Skeletal muscle activity affects the deformity of long bone morphology in lathyritic chick embryo.

Embryonic muscle activity is involved in various aspects of bone morphogenesis and growth. Normal mechanical stimuli of muscle contraction are important in most cases, and when the muscles are immobilized, the developing bones are abnormally shaped. In chick embryos, a characteristic curved deformity is reproducibly induced in the developing tibiotarsus using the bone-weakening agent, beta-aminopropionitrile (bAPN). In this study, we applied decamethonium bromide (DMB), a well-established neuromuscular blocking agent, to embryos treated with bAPN, to test the hypothesis that the deformity is triggered and formed depending on the balance between the decrease in stiffness of the bAPN-affected tibiotarsus and the normal physiological increase in embryonic skeletal muscle activity. The occurrence of curved morphology induced by bAPN administered at 4 or 8 days of incubation (embryonic day [ED]) was temporally consistent with the posterior displacement of the leg muscles, which occurred just before ED8. The displaced muscles were assumed to exert a contraction force comparable to that of untreated normal muscles. When treated with DMB at ED8, the muscles atrophied and exhibited degenerative changes, and the degree of curved morphology was alleviated and reduced to 50% or more in the morphometric evaluation at ED10. These findings indicated that the coordinated development of skeletal element stiffness and muscle activity must be temporally regulated, particularly during the early stages of skeletogenesis.

The teeth and dentition of the filefish (Stephanolepis cirrhifer) revisited tomographically.

The upper and lower tooth-bearing jaws of the filefish (Stephanolepis cirrhifer) were scanned using a micro-CT system in order to address the existing gaps between the traditional pictures of the morphology and histology. 2D tomograms, reconstructed 3D models and virtual dissection were employed to examine and evaluate the in situ geometry of tooth implantation and the mode of tooth attachment both separately and collectively. No distinct sockets comparable to those in mammals were evident, but shallow depressions were observed in the premaxillary and the dentary. The opening of the tooth pulp cavity was not simply oriented towards the apparent tooth base in a direction opposite to the tooth apex. The opening was distorted basoposteriorly or basoanteriorly depending on the position of the tooth, and the edge of the pulp cavity opening was barely ankylosed; i.e. the sites of pleurodont ankylosis along the basoposterior or basoanterior edge of the opening appeared to closely match the contour of the shallow depression in the bone. These 3D findings appear to be very informative when considering the phylogeny of tooth attachment, suggesting that micro-CT would be a useful modality concurrent with or in advance of histological investigations.

Temporospatial distribution of osteogenic and osteoclastic cells during development of the tarsometatarsal skeleton in the chick embryo (Gallus gallus).

The morphogenesis of long bones is a multistep process that generates a variety of genetically defined forms. The tarsometatarsal (TMT) long bone morphology in birds develops through lateral fusion of three initially independent periosteal bone cylinders (BCs). Previous studies have clarified the histological details and chronology of the changes occurring during development. The present study investigated the temporospatial distribution of osteogenic and osteoclastic cells in the embryonic chicken using histochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, with particular reference to the radial growth of BCs and their subsequent fusion process. Osteogenic cells were localized preferentially in the periosteum of radially growing BCs, leaving open cancellous spaces in the BC wall. Osteoclasts observed later than embryonic day 10 were localized preferentially in the endosteal surface, and therefore the radial growth of BCs resulting from osteoblast activity was accompanied by endosteal resorption by osteoclasts, with progressive enlargement of the bone marrow spaces. During BC fusion, trabecular bridges were formed by periosteal osteogenic cells, with removal of the bone septum by endosteal osteoclasts. These findings suggest that fusion of BCs in the embryonic chicken is mediated by cellular events constituting ordinary long bone development, and not through a defined mechanism specific for fusion.

Highly reproducible skeletal deformities induced by administration of ß-aminopropionitrile to developing chick embryos.

The formation of cross-linkages between and within collagen is catalyzed by lysyl oxidase, which can be inhibited by ß-aminopropionitrile (BAPN), a lathyrogen from sweet pea (Lathyrus odoratus) seeds. The quality and integrity of the collagenous template of skeletal elements depend on an appropriate concentration of collagen cross-links. In this study, chick embryos treated in ovo with BAPN on embryonic days (ED) 4-9 were found to develop multiple skeletal deformities. The most readily discernible and highly reproducible deformity was evident in the tibiotarsus, on which we focused to explore the chronology of the malformation process. Several lines of observation indicated that the bending deformity observable at ED10 in the tibiotarsus was inducible by BAPN administered on ED4-8; in other words, administration of BAPN on ED8 was sufficient to induce the deformity by ED10, whereas administration on ED9 was ineffective. Ultrastructurally, osteoclasts appeared to show enhanced activity in the medullary surface of the bone collar after BAPN administration. In addition, bone hyperplasia associated with the bending deformity was suggested to be correlated with higher osteoblast activity on the concave (or flexor) side of the tibiotarsal skeleton. These findings indicate that the bending deformity due to reduced mechanical integrity of the collagenous template is also associated with aberrant bone remodeling. (J Oral Sci 58, 255-263, 2016).

Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth.

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

Progressive bundling of fibrillin microfibrils into oxytalan fibers in the chick presumptive dermis.

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurle's group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well-oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4-6 (ED4-6). Immunohistochemically, fibrillin-positive dots representing cross-sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ∼15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell-fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell-associated process.

Development of the tarsometatarsal skeleton by the lateral fusion of three cylindrical periosteal bones in the chick embryo (Gallus gallus).

An avian tarsometatarsal (TMT) skeleton spanning from the base of toes to the intertarsal joint is a compound bone developed by elongation and lateral fusion of three cylindrical periosteal bones. Ontogenetic development of the TMT skeleton is likely to recapitulate the changes occurred during evolution but so far has received less attention. In this study, its development has been examined morphologically and histologically in the chick, Gallus gallus. Three metatarsal cartilage rods radiating distally earlier in development became aligned parallel to each other by embryonic day 8 (ED8). Calcification initiated at ED8 in the midshaft of cartilage propagated cylindrically along its surface. Coordinated radial growth by fabricating bony struts and trabeculae resulted in the formation of three independent bone cylinders, which further became closely apposed with each other by ED13 when the periosteum began to fuse in a back-to-back orientation. Bone microstructure, especially orientation of intertrabecular channels in which blood vasculature resides, appeared related to the observed rapid longitudinal growth. Differential radial growth was considered to delineate eventual surface configurations of a compound TMT bone, but its morphogenesis preceded the fusion of bone cylinders. Bony trabeculae connecting adjacent cylinders emerged first at ED17 in the dorsal and ventral quarters of intervening tissue at the mid-diaphyseal level. Posthatch TMT skeleton had a seemingly uniform mid-diaphysis, although the septa persisted between original marrow cavities. These findings provide morphological and histological bases for further cellular and molecular studies on this developmental process.

Late deposition of elastin to vertical microfibrillar fibers in the presumptive dermis of the chick embryonic tarsometatarsus.

Fibrillin microfibrils are integral components of elastic fibers and serve as a scaffold for elastin deposition. However, microfibrillar fibers (MFs) are not necessarily committed to develop into so-called elastic fibers. In dermis, elastin-free oxytalan MFs originating from the dermoepidermal junction are continuous to elaunin-type MFs (with a small amount of elastin) in the deeper papillary dermis, whereas the reticular dermis contains elastic fibers, or MFs embedded largely in elastin. In this study, we have investigated temporospatial patterns of elastin deposition on the MFs in tarsometatarsal presumptive dermis. While the earliest expression of elastin was demonstrated immunohistochemically as early as embryonic day 4 (ED4) in the wall of cardiac outflow and pharyngeal arch arteries, its deposition in the tarsometatarsus was first detected at ED6 in the deeper mesenchyme and at ED13 in the subectodermal mesenchyme. In the latter tissue, MFs had been organized perpendicularly to the covering ectoderm by ED4, well before an overt accumulation of collagenous matrix. Elastin deposition was observed initially in a punctate manner at ED13 and afterward became continuous along MFs. However, a characteristic spaced array of subectodermal vertical MFs was disorganized by ED17. These findings suggest that elastin deposition in the subectodermal MFs is not deployed by continuous, orderly propagation from elastic fibers in the deeper mesenchyme but occurs de novo in multiple foci along vertical MFs. Moreover, the present chronology of elastin deposition indicates that subectodermal, elastin-free MFs function as a transient, but primary fibrous structure in the presumptive dermis before the accumulation of collagenous matrix.

Cellular origin of microfibrils explored by monensin-induced perturbation of secretory activity in embryonic primary cultures.

Fibrillin is a primary component of elastin-associated microfibrils. Since microfibrils are distributed rather ubiquitously in embryonic tissues, attention has focused on the types of cells responsible for producing fibrillin. To clarify this issue, we employed monensin-induced perturbation of secretory activity in embryonic primary cultures, as this would allow examination of both the secreted protein and the formation of extracellular fibrils in the same culture. Micromasses of avian limb bud mesoderm, its ectodermal covering and several explants from other sources were cultured in the presence and absence of monensin, and evaluated immunohistochemically using antibodies against fibrillin and cell lineage markers. The results indicated that monensin perturbation induced intracellular accumulation of fibrillin and prevented the formation of microfibrils. It was shown specifically that not only mesodermally derived fibrogenic cells and myogenic cells of skeletal and smooth muscle cell lineage, but also epithelial-type cells such as endothelial and ectodermal cells, are producers of fibrillin. This dual cellular origin of fibrillin at the ectomesenchymal interface is considered significant for understanding the formation and remodeling of microfibrils originating from the basal lamina.

Metal-assisted stabilization and probing of collagenous triple helices.

Collagen model peptides that contain 2,2'-bipyridyl (bpy) ligands were designed and synthesized. The thermal stability of the collagenous triple helix was increased by forming an Fe(II)(bpy-peptide)(3) complex. The chirality of the metal center was shifted to form right-handed Delta-isomers induced by the supercoiling of the peptide moiety. Moreover, the refolding rate of the triple helix was increased in the presence of Fe(II). This metal-coordinating system possesses potential to be used to stabilize the triple-helical conformation as well as to probe the folding status of collagen model peptides.