 17 years and occasionally also from subjects above this age.     

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.     

Comparison of the effects of hydrostatic compressive force on glycosaminoglycan synthesis and proliferation in rabbit chondrocytes from mandibular condylar cartilage, nasal septum, and spheno-occipital synchondrosis in vitro

We have developed a simple in vitro model whereby precise quantities of compressive force can be applied to cultured chondrocytes from craniofacial cartilage: mandibular condylar cartilage (MCC), nasal septal cartilage (NSC), and spheno-occipital synchondrosis (SOS). Using this model, we found that hydrostatic compressive force stimulated glycosaminoglycan (GAG) synthesis, a cartilage phenotype, in MCC and SOS chondrocytes and DNA synthesis in MCC, NSC, and SOS chondrocytes. These stimulations were dependent on force magnitude and duration, reaching maximal GAG synthesis at 27 hours and maximal DNA synthesis at 20 hours after application of force. The maximal increase of GAG synthesis induced by compressive force was about 60% at 100 gm/cm2 for 5 minutes in nonstimulated MCC chondrocytes and 40% at 50 gm/cm2 for 1 minute in nonstimulated SOS chondrocytes. The maximal increase in DNA synthesis, produced by a compressive force of 50 gm/cm2 for 1 minute, was 50% in NSC chondrocytes, 50% in SOS chondrocytes, and 30% in MCC chondrocytes. There was no stimulation of GAG synthesis in NSC chondrocytes. These observations suggest that extrinsic force regulates craniofacial growth by controlling the differentiation and proliferation of chondrocytes in the craniofacial skeleton and that the difference in their responses to compressive force may reflect differences in the characteristics of these cells and their physiologic function in vivo.     

The Effect of α‐Tocopherol and Selenium on Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts In Vitro

Background: The aim of the present study is to evaluate the effect of α‐tocopherol and selenium on gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) in terms of proliferation, basic fibroblast growth factor (bFGF) release, collagen type I synthesis, and wound healing. Methods: Primary cultures of human GFs and PDLFs were isolated. Four test groups and a control group free of medication was formed. In group E, 60 μM α‐tocopherol was used, and in groups ES1, ES2, and ES3, the combination of 60 μM α‐tocopherol with 5 × 10^?9 M, 10 × 10^?9 M, and 50 × 10^?9 M selenium was used, respectively. Viability, proliferation, bFGF, and collagen type I synthesis from both cell types were evaluated at 24, 48, and 72 hours, and healing was compared on a new wound‐healing model at 12, 24, 36, 48, and 72 hours. Results: α‐Tocopherol alone significantly increased the healing rate of PDLFs at 12 hours and increased bFGF and collagen type I release from GFs and PDLFs at 24, 48, and 72 hours. The α‐tocopherol/selenium combination significantly enhanced the proliferation rate of both cells at 48 hours, decreased the proliferation of PDLFs at 72 hours, and increased the healing rate of GFs at 12 hours and PDLFs at 12 and 48 hours. bFGF and collagen type I synthesis was also increased in both cell types at 24, 48, and 72 hours by α‐tocopherol/selenium combination. Conclusion: α‐Tocopherol and α‐tocopherol/selenium combination is able to accelerate the proliferation rate and wound‐healing process and increase the synthesis of bFGF and collagen type I from both GFs and PDLFs.     

生长期兔髁突纤维软骨细胞和骨骺透明软骨细胞生物学特性的比较研究

目的 建立软骨细胞体外培养模型,研究纤维软骨和透明软骨的生物学差异。 方法 分离4周龄雌性兔双侧髁突软骨和膝关节骨骺软骨,采用胰酶和胶原酶联合消化的方法收集软骨细胞,分别进行体外培养并连续传代至P10。使用倒置显微镜观察细胞形态;采用阿利新蓝和Ⅱ型胶原免疫组化染色的方法分别对髁突纤维软骨细胞和骨骺透明软骨细胞进行鉴定;用细胞计数的方法绘制生长曲线;运用荧光定量PCR和Western印迹技术对软骨特异性指标Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan进行检测,并使用SPSS 13.0软件包对数据进行统计学分析。 结果 倒置显微镜观察示,髁突纤维软骨细胞和骨骺透明软骨细胞形态均会随细胞传代而发生改变,且P2代以后改变明显;基因和蛋白水平的检测均证实P2代后软骨细胞去分化明显。阿利新蓝和Ⅱ型胶原免疫组化染色鉴定结果均为阳性,对照组为阴性。实时定量荧光PCR和Western印迹结果证明：在纤维软骨中,Ⅰ型胶原的表达量高于透明软骨;而Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达量却明显低于透明软骨。 结论 使用本方法培养软骨细胞简单有效;髁突纤维软骨细胞和骨骺透明软骨细胞体外培养均存在去分化现象,且两者Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达存在显著差异,生物学特性并不相同。     

Functional appliance therapy accelerates and enhances condylar growth.

The present study was designed to quantitatively assess the temporal pattern of expression of Sox 9, the regulator of chondrocyte differentiation and type II collagen, the major component of the cartilage matrix during forward mandibular positioning, and compare it with the expression during natural growth. Female Sprague-Dawley rats, 5 weeks old, were used. Results showed that the expression of Sox 9 and type II collagen are accelerated and enhanced when the mandible is positioned forward. Furthermore, we monitored the amount of new bone formation during mandibular advancement and after the removal of bite-jumping appliances. A substantial increase was observed in the amount of newly formed bone when the mandible was positioned forward. No significant difference in new bone formation could be found after the appliance was removed when compared with natural growth. Thus, functional appliance therapy accelerates and enhances condylar growth by accelerating the differentiation of mesenchymal cells into chondrocytes, leading to an earlier formation and increase in amount of cartilage matrix. This enhancement of growth did not result in a subsequent pattern of subnormal growth for most of the growth period; this indicates that functional appliance therapy can truly enhance condylar growth.     

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.     

The effect of rhombencephalic hypoplasia on posterior cranial base elongation in rodents

A single intraperitoneal injection of methylazoxymethanol acetate in circumnatal rats produces a dose dependent cerebellar hypoplasia. All other tissues, including skeletal, are unaffected. Consequent decrease, in the ventral length attained by the growing rhombencephalon is accompanied by a corresponding decrease in the length of the basioccipital bone relative to that of the normally dimensioned basisphenoid bone. The width of the sphenooccipital synchondrosis is also proportionally decreased, with characteristic alterations in the structure of the several zones of this synchondrosis as compared to controls.Three autosomal recessive mutant mice strains were also studied in which the defect is manifested solely as decreased cerebellar neuronal neogenesis. Again, proportional linear decreases occurred in the ventral length of the rhombencephalon, the basioccipital bone and in width of spheno-occipital synchondrosis. The histological changes observed between normal and mutant mice were related to these differences. These data suggest that posterior cranial base elongation and the processes of endochondral ossification in the spheno-occipital synchondrosis of rodents are significantly regulated by the demands of functionally related growing neural masses.     

生长因子对人髁突软骨细胞Ⅰ、Ⅱ、Ⅲ型前胶原基因表达的调控研究

目的：研究低血清培养条件下转化生长因子－β（ＴＧＦ－β）,胰岛素生长因子－Ⅰ（ＩＧＦ－Ⅰ）,碱性成纤维生长因子（ｂＦＧＦ）对人髁突软骨细胞Ⅰ,Ⅱ,Ⅲ型前胶原基因表达的调控作用。方法：采用体外细胞培养及ｍＲＮＡ狭缝杂交的方法。结果：ＩＧＦ－Ⅰ对３种前胶原的ｍＲＮＡ水平无显著影响。ｂＧＦＧ,ＴＧＦ－β均呈不同程度地抑制Ⅱ型胶原的基因表达,分别为对照组的０．３５２及０．６５８倍同时ＴＧＦ－β显著增加了Ⅰ