The migration and distribution of somite cells after labelling with the carbocyanine dye, Dil: the relationship of this distribution to segmentation in the vertebrate body

Summary The cells of individual somites in 2-day-old chick embryos were marked by injecting a fluorescent dye into the somitocoele. This procedure permanently marked the cells and allowed their subsequent development and distribution to be followed. The cells were found to remain in close association with each other within limited boundaries and did not mix to any great extent with similar cells from adjacent somites. Fluorescent cells from single somites were found in the intervertebral disc, connective tissue surrounding two adjacent neural arches, all the tissues between the neural arches, the dermatome, and the associated myotome. No fluorescent cells were found in the notochord or in any nervous tissue apart from accompanying connective tissue. Surprisingly, the vertebral bodies and neural arches did not contain any fluorescent cells apart from those in the connective tissue surrounding them, but this absence of fluorescent cells was thought to be due to the dilution of the fluorescence following cell proliferation. These results provide further experimental support for the theory of resegmentation in vertebral formation, and also provide evidence of a compartmental method of development along the rostrocaudal axis in vertebrates, similar to that already discovered in insects. On the basis of cell lineage criteria, the sclerotome might be considered as a developmental compartment.     

Developmental Failure of Segmentation in a Caudal Vertebra of Apatosaurus (Sauropoda)

A vertebral element assigned to an Apatosaurus cf. ajax from the Late Jurassic Morrison Formation is described. The specimen exhibits an unusual morphology where two vertebrae are nearly seamlessly fused together, including the haemal arch that spans them. This morphology is thought be the result of a developmental abnormality. CT scans of the specimen reveal a thin zone of dorsoventral thickening between the two neural arches consistent with cortical bone. Contrast in internal morphology differentiates the anterior and posterior vertebral bodies with the anterior expressing greater porosity, which increased accommodation for barite‐rich calcite precipitation. No vacuities are observed to suggest the former presence of an intervertebral disk or intervertebral joints: the absence of an intervertebral disc or intervertebral joints is indicative of a condition known as block vertebra. Block vertebrae occur with the loss, or inhibition, of somitocoele mesenchyme early in embyogenesis (i.e., during resegmentation of the somites responsible for the formation of the affected vertebra). The derivatives of somitocoele mesenchyme include the intervertebral disc and joints. Although vertebral paleopathologies are not uncommon in the fossil record, this specimen is the first recognized congenital malformation within Sauropoda. Anat Rec, 297:1262–1269, 2014. © 2014 Wiley Periodicals, Inc.     

Increases in the number of cells in different areas of epithelial somites related to changes in morphology and development

There are two distinct groups of cells in the epithelial somite: cells in the epithelial ball that form the periphery, and loose mesenchymal cells found in the central cavity (somitocoele). Recent work has produced evidence to show that these two groups of cells have significant differences (morphology, origin, fibronectin content, reaction to peanut lectin, communication properties) but the significance of these differences has yet to be established. It is not yet clear whether the epithelial somite stage of development is merely a time for cell proliferation, or whether it is a time when significant differences develop which have consequences in subsequent morphogenesis. Certainly, there are indications that the two groups of cells might form different structures related to the vertebral column based on their position in the subsequent sclerotome. In this study, we have examined the number of cells that are present in both the epithelial ball and the somitocoele at various stages of maturity. The results show that later-formed somites contain significantly more cells in both the epithelial ball and the somitocoele. Furthermore, while the density of cells in the epithelial ball remains constant (accounting for an increase in dimensions of the somite), there is a significant increase in density of cells in the somitocoele. This suggests that there is an important distinction being created between the cells of the epithelial ball and those in the somitocoele. The results also illustrate that somite development is not the same at all segmental levels and that development of each might need to be considered on an individual basis, especially as the later-formed somites are known not to remain in this stage of development for as long as the earlier-formed somites.     

Kinetics and differentiation of somite cells forming the vertebral column: studies on human and chick embryos

We have studied the kinetics of somite cells with an antibody against proliferating cell nuclear antigen (PCNA/cyclin) in human and chick embryos, and with the BrdU anti-BrdU method in chick embryos, to investigate whether the metameric pattern of the developing vertebral column can be explained by different proliferation rates. Furthermore we applied antibodies against differentiation markers of chondrogenic and myogenic cells of the somites in order to study the correlation between proliferation and differentiation. There are no principal differences in the proliferation pattern of the vertebral column between human and chick embryos. In all stages examined, the cell density is higher in the caudal sclerotome halves than in the cranial halves. Laterally, the caudal sclerotome halves, which give rise to the neural arches, are characterized by a higher proliferative activity than the cranial halves. Although there is a high variability, the labelling indices show significant differences between the two halves with both proliferation markers. With the onset of chondrogenic differentiation, only the perichondrial cells retain a high proliferation rate. During fetal development, the neural arches and their processes grow appositionally. Even at the earliest stages, there is practically no immunostaining for PCNA or BrdU in the desmin-positive myotome cells of human and chick embryos. Axially, a higher proliferation rate is found in the condensed mesenchyme of the anlagen of the intervertebral discs than in the anlagen of the vertebral bodies. During fetal development, cells at the borders between vertebral bodies and intervertebral discs proliferate, indicating appositional growth. Our results show that local differences in the proliferation rates of the paraxial mesoderm exist, and may be an important mechanism for the establishment of the metameric pattern of the vertebral column in human and chick embryos.     

The fate of somitocoele cells in avian embryos

The early somite of avian embryos is made up of an epithelial wall and mesenchymal cells located within the somitocoele. We have studied the fate of somitocoele cells for a period of up to 6 days, using the quailchick marker technique. We also applied the QH-1 antibody, which specifically stains hemangiopoietic cells of quail origin, and studied the proliferative activity of epithelial somites with the BrdU anti-BrdU method. Our results show that somitocoele cells mainly give rise to the ribs and peripheral parts of the intervertébral discs. After 1 and 2 days of reincubation, the grafted somitocoele cells were located in the lateral part of the sclerotome, and only a few cells migrated axially towards the notochord. In frontal sections, the cells were located in a triangular area within the cranial part of the caudal sclerotome half. After 3 days of reincubation, some of the cells had migrated cranially along the myotome. After longer reincubation periods, cells grafted into one somite could be found in two adjacent ribs. The studies with the QH-1 antibody show that a subpopulation of somitocoele cells has angiogenic potency. Endothelial cells originating from the mesenchyme of the somitocoele migrated actively and even invaded the ipsilateral half of the neural tube. In the epithelial wall of the somite, BrdU-labelled nuclei were found basally, whereas more apically the nuclei were not stained, but mitotic figures were frequently present. The somitocoele cells also showed a high proliferative activity with about 26% of nuclei labelled with BrdU.Supported by grants (Ch 44/9-2, Ch 44/12-1) from the Deutsche Forschungsgemeinschaft     

Arthritis and ankylosis in twy mice with hereditary multiple osteochondral lesions: With special reference to calcium deposition

A murine autosomal recessive mutant named twy (tiptoe walking-Yoshimura) mouse showing multiple osteochondral lesions including ankylosis of the vertebral column and limb joints underwent sequential histopathological analysis of posterior limb joint lesions and intervertebral disc lesions. In the articular cartilage, a decrease in alcian blue-positive extracellular matrix and the presence of degenerated collagen fibers were found at the age of around 4-8 weeks. Calcium deposits in the articular cartilage were found at that time and later in the articular space and synovial tissue. Calcium deposits were also found in the intervertebral discs at 4 weeks. Using electron microscopy, some of the crystals were seen inside small vesicles. In both joints, degeneration of, and calcium deposition in, the articular cartilage progressed with age, finally producing bony ankylosis. These histological observations suggest that calcification and degeneration of the articular cartilage are the major factors in the pathogenesis of joint disorders in the twy mouse, and this mutant mouse provides a good model for studying the process and mechanism of osteoarthritic lesions, destructive arthritis and ankylosis.     

Pax-1 in the development of the cervico-occipital transitional zone

The Pax-1 gene has been found to play an important role in the development of the vertebral column. The cervico-occipital transitional zone is a specialized region of the vertebral column, and malformations of this region have frequently been described in humans. The exact embryonic border between head and trunk is a matter of controversy. In order to determine a possible role of Pax-1 in the development of the cervico-occipital transitional zone we studied the expression of this gene in a series of quail embryos and murine fetuses with in situ hybridization and immunohistochemistry. Pax-1 is expressed in all somites of the embryo, including the first five occipital ones. During embryonic days 3–5 the gene is down-regulated in the caudal direction within the first five somites, whereas more caudally Pax-1 is strongly expressed in the cells of the perinotochordal tube. In 5-day-old quail embryos, the cartilaginous anlage of the basioccipital bone has developed and there is no more expression of Pax-1 in this region. The fusion of the dens axis with the body of the axis also coincides with switching off of the Pax-1 gene. More caudally, the gene is continuously expressed in the intervertebral discs of murine embryos and therefore seems to be important for the process of resegmentation. Quail embryos do not possess permanent intervertebral discs. “Hyper-” or “hyposegmentation” defects may be explained by an over- or under-expression of Pax-1 during development. We also reinvestigated the border between the head and trunk in chick embryos by performing homotopical grafting experiments of the 5^th somite between chick and quail embryos. Grafted quail cells formed mainly the caudal end of the basioccipital bone. They were also located in the cranial half of the ventral atlantic arch, and only a few cells were found in the tip of the dens axis.     

Closure of the vertebral canal in human embryos and fetuses

The vertebral column is the paradigm of the metameric architecture of the vertebrate body. Because the number of somites is a convenient parameter to stage early human embryos, we explored whether the closure of the vertebral canal could be used similarly for staging embryos between 7 and 10 weeks of development. Human embryos (5–10 weeks of development) were visualized using Amira 3D^® reconstruction and Cinema 4D^® remodelling software. Vertebral bodies were identifiable as loose mesenchymal structures between the dense mesenchymal intervertebral discs up to 6 weeks and then differentiated into cartilaginous structures in the 7th week. In this week, the dense mesenchymal neural processes also differentiated into cartilaginous structures. Transverse processes became identifiable at 6 weeks. The growth rate of all vertebral bodies was exponential and similar between 6 and 10 weeks, whereas the intervertebral discs hardly increased in size between 6 and 8 weeks and then followed vertebral growth between 8 and 10 weeks. The neural processes extended dorsolaterally (6th week), dorsally (7th week) and finally dorsomedially (8th and 9th weeks) to fuse at the midthoracic level at 9 weeks. From there, fusion extended cranially and caudally in the 10th week. Closure of the foramen magnum required the development of the supraoccipital bone as a craniomedial extension of the exoccipitals (neural processes of occipital vertebra 4), whereas a growth burst of sacral vertebra 1 delayed closure until 15 weeks. Both the cranial‐ and caudal‐most vertebral bodies fused to form the basioccipital (occipital vertebrae 1–4) and sacrum (sacral vertebrae 1–5). In the sacrum, fusion of its so‐called alar processes preceded that of the bodies by at least 6 weeks. In conclusion, the highly ordered and substantial changes in shape of the vertebral bodies leading to the formation of the vertebral canal make the development of the spine an excellent, continuous staging system for the (human) embryo between 6 and 10 weeks of development.     

From somites to vertebral column.

We report on the development and differentiation of the somites with respect to vertebral column formation in avian and human embryos. The somites, which are made up of different compartments, establish a segmental pattern which becomes transferred to adjacent structures such as the peripheral nervous system and the vascular system. Each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of vertebrae, whereas the vertebral body and the intervening disc develop from the axially-located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs. The neural arches form a frame for the neural tube and spinal ganglia. The boundary between head and vertebral column is located between the 5th and 6th somites. In the human embryo, proatlas, body of the atlas segment, and body of the axis fuse to form the axis.     

Pax-1 in the development of the cervico-occipital transitional zone

The Pax-1 gene has been found to play an important role in the development of the vertebral column. The cervico-occipital transitional zone is a specialized region of the vertebral column, and malformations of this region have frequently been described in humans. The exact embryonic border between head and trunk is a matter of controversy. In order to determine a possible role of Pax-1 in the development of the cervico-occipital transitional zone we studied the expression of this gene in a series of quail embryos and murine fetuses with in situ hybridization and immunohistochemistry. Pax-1 is expressed in all somites of the embryo, including the first five occipital ones. During embryonic days 3–5 the gene is down-regulated in the caudal direction within the first five somites, whereas more caudally Pax-1 is strongly expressed in the cells of the perinotochordal tube. In 5-day-old quail embryos, the cartilaginous anlage of the basioccipital bone has developed and there is no more expression of Pax-1 in this region. The fusion of the dens axis with the body of the axis also coincides with switching off of the Pax-1 gene. More caudally, the gene is continuously expressed in the intervertebral discs of murine embryos and therefore seems to be important for the process of resegmentation. Quail embryos do not possess permanent intervertebral discs. Hyper- or hyposegmentation defects may be explained by an over- or under-expression of Pax-1 during development. We also reinvestigated the border between the head and trunk in chick embryos by performing homotopical grafting experiments of the 5^th somite between chick and quail embryos. Grafted quail cells formed mainly the caudal end of the basioccipital bone. They were also located in the cranial half of the ventral atlantic arch, and only a few cells were found in the tip of the dens axis.     

Factors regulating viable cell density in the intervertebral disc: blood supply in relation to disc height

The intervertebral disc is an avascular tissue, maintained by a small population of cells that obtain nutrients mainly by diffusion from capillaries at the disc–vertebral body interface. Loss of this nutrient supply is thought to lead to disc degeneration, but how nutrient supply influences viable cell density is unclear. We investigated two factors that influence nutrient delivery to disc cells and hence cell viability: disc height and blood supply. We used bovine caudal discs as our model as these show a gradation in disc height. We found that although disc height varied twofold from the largest to the smallest disc studied, it had no significant effect on cell density, unlike the situation found in articular cartilage. The density of blood vessels supplying the discs was markedly greater for the largest disc than the smallest disc, as was the density of pores allowing capillary penetration through the bony endplate. Results indicate that changes in blood vessels in the vertebral bodies supplying the disc, as well as changes in endplate architecture appear to influence density of cells in intervertebral discs.     

Calcium pyrophosphate dihydrate (CPPD) deposition in ochronotic arthropathy

The post-mortem examination of an unsuspected case of alkaptonuria revealed extensive ochronosis. Histological examination of undecalcified sections of tracheal, costal, femoral and patellar cartilage revealed, in addition to ochronotic pigment, extensive calcium pyrophosphate dihydrate (CPPD) deposition. Similar deposits were present in intervertebral discs and were related to ossification of the discs resulting in partial or complete ankylosis. The calcific deposits were present around chondrocytes in the articular cartilage and this may be an important factor in the initiation of the osteoarthrotic process which characterises ochronotic arthropathy as it affects large diarthrodial joints.     

Communication compartments in the axial mesoderm of the chick embryo

Summary Intracellular microinjection of the fluorescent tracer Lucifer Yellow into mesoderm cells along the rostrocaudal axis of the early chick embryo has revealed compartments where the intercellular diffusion of dye, presumably via gap junctions, is restricted at the borders between groups of cells. Cells in the segmental plate were dye-coupled, as were cells forming the epithelial somites. However, dye-coupling was not observed between different somites, nor was it observed between the outer epithelial cells and the cells in the somitocoele. On dispersal of the somite, dermatome cells were dye-coupled. However, sclerotome cells were found to be divided into rostral and caudal compartments separated by a group of cells bordering the intrasclerotomal fissure (of von Ebner) that also exhibited dye-coupling, restricted primarily to cells along the fissure. Some of these compartment borders can be accounted for by the presence of a morphological barrier which reduces cell-cell contact, but others are more difficult to explain, as there appears to be extensive cell-cell contact across the border. This would be analogous to some compartments found in insects. Some of the compartments also have borders similar to those described by cell lineage studies. The results also indicate that dye-coupling becomes restricted in a spatial and temporal manner as the mesodermal cells mature.     

Morphological analysis of the role of the neural tube and notochord in the development of somites

The differentiation of avian somites and skeletal muscles, which themselves are derived from somites, was studied in ovo after the isolation of the unsegmented segmental plate from the notochord and/or neural tube by surgical operations at the level of the segmental plate. In each experiment, the newly formed somites had a normal histological structure, with an outer epithelial somite and core cells in the somitocoeles. Thereafter, the three derivatives of the somites (dermatome, myotome and sclerotome) reacted differently to the different operations. When the somites developed without the notochord, only somitocoele cells showed massive cell death, and muscles developed regardless of the presence or absence of the neural tube. On the contrary, no cell death was observed in any part of the somites that were formed with the neural tube or the notochord present, and muscle cells developed. However, in those embryos that retained only the notochord, striated muscles developed only in the lateral body wall. In each of the experimental operations, the surface ectoderm always covered the somites, and, regardless of the state of sclerotome and/or myotome differentiation, the dermatome always survived. These histological observations indicate that the first step in somite formation is independent of axial structures. The results further suggest that the notochord may produce diffusible factors that are necessary for the survival and further development of sclerotomal cells, and that both the neural tube and notochord can support muscle differentiation. However, it is likely that each structure has a relationship to the development of epaxial muscles and hypaxial muscles respectively. Furthermore, an intimate relationship may also exist between the surface ectoderm and the development of the dermatome.     

退变性脊柱侧弯的生物力学有限元分析

背景：脊柱在结构、形状、材料特性以及承受载荷方面都比较复杂,传统的生物力学方法不能完全解决这些特性问题。 目的：探讨退变性脊柱侧弯椎间盘、关节突关节、椎体等的应力分布,为其发生、发展的生物力学机制提供依据。 方法：基于退变性脊柱侧弯患者T₁₂-S₁上段连续的CT扫描图像,赋予模型特定的材料属性,建立完整、有效的退变性脊柱侧弯三维有限元模型。在前屈、后伸、左侧弯、右侧弯、左旋转、右旋转6种工况下对模型进行加载,计算和分析脊柱的活动度、椎间盘、椎体及关节突关节软骨的应力分布。 结果与结论：退变性脊柱侧弯有限元模型比正常腰椎的活动度要小,椎间盘应力分布趋向于椎间盘的四周,后伸运动时各椎间盘应力最大,侧弯顶点椎体容易出现应力集中的情况,在旋转工况下关节突软骨的应力集中最明显,后伸工况下次之,尤其以侧弯顶点节段的关节突软骨影响最大。退变性脊柱侧弯侧弯顶点容易出现应力集中,后伸、旋转运动可加重退变性脊柱侧弯发展。     

The effects of somite removal on vertebral formation in the chick

We have examined the effects on vertebral development of various combinations of somite removal in two day old chick embryos as shown by vertebral formation after a further seven days of incubation. Each combination produced one of a variety of results ranging from completely normal vertebral formation, through fusion of various vertebral elements, to the absence of complete vertebral halves and the formation of hemivertebrae. Assessment of our operating ability showed that we were removing at least 90% of the somitic material and therefore these results suggest that there is a regulating mechanism available to the embryo, at least with regards to vertebral development. When two consecutive somites were removed, vertebrae frequently developed that were lacking certain elements. This suggests that the somitic cells are already determined with regards to formation of specific vertebral elements. Experiments involving the removal of a bilateral pair of somites (a repetitive unit) also provided evidence of a counting mechanism which ensures that the correct number of total vertebrae are present.     

椎体后凸成形椎间盘骨水泥渗漏时行相邻椎体预防性强化的有限元分析◆

背景：椎体后凸成形术后相邻椎体发生骨折时有报道,很多学者分析椎间盘骨水泥渗漏是相邻椎体继发骨折的重要原因之一。 目的：以有限元方法分析椎体后凸成形并发椎间盘骨水泥渗漏时其相邻椎体生物力学的变化,进而分析相邻椎体继发骨折的原因并寻求补救措施。 方法：利用MIMICS、ABAQUS等软件重建胸腰段(T11~L2)三维有限元模型,模拟L1椎体骨质疏松性压缩性骨折及椎体后凸成形治疗,观察有无T12~L1椎间盘骨水泥渗漏对骨折相邻椎体的生物力学影响;进一步分别模拟以2,3,4 mL骨水泥对T12椎体行预防性强化,观察不同载荷下不同模型整体及各部分的Von Mises应力。 结果与结论：成功建立了相关三维有限元模型。相邻椎体T12及其下终板的最大应力在T12~L1椎间盘有骨水泥渗漏组比无渗漏组的明显增加;T12椎体不同剂量骨水泥预防性强化后最大应力不同程度降低,且小剂量(2~4 mL)骨水泥预防性强化并不明显影响其余椎体、椎间盘的生物力学行为。提示椎体后凸成形并发椎间盘骨水泥渗漏可能会导致相邻椎体继发骨折,同时行相邻椎体预防性强化可能会减少继发骨折的发生。      

Finite element modeling of the cervical spine: role of intervertebral disc under axial and eccentric loads

An anatomically accurate, three-dimensional, nonlinear finite element model of the human cervical spine was developed using computed tomography images and cryomicrotome sections. The detailed model included the cortical bone, cancellous core, endplate, lamina, pedicle, transverse processes and spinous processes of the vertebrae; the annulus fibrosus and nucleus pulposus of the intervertebral discs; the uncovertebral joints; the articular cartilage, the synovial fluid and synovial membrane of the facet joints; and the anterior and posterior longitudinal ligaments, interspinous ligaments, capsular ligaments and ligamentum flavum. The finite element model was validated with experimental results: force–displacement and localized strain responses of the vertebral body and lateral masses under pure compression, and varying eccentric anterior-compression and posterior-compression loading modes. This experimentally validated finite element model was used to study the biomechanics of the cervical spine intervertebral disc by quantifying the internal axial and shear forces resisted by the ventral, middle, and dorsal regions of the disc under the above axial and eccentric loading modes. Results indicated that higher axial forces (compared to shear forces) were transmitted through different regions of the disc under all loading modes. While the ventral region of the disc resisted higher variations in axial force, the dorsal region transmitted higher shear forces under all loading modes. These findings may offer an insight to better understand the biomechanical role of the human cervical spine intervertebral disc.