大鼠生长发育过程中颅底软骨联合细胞增殖和凋亡的研究

目的研究大鼠生长发育过程中颅底细胞的增殖和凋亡现象,探讨颅底生长发育的变化规律。方法选用出生后4d、8d、16d、32d、48d、64d及128d的SD大鼠,取颅底软骨联合组织,行增殖细胞核抗原(PCNA)免疫组织化学染色标记增殖细胞,末端脱核苷酸转移酶介导的缺口末端标记技术(TUNEL法)标记凋亡细胞,比较观察不同时间段大鼠颅底的细胞增殖和凋亡的表达情况。结果在出生后4d、8d及16d的大鼠可见大量的PCNA阳性细胞存在于软骨联合区,而32d、48d、64d及128d大鼠的软骨联合区仅有少量的PCNA阳性细胞,而在软骨联合区两侧的骨小梁之间的PCNA阳性细胞较多。在出生后4d、8d的大鼠软骨联合的肥大区,交界区发现少数细胞凋亡现象;而在出生后16d、32d、48d及64d的大鼠颅底软骨联合组织的肥大区、交界区和两侧的骨小梁之间发现较多散在的黄色荧光凋亡细胞,并随时间推移凋亡细胞逐渐增多;但在128d的大鼠又几乎未见凋亡细胞。结论在大鼠颅底生长发育过程中,细胞增殖和凋亡存在特定的时空变化规律。     

Sphenoidal-clival neoformation: an endoscopic approach

The cranial base has distinct embryologic origins. The anterior cranial base is derived solely from the neural crest, similar to other facial bones, whereas the posterior cranial base is formed by the paraxial mesoderm. Both these parts also develop and grow with distinct features. Unlike other craniofacial bones that are mostly formed through intramembraneous ossification, the cranial base is formed through endochondral ossification, in which a cartilage plate, known as the chondrocranium, is formed first and soon replaced by bones. Individual bones are then connected by cartilaginous structures, termed synchondroses, which are morphologically similar to long-bone growth plates.These processes justify the presence of a disembryogenic cyst in the sphenoid bone. The authors present a case of a clival-sphenoidal region neoformation treated with a transnasal-endoscopic approach.     

甲状旁腺相关蛋白及其受体PTH1R及Ihh信号在鼠胚髁突软骨中的分布特征

目的探讨甲状旁腺相关蛋白(PTHrP)及其受体PTH1R及Ihh信号在髁突软骨发育过程中的分布特征及意义。方法取E14~18 d鼠胚,以免疫组化及原位杂交染色方法,检测PTH1R抗体及PTHrPI、hh mRNA在下颌髁突软骨发生过程中的分布特征。结果在鼠胚下颌髁突软骨发育过程中,PTHrP、PTH1R主要表达于软骨膜间质细胞、软骨细胞及大部分肥大软骨细胞中,Ihh主要位于肥大软骨细胞上层及其与扁平细胞层交界处,PTHrP及Ihh信号可能通过自分泌和(或)旁分泌作用调控髁突软骨细胞的增殖和分化。结论鼠胚下颌髁突软骨发育过程中,PTHrP、PTH1RI、hh的分布特征与其在生长板分布不同,提示PTHrP可能对髁突全层软骨细胞均具有增殖促进作用。     

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)     

TMJ development and growth require primary cilia function

Primary cilia regulate limb and axial skeletal formation and hedgehog signaling, but their roles in temporomandibular joint (TMJ) development are unknown. Thus, we created conditional mouse mutants deficient in ciliary transport protein Kif3a in cartilage. In post-natal wild-type mice, primary cilia were occasionally observed on the superior, inferior, or lateral side of condylar cells. Cilia were barely detectable in mutant chondrocytes but were evident in surrounding tissues, attesting to the specificity of chondrocyte Kif3a ablation. Mutant condyles from 3-month-old mice were narrow and flat along their antero-posterior and medio-lateral axes, were often fused with the articular disc, and displayed an irregular bony surface. The polymorphic layer in P15 mutants contained fewer Sox9-expressing chondroprogenitor cells because of reduced mitotic activity, and newly differentiated chondrocytes underwent precocious hypertrophic enlargement accompanied by early activation of Indian hedgehog (Ihh). Interestingly, there was excessive intramembranous ossification along the perichondrium, accompanied by local expression of the hedgehog receptor Patched-1 and up-regulation of Osterix and Collagen I. In summary, Kif3a and primary cilia are required for coordination of chondrocyte maturation, intramembranous bone formation, and chondrogenic condylar growth. Defects in these processes in Kif3a condylar cartilage are likely to reflect abnormal hedgehog signaling topography and dysfunction.     

Ihh signaling regulates mandibular symphysis development and growth

Symphyseal secondary cartilage is important for mandibular development, but the molecular mechanisms underlying its formation remain largely unknown. Here we asked whether Indian hedgehog (Ihh) regulates symphyseal cartilage development and growth. By embryonic days 16.5 to 18.5, Sox9-expressing chondrocytes formed within condensed Tgfβ-1/Runx2-expressing mesenchymal cells at the prospective symphyseal joint site, and established a growth-plate-like structure with distinct Ihh, collagen X, and osteopontin expression patterns. In post-natal life, mesenchymal cells expressing the Ihh receptor Patched1 were present anterior to the Ihh-expressing secondary cartilage, proliferated, differentiated into chondrocytes, and contributed to anterior growth of alveolar bone. In Ihh-null mice, however, symphyseal development was defective, mainly because of enhanced chondrocyte maturation and reduced proliferation of chondroprogenitor cells. Proliferation was partially restored in dual Ihh;Gli3 mutants, suggesting that Gli3 is normally a negative regulator of symphyseal development. Thus, Ihh signaling is essential for symphyseal cartilage development and anterior mandibular growth.     

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.     

The cranial base in craniofacial development: a gene therapy study

The etiology of midface retrusion remains largely unclear. We hypothesized that the cranial base synchondroses play a key role in the development of the craniofacial skeleton in the Sandhoff mouse model. We observed that developmental abnormalities of the cranial base synchondroses involving proliferative chondrocytes are important in craniofacial growth and development. Neonatal restitution of beta-hexosaminidase in mutant mice by gene therapy successfully ameliorated the attendant skeletal defects and restored craniofacial morphology in vivo, suggesting this as a critical temporal window in craniofacial development. Analysis of our data implicates parathyroid-related peptide (PTHrP) and cyclo-oxygenase-2 (COX-2) as possible factors underlying the development of the aforementioned skeletal defects. Hence, timely restitution of a genetic deficiency or, alternatively, the restoration of PTHrP or cyclo-oxygenase activity by the administration of PTH and/or non-steroidal anti-inflammatory drugs or COX-2 selective inhibitors to affected individuals may prove beneficial in the management of midface retrusion.     

Effect of excessive methionine on the development of the cranial growth plate in newborn rats

ObjectiveMethionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats.DesignTwenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis.ResultsThe weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group.ConclusionsIn summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia.     

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.     

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.     

Cranial base in craniofacial development: developmental features, influence on facial growth, anomaly, and molecular basis

The cranial base is of crucial importance in integrated craniofacial development. As distinct from facial bones, it is formed through endochondral ossification. The posterior and anterior cranial bases are derived from distinct embryologic origins and grow independently--the anterior cranial base solely from the neural crest, the posterior cranial base from the paraxial mesoderm. The anterior cranial base has more prolonged and active growth and exerts more influence on facial growth than does the posterior cranial base. Cranial base angulation is a unique feature in modern human beings. Cranial base anomalies have been identified in many genetic and developmental disorders. The molecular basis of cranial base development and growth is being clarified. In this review, these aspects of cranial base are discussed in detail, with a focus on developmental features, roles in craniofacial growth, anomalies, and the genetic basis of development.     

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.     

Cranial base in craniofacial development: Developmental features,influence on facial growth,anomaly, and molecular basis

The cranial base is of crucial importance in integrated craniofacial development. As distinct from facial bones, it is formed through endochondral ossification. The posterior and anterior cranial bases are derived from distinct embryologic origins and grow independently — the anterior cranial base solely from the neural crest, the posterior cranial base from the paraxial mesoderm. The anterior cranial base has more prolonged and active growth and exerts more influence on facial growth than does the posterior cranial base. Cranial base angulation is a unique feature in modern human beings. Cranial base anomalies have been identified in many genetic and developmental disorders. The molecular basis of cranial base development and growth is being clarified. In this review, these aspects of cranial base are discussed in detail, with a focus on developmental features, roles in craniofacial growth, anomalies, and the genetic basis of development.     

Wnt/beta-catenin inhibits dental pulp stem cell differentiation

Dental pulp stem cells (DPSCs) are a unique precursor population isolated from postnatal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through beta-catenin. In this study, the regulation of odontoblast-like differentiation of DPSCs by canonical Wnt signaling was examined. DPSCs were stably transduced with canonical Wnt-1 or the active form of beta-catenin, with retrovirus-mediated infection. Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin, and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules in DPSCs. Moreover, over-expression of beta-catenin was also sufficient to suppress the differentiation and mineralization of DPSCs. In conclusion, our results suggest that canonical Wnt signaling negatively regulates the odontoblast-like differentiation of DPSCs.     

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。     

Bone development and function: genetic and environmental mechanisms

Bone manipulation is fundamental to orthodontics and dentofacial orthopedics. This clinically oriented overview of bone biology is related to the fundamental genetic and environmental mechanisms of bone development. The genome codes for growth factors, ischemic agents, vascular induction/invasion mechanisms, and mechanically induced inflammation. These biological mechanisms interact with the physical factors of diffusion limitation and mechanical loading to produce bone morphology. The patterning of facial ossification centers, the nasal cartilage, and the secondary facial cartilages is genetically determined. Growth against pressure gradients is accomplished by secondary cartilages: condylar cartilage, angular synchondrosis, symphyseal synchondrosis, nasal septum, and posterior palatal synchondrosis. Once a functional occlusion is established (∼12–18 months in humans) the symphyseal synchondrosis fuses, and the palatal synchondrosis reverts to a midpalatal suture. Thereafter, facial growth is dictated by oropharyngeal function and manifest at secondary growth sites: mandibular condyle, facial sutures, and subperiosteal bone modeling. The basic genetic mechanisms for bone adaptation are vascular invasion, inflammatory response, and localized cellular control agents (cytokines, growth factors). Approximately 90 genes are known to be involved in craniofacial development.     

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。