Ontogeny of the Shell Bones of Embryos of Podocnemis unifilis (Troschel, 1848) (Testudines,Podocnemididae)

The aim of the present study was to investigate the sequence of shell bone formation in the embryos of the Pleurodira, Podocnemis unifilis. Their bones and cartilage were collected and cleared before staining. The shell was also examined by obtaining a series of histological slices. All the bony elements of the plastron have independent ossification centers, which subsequently join together and retain two fontanelles until the period of hatching. This turtle has a mesoplastra, which is characteristic of the Podocnemididae. The carapace begins to form concurrently with the ossification of the ribs at the beginning of stage 20. All the plates, except the suprapygal, initiate ossification during the embryonic period. The main purpose of the histological investigation was to highlight the relationship between the formation of the carapace and ribs with that of the neural plates. The costal and neural plates were found not to independent ossification centers, but to be closely related to components of the endoskeleton, originating as expansions of the perichondral collar of the ribs and the neural arches, respectively. Considering the ribs as an endoskeletal element of the carapace, the carapace and plastron begin ossification at the same stage in P. unifilis. This pattern reveals similarities with other Pleurodira, as well as evident variations, such as the presence of the seven neural bones and the presence of only one ossification center in the nuchal plate. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

The pisiform growth plate is lost in humans and supports a role for Hox in growth plate formation

The human pisiform is a small, nodular, although functionally significant, bone of the wrist. In most other mammals, including apes and Australopithecus afarensis, pisiforms are elongate. An underappreciated fact is that the typical mammalian pisiform forms from two ossification centers. We hypothesize that: (i) the presence of a secondary ossification center in mammalian pisiforms indicates the existence of a growth plate; and (ii) human pisiform reduction results from growth plate loss. To address these hypotheses, we surveyed African ape pisiform ossification and confirmed the presence of a late-forming secondary ossification center in chimpanzees and gorillas. Identification of the initial ossification center occurs substantially earlier in apes relative to humans, raising questions concerning the homology of the human pisiform and the two mammalian ossification centers. Second, we conducted histological and immunohistochemical analyses of pisiform ossification in mice. We confirm the presence of two ossification centers separated by organized columnar and hypertrophic chondrocyte zones. Flattened chondrocytes were highly mitotic, indicating the presence of a growth plate. Hox genes have been proposed to play a fundamental role in growth plate patterning. The existence of a pisiform growth plate presents an interesting test case for the association between Hox expression and growth plate formation, and could explain the severe effects on the pisiform observed in Hoxa11 and Hoxd11 knockout mice. Consistent with this hypothesis, we show that Hoxd11 is expressed adjacent to the pisiform in late-stage embryonic mouse limbs supporting a role for Hox genes in growth plate specification. This raises questions concerning the mechanisms regulating Hox expression in the developing carpus.     

Multiple growth cartilages in the neural arch

The origin of growth cartilages in the neural arch of human fetal vertebrae was investigated in an attempt to resolve the conflicting opinion on ossification in this region. Serial sections of developing vertebrae were examined, and three growth cartilages for each half of the neural arch were found. They have their origin from a single center of ossification. Endochondral bone formation similar to ossification in the diaphysis of a long bone occurs in the neural arch. This information may help in the understanding of how vertebral malformations such as scoliosis and spondylolysis occur during postnatal life.     

The cast of clasts: catabolism and vascular invasion during bone growth,repair, and disease by osteoclasts,chondroclasts, and septoclasts

Three named cell types degrade and remove skeletal tissues during growth, repair, or disease: osteoclasts, chondroclasts, and septoclasts. A fourth type, unnamed and less understood, removes nonmineralized cartilage during development of secondary ossification centers. “Osteoclasts,” best known and studied, are polykaryons formed by fusion of monocyte precursors under the influence of colony stimulating factor 1 (CSF)-1 (M-CSF) and RANKL. They resorb bone during growth, remodeling, repair, and disease. “Chondroclasts,” originally described as highly similar in cytological detail to osteoclasts, reside on and degrade mineralized cartilage. They may be identical to osteoclasts since to date there are no distinguishing markers for them. Because osteoclasts also consume cartilage cores along with bone during growth, the term “chondroclast” might best be reserved for cells attached only to cartilage. “Septoclasts” are less studied and appreciated. They are mononuclear perivascular cells rich in cathepsin B. They extend a cytoplasmic projection with a ruffled membrane and degrade the last transverse septum of hypertrophic cartilage in the growth plate, permitting capillaries to bud into it. To do this, antiangiogenic signals in cartilage must give way to vascular trophic factors, mainly vascular endothelial growth factor (VEGF). The final cell type excavates cartilage canals for vascular invasion of articular cartilage during development of secondary ossification centers. The “clasts” are considered in the context of fracture repair and diseases such as arthritis and tumor metastasis. Many observations support an essential role for hypertrophic chondrocytes in recruiting septoclasts and osteoclasts/chondroclasts by supplying VEGF and RANKL. The intimate relationship between blood vessels and skeletal turnover and repair is also examined.     

Prenatal and postnatal development of the cervical portion of the spine in the short-finned pilot whale Globicephala macrorhyncha

Fusion of the cervical spine in Globicephala macrorhyncha is a prenatal rather than postnatal phenomenon which encompasses all cervical vertebra. This results in a relatively short, nonarticulated, composite cervical spine in this particular species. Cervicothoracic spine segments removed from fetuses demonstrated complete fusion of all cervical vertebra commencing during early prenatal development. C1 and C2 initially developed as a composite central cartilaginous unit, although laterally there was some separation through rudimentary interzone formation. However, C3 through C7 formed individual cartilaginous centra which were divided from each other by thin, well-demarcated interzones, but without the formation of intervertebral discs (which were concomitantly evident dividing the thoracic, lumbar, and caudal vertebra, and were also present between the seventh cervical and first thoracic vertebra, although this was a very rudimentary intervertebral region). The first primary ossification center appeared in C2. Subsequently, primary ossification occurred in C7, and finally in C2 through C6, with ossification progressing in a craniocaudal fashion in these four vertebra. The centra ossification centers then progressively coalesced in the midline, from C2 to C7, in a craniocaudal sequence. This entire chondroosseous fusion process was completed during early gestation (probably less than 2 to 3 months of prenatal development), so that a composite “single” cervical vertebra developed that characterizes this species at birth and throughout postnatal development. Postnatally, ossification spreads laterally within each centrum, and also progressively removes the vestiges of the intervertebral material. C7 also develops a secondary ossification center, but only in the caudal region. The cranial end of C7 and the remainder of the cervical vertebra do not form secondary centers. An extensive fibrocartilaginous/hyaline cartilage bridge remains between C1 and C2, even after closure of the vertebral physes. Undoubtedly, this allows continued growth in C1 and C2, which become the dominant portion of the cervical unitary vertebra. Eventually, even this synchondrosis will disappear to form a completely osseous cervical mass.     

Maxillary ossification in a series of six human embryos and fetuses aged from 9 to 12 weeks of amenorrhea: clinical implications

An anatomic controversy exists concerning the number of nuclei of ossification of the maxilla in human embryos and fetuses. Many authors have described two maxillary ossification nuclei, an incisor nucleus and a maxillary nucleus properly so-called. Some of these have explained congenital labiomaxillary clefts by a defect of bony fusion between these two ossification nuclei. Others consider that there is only a single nucleus for maxillary ossification. In order to settle this question we performed a histologic study in six heads of embryos and fetuses aged from 9 to 12 weeks of amenorrhea (WA) to demonstrate the presence of one or more sites of maxillary ossification at the earliest stages. Our study revealed, on each side, a single zone of maxillary ossification, present from 9 WA, in the form of a sheet of osteoid tissue situated in close contact with the dental lamina. Maxillary ossification then progressed in the latero-medial direction, preserving a median transverse and ventral mesenchymal septum. This septum may constitute the phylogenetic remnant of an ancestral premaxilla.     

Development of the microcirculation of the secondary ossification center in rat humeral head

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed.     

Deviations in the sequence of appearance of primary centers of ossification in the feet of human fetuses with cleft lip and/or palate

The feet of 35 human fetuses with cleft lip and/or palate and 208 human fetuses without cleft Lip or palate were cleared and stained, and their conformity to the “normal” sequence of appearance of the primary centers of ossification in the 19 post-tarsal bones was noted. An arbitrary definition of a significant deviation from the normal sequence was established and a system of evaluating the relative severity of deviations was set up. The relative number of cleft fetuses showing deviations from the accepted sequence (42.9% ) was significantly higher than of non-cleft fetuses (10.6%). The cleft and non-cleft deviant fetuses were not significantly different in relative severity. There was no difference between the two series in the relative frequency with which specific bones were affected. In each series the fifth distal phalanx was most frequently affected. In both series, deviations in the order of initial ossification more frequently involved the seventh to tenth phalanges in the sequence. Asymmetry was of common occurrence in deviants of both the cleft and non-cleft series. There was no significant difference in the number of deviant centers per fetus in the cleft and non-cleft series. The biological mechanisms underlying the orderly and regular sequence of appearance of ossification centers in the foot are not known, hence, the nature of the interference with the “normal” sequence cannot be described.     

A re-evaluation of the premaxillary bone in humans

The discovery of the premaxillary bone (os incisivum, os intermaxillare or premaxilla) in humans has been attributed to Goethe, and it has also been named os Goethei. However, Broussonet (1779) and Vicq dAzyr (1780) came to the same result with different methods. The first anatomists described this medial part of the upper jaw as a separate bone in the vertebrate skull, and, as we know, Coiter (1573) was the first to present an illustration of the sutura incisiva in the human. This fact, and furthermore its development from three parts:—(1) the alveolar part with the facial process, (2) the palatine process, and (3) the processus Stenonianus—can no longer be found in modern textbooks of developmental biology. At the end of the nineteenth and in the early twentieth century a vehement discussion focused on the number and position of its ossification centers and its sutures. Therefore, it is hard to believe that the elaborate work of the old embryologists is ignored and that the existence of a premaxillary bone in humans is even denied by many authors. Therefore this re-evaluation was done to demonstrate the early development of the premaxillary bone using the reconstructions of Felber (1919), Jarmer (1922) and data from our own observations on SEM micrographs and serial sections from 16 mm embryo to 68 mm fetus. Ossification of a separate premaxilla was first observed in a 16 mm embryo. We agree with Jarmer (1922), Peter (1924), and Shepherd and McCarthy (1955) that it develops from three anlagen, which are, however, not fully separated. The predominant sutura incisiva (rudimentarily seen on the facial side in a prematurely born child) and a shorter sutura intraincisiva argue in this sense. The later growth of this bone and its processes establish an important structure in the middle of the facial skull. Its architecture fits well with the functional test of others. We also focused on the relation of the developing premaxilla to the forming nasal septum moving from ventral to dorsal and the intercalation of the vomer. Thus the premaxilla acts as a stabilizing element within the facial skeleton comparable with the keystone of a Roman arch. Furthermore, the significance of the premaxillary anlage for the closure of the palatine was documented by a synopsis made from a stage 16, 10.2 mm GL embryo to a 49 mm GL fetus. Finally the growth of the premaxilla is closely related to the development of the human face. Abnormal growth may be correlated to characteristic malformations such as protrusion, closed bite and prognathism. Concerning the relation of the premaxillary bone to cleft lip and palate we agree with others that the position of the clefts is not always identical with the incisive suture. This is proved by the double anlagen of an upper–outer incisor in a 55 mm fetus and an adult.     

Skeletal changes in murine runt disease

Runt disease was produced in neonatal F₁ hybrid mice by the injection of spleen cells from a mature donor of the parental strain. Clinically apparent runting appeared after the tenth day. Mice were serially sacrificed when 11–19 days old, and their long bones were examined histologically and histochemically. In runt disease, development of the epiphyseal secondary centers of ossification was retarded. The cells and protein polysaccharides did not mature normally, and the tissue contained acellular cartilage. Accumulations of hypertrophic chondrocytes signified impaired endochondral ossification. While the osteogenic cells in the medullary spaces had been replaced largely by mononuclear lymphoid cell infiltrates, bone collagen was present on unremodeled metaphyseal trabeculae which possessed wide cartilage cores. The intertrabecular spaces were moderately fibrotic. Large remnants of unossified unresorbed cellular cartilage were frequently observed in the metaphysis. It appeaed that the generalized graft-versus-host reaction induced by the spleen cell “grafts” was extended to the skeleton, but that the changes in the cartilage and bone tissue had occurred largely before the onset of clinical runting.     

The Neonatal Ilium—Metaphyseal Drivers and Vascular Passengers

At birth the newborn is equipped with a developing locomotor apparatus, which will ultimately become involved in load transfer from the period when the child adopts a sitting posture through to the attainment of a bipedal gait. This load transfer has been considered to influence trabecular bone structural organization by setting up forces, which remodel the internal architecture into a functionally optimized form. However, during the neonatal developmental period the locomotor apparatus is nonweight bearing and instead only supports reflexive movements. Surprisingly, a structural organization has been identified within the internal trabecular architecture and external cortical morphology of the neonatal ilium, which appears to mimic the structural composition of the more mature bone. This study aims to build upon previous qualitative and quantitative investigation of this apparently precocious patterning by further examining structural data obtained from selected volumes of interest within the ilium. Analysis has revealed statistically significant differences in regional trabecular and cortical bone characteristics, which have formed the basis of a possible growth model for the ilium. Volumetric comparison has demonstrated the presence of three progressive “growth regions” and three “restricted growth regions,” which appear to relate to metaphyseal and nonmetaphyseal borders of the ilium. Therefore, the structural data and statistical analysis presented in this study challenge the current concept of implied centrifugal ossification within the human ilium and present evidence of an alternative pattern of ossification that is largely dictated and controlled by vascular distribution and growth plate position. Anat Rec 293:1297–1309, 2010. © 2010 Wiley‐Liss, Inc.     

Cross-sectional study of the ossification center of the C1–S5 vertebral bodies

Purpose

Knowledge on the normative growth of the spine is relevant in the prenatal detection of its abnormalities. This study describes the size of the ossification center of C1–S5 vertebral bodies.

Materials and methods

Using CT, digital-image analysis, and statistics, the size of the ossification center of C1–S5 vertebral bodies in 55 spontaneously aborted human fetuses aged 17–30 weeks was examined.

Results

No sex significant differences were found. The body ossification centers were found within the entire presacral spine and in 85.5 % of S1, in 76.4 % of S2, in 67.3 % of S3, in 40.0 % of S4, and in 14.5 % of S5. All the values for the atlas were sharply smaller than for the axis. The mean transverse diameter of the body ossification center gradually increased from the axis to T12 vertebra, so as to stabilize through L1–L3 vertebrae, and finally was intensively decreasing to S5 vertebra. There was a gradual increase in sagittal diameter of the body ossification center from the axis to T5 vertebra and its stabilization for T6–T9 vertebrae. Afterward, an alternate progression was observed: a decrease in values for T10–T12 vertebrae, an increase in values for L1–L2 vertebrae, and finally a decrease in values for L3–S5 vertebrae. The values of cross-sectional area of ossification centers were gradually increasing from the axis to L2 vertebra and then started decreasing to S5 vertebra. The following cross-sectional areas were approximately equivalent to each other: for L5 and T3–T5, and for S4 and C1. The volumetric growth of the body ossification center gradually increased from the axis to L3 vertebra and then sharply decreased from L4 to S5.

Conclusions

No male–female differences are found in the size of the body ossification centers of the spine. The growth dynamics for morphometric parameters of the body ossification centers of the spine follow similarly with gestational age.     

Leptin increases growth of primary ossification centers in fetal mice

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg⁻¹) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.     

The Role of Type X Collagen in Endochondral Ossification as Deduced by Fourier Transform Infrared Microscopy Analysis

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice which expressed a truncated collagen X protein were reported to exhibit morphologic alterations in all tissues arising through EO. Fourier Transform InfraRed (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine the role of collagen X in mineralization, the “quality” of mineral crystals was analyzed in thin sections of calcified cartilage from tibia obtained from several independent transgenic mouse lines showing varying degrees of the mutant phenotype and mice without type X collagen expression, by means of Fourier Transform InfraRed microscopy (FTIRM). In the present paper, the term “mineral quality” is employed to describe crystallinity/crystal maturation, and acid phosphate content. The results indicate significant differences between normal and transgenic mice bone mineral, both in the amount present and the “quality” of the crystals. In contrast, the analysis of the mineral in mice without type X collagen expression was not different from their age/sex-matched controls.     

Age changes in the human laryngeal cartilages

Various changes in the human laryngeal cartilages have been studied by the naked eye, radiology, and histology in 28 dissecting room and 20 autopsy specimens (21 male and 27 female) ranging from 14 to 101 years. Except for one 14- and one 20-year-old specimen, radiographic signs of calcification occurred in all and were found in hyaline cartilaginous tissue of the thyroid, cricoid, arytenoid, and the variably occurring triticiate cartilages. A series of stages has been established indicating the pattern of spread of the process in the thyroid cricoid and arytenoid cartilages. Minor differences between the sexes were found in the thyroid and the cricoid, and some correlation was noted between sites of calcification with sites of muscle attachment and sites of greatest mass. The degree of involvement was not, however, found to be a reliable index of age, with wide variations occurring between individuals. Histology showed various degenerative processes, including calcification, but also revealed the occurrence of actual ossification even in some younger specimens. However, some foci of cartilage always persisted even when ossification was well advanced. Naked eye examination detected regions within the cartilages where ossification was well established, dark areas being produced by the presence of marrow. The remaining tissue was found histologically to be composed of cartilage, which might be calcified or might even contain small foci of bone. Radiology was an accurate method for detection of calcification and ossification, which were sometimes distinguishable, larger masses of calcified cartilage having a dense uniform radiographic appearance unlike the trabecular pattern of bone. © 1993 Wiley-Liss, Inc.     

Histopathological abnormalities in painful bipartite patellae in adolescents

The purpose of this study was to clarify the etiology of painful bipartite patella in adolescents by histopathological examination of excised specimens. We performed excision of a fragment of painful bipartite or tripartite patella from six patients (six knees). The articular cartilage, interposed tissue, bone, and bone marrow of the excised specimens were histologically examined. The articular cartilage was intact in all but two patellae. The predominant composition of the interposed tissue was fibrous tissue in one patient; fibrous tissue and fibrocartilage in four patients; and fibrous tissue, fibrocartilage, and hyaline cartilage in one patient. In the interposed tissue, diffuse degenerative and necrotic fibrocartilage was observed in four patients and focal necrotic fibrocartilage was seen in two patients. In all patients, the central region of the interposed tissue almost completely lacked blood vessels. Other histological features of the interposed tissue included necrosis of the trabecular bone in three patients, irregularly shaped spicules of immature bone in three patients, and fragments of hyaline cartilage in two patients. In all patients the bone marrow adjacent to the interposed tissue showed numerous small blood vessels, and trabecular bone surfaces and the fibrocartilage surface adjacent to this bone marrow was scalloped and lined with numerous osteoclasts. The striking histopathological features of the interposed tissue were fibrous tissue and necrosis of the fibrocartilage. These abnormalities may ultimately lead to the failure of an accessory ossification center to unite with the main portion of the patella.     

听骨发育的研究及其临床意义

本文对325侧胚胎第11周至成人颞骨标本按矢状位、垂直位和水平位作连续组织切片。观测结果:砧骨在胚胎第11周,锤骨在胚胎第15周、镫骨在胚胎第16周分别出现软骨化骨中心。各听骨仅有一个骨化中心,各有自身的生长规律,约在胚胎第28周,软骨化骨完成,无骺板存在,听骨大小形态与成人相近。提出胎儿听骨代替成人听骨作同种异体听骨移植重建的理论依据。     

Simpson-Golabi-Behmel syndrome: Congenital diaphragmatic hernia and radiologic findings in two patients and follow-up of a previously reported case

This report suggests the association of congenital diaphragmatic hernia in Simpson-Golabi-Behmel syndrome by describing two unrelated males with this malformation. One male was the maternal half-nephew of our previously reported 8-year-old boy with this syndrome. Review of the skeletal roentgenograms of these 2 affected males, and those of the previously reported 8-year-old, documents flare of the iliac wings, narrow sacroiliac notches, and the presence of two carpal ossification centers as a newborn (“advanced bone age”). We also report the follow-up of the 8-year-old boy, now 16 years old, who continues to have significant overgrowth and speech, dental, developmental, and adjustment problems. © 1993 Wiley-Liss, Inc.