The anterior ligament of the human malleus.

The authors have studied the anterior ligament of the malleus (ALM) from a morphological and embryological point of view. Classical textbooks of anatomy stress the correlation between the ALM and the anterior pin of the sphenoid and define the ligament as a residual of Meckel's cartilage. This study demonstrates the y-shaped form of the ligament, one arm of which reaches the capsule of the temporomandibular joint and the other the pin of the sphenoid bone. Meckel's cartilage pilots the fibres of the ligament itself. Several clinical implications may be hypothesised on the basis of this study.     

Fibroblast growth factor 10 regulates Meckel's cartilage formation during early mandibular morphogenesis in rats

Fibroblast growth factors (FGF) are pluripotent growth factors that play pivotal roles in the development of various organs. During mandibular organogenesis, Meckel's cartilage, teeth, and mandibular bone differentiate under the control of various FGF. In the present study, we evaluated the role of FGF10 in rat mandibular chondrogenesis and morphogenesis using mandibular organ culture and mandibular cell micromass culture systems. The overexpression of Fgf10 induced by the electroporation of an FGF10 expression vector not only altered the size and shape of Meckel's cartilage, but also upregulated the expression of the cartilage characteristic genes Col2a1 and Sox9 in a mandibular organ culture system. Meckel's cartilage was deformed, and its size was increased when Fgf10 was overexpressed in the lateral area of the mandible. Meanwhile, no effect was found when Fgf10 was overexpressed in the medial portion. In the mandibular cell micromass culture, recombinant FGF10 treatment enhanced chondrogenic differentiation and endogenous ERK (extracellular signal-regulated kinase) phosphorylation in cells derived from the lateral area of the mandible. On the other hand, FGF10 did not have significant effects on mandibular cell proliferation. These results indicate that FGF10 regulates Meckel's cartilage formation during early mandibular morphogenesis by controlling the cell differentiation in the lateral area of the mandibular process in rats.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Endocrine regulation of the growth plate

Longitudinal bone growth occurs at the growth plate by endochondral ossification. Within the growth plate, chondrocyte proliferation, hypertrophy, and cartilage matrix secretion result in chondrogenesis. The newly formed cartilage is invaded by blood vessels and bone cells that remodel the newly formed cartilage into bone tissue. This process of longitudinal bone growth is governed by a complex network of endocrine signals, including growth hormone, insulin-like growth factor I, glucocorticoid, thyroid hormone, estrogen, androgen, vitamin D, and leptin. Many of these signals regulate growth plate function, both by acting locally on growth plate chondrocytes and also indirectly by modulating other endocrine signals in the network. Some of the local effects of hormones are mediated by changes in paracrine factors that control chondrocyte proliferation and differentiation. Many human skeletal growth disorders are caused by abnormalities in the endocrine regulation of the growth plate. This review provides an overview of the endocrine signals that regulate longitudinal bone growth, their interactions, and the mechanisms by which they affect growth plate chondrogenesis.     

Absolute strength of the muscles attached to the mandible

In the course of an anatomical investigation of the muscles, securing movements of the mandible, performed on 10 human corpses, the muscle fibre length, volume and weight of each muscle has been estimated. Owing to the formula suggested by P. F. Leshaft (1880)--q = v/e, the physiological diameter of the muscles has been determined. Since the muscle with the diameter equal to 1 cm2 develops an absolute forse of 10 kg, the absolute muscle force value of the anterior group of muscles has been obtained for the first time (venter anterior musculi digastri--4.8 kg, musculus mylohyoideus--10.7 kg and musculus geniohyoideus--6.3 kg). The data on the absolute force of the posterior group of muscles has been verified (musculus masseter--24.2 kg, musculus temporalis--28 kg, musculus pterygoideus medialis--15.6 kg and musculus pterygoideus lateralis--15.5 kg). Analysing the interaction of forces of the muscles participating in the mandible movements, direction and value of displacements of the mandibular fragments have been explained and confirmed on some clinical examples. The data on the absolute force of the muscles studied can be used for investigating the displacement mechanism of the mandibular fragments after its resection and when its integrity is broken.     

A comparative microscopic and ultrastructural study of perichondrial tissue in cartilage of Octopus vulgaris (Cephalopoda, Mollusca)

The microscopic and submicroscopic structures of perichondrial tissues in the head cartilages of Octopus vulgaris were studied by polarized light and transmission electron microscopy. The orbital cartilages possess a birefringent layer parallel to the surface of the cartilage; ultrastructurally, this layer, which may be considered perichondrial tissue, has the typical organisation of connective tissue but does not possess the stratification of collagen laminae found in vertebrate perichondria. Perichondrial extracellular matrix is clearly distinct from that of cartilage because its collagen fibrils are of a larger diameter than collagen fibrils from cartilage. In addition, perichondrial fibroblasts are characteristically located at the center of collagen fibers. In the cerebral cartilage, the perichondrium is absent or discontinuous in relation to complex interconnections between cartilage and connective fibres, muscle fibres, blood vessels and nerve. Distinctive cartilage-lining cells, rich in electron dense cytoplasmatic granules, are stratified either along the cartilage surface or along vessels and muscle fibres that penetrate within the cartilage. The perichondrium of cephalopod cartilage, whose structure varies according to the location and function of its skeletal segments, mimics that of vertebrate perichondrium, exemplifying the high level of tissue differentiation attained by cephalopods.     

Development of the mandibular skeleton in the embryonic chick as evaluated using the DNA-inhibiting agent 5-fluoro-2'-deoxyuridine

Mandibular development was examined in embryonic chicks following administration of 5-fluoro-2'-deoxyuridine (FUDR, 0.001-1.0 microgram/egg), an inhibitor of both DNA synthesis and of cell division. FUDR was injected in ovo at one of three developmental stages corresponding to 1) the migration of mandible-destined, midbrain-level neural crest cells (Hamburger and Hamilton [H.H.] stage 10); 2) midway through the epithelial-mesenchymal interaction required to initiate mandibular osteogenesis (H.H. stage 22), which is also after the epithelial-neural crest cell interaction required for the initiation of chondrogenesis in Meckel's cartilage; and 3) when prechondroblasts of Meckel's cartilage are beginning to differentiate (H.H. stage 25). Micromelia was induced following the administration of FUDR at either H.H. stages 22 or 25 but not when FUDR was given at H.H. stage 10. Although the micromelic mandibles were shorter than normal, Meckel's cartilage and the mandibular membrane bones both differentiated and grew along the full proximodistal length of the shortened mandibles. In contrast to the situation previously described by Ferguson for alligator embryos exposed to FUDR, the migration of neural crest cells in the embryonic chick was not inhibited by FUDR. In contrast to the situation previously described for rat embryos exposed to FUDR, differentiation of Meckel's cartilage was not inhibited in embryonic chicks exposed to FUDR. Differentiation of the membrane bones was also normal following either in ovo administration of FUDR or when mandibular processes were maintained in FUDR in vitro. Therefore, FUDR does not produce micromelia in the embryonic chick by interfering with the epithelial-mesenchymal/neural crest cell interactions, which are prerequisites or differentiation of cartilage or bone, nor by inhibiting the differentiation of chondrogenic or osteogenic mesenchymal cells after completion of these tissue interactions. Neither did the growth-inhibiting action of FUDR result from an inhibition of growth of Meckel's cartilage during the several days following initial chondrogenic differentiation. Rather, subsequent growth of the entire mandibular process was delayed. This mechanism of action differs from that in the alligator embryo, in which FUDR inhibits mandibular growth by removing mandible-destined, migrating neural crest cells, and in the rat, in which FUDR inhibits the differentiation of Meckel's cartilage but catch-up growth restores growth of the mandible to normal.     

Roles of FGFR3 during morphogenesis of Meckel's cartilage and mandibular bones

To address the functions of FGFR2 and FGFR3 signaling during mandibular skeletogenesis, we over-expressed in the developing chick mandible, replication-competent retroviruses carrying truncated FGFR2c or FGFR3c that function as dominant negative receptors (RCAS-dnFGFR2 and RCAS-dnFGFR3). Injection of RCAS-dnFGFR3 between HH15 and 20 led to reduced proliferation, increased apoptosis, and decreased differentiation of chondroblasts in Meckel's cartilage. These changes resulted in the formation of a hypoplastic mandibular process and truncated Meckel's cartilage. This treatment also affected the proliferation and survival of osteoprogenitor cells in osteogenic condensations, leading to the absence of five mandibular bones on the injected side. Injection of RCAS-dnFGFR2 between HH15 and 20 or RCAS-dnFGFR3 at HH26 did not affect the morphogenesis of Meckel's cartilage but resulted in truncations of the mandibular bones. RCAS-dnFGFR3 affected the proliferation and survival of the cells within the periosteum and osteoblasts. Together these results demonstrate that FGFR3 signaling is required for the elongation of Meckel's cartilage and FGFR2 and FGFR3 have roles during intramembranous ossification of mandibular bones.     

Anatomo-topographic characteristics of the masseter muscle]

The musculus masseter, ensuring movements of the mandible, displace the osseous pieces at its fracture up/down in the lateral and medial sides. Morphometrical investigation of the musculi depressores++ mandibulae has been performed. As a whole 33 corpses (29-78 years of age) of normosthenic++ complexion have been studied. The measurements have been performed by means of a special compasses and a ruler with an approximation to 1 mm and 1 degree. The length of the digastric muscle belly is 55.3 +/- 1.1 mm. The length of the geniohyoid muscle is 44.5 +/- 0.9 mm. The distance between the centers, where the digastric muscle are fixed on the hypoglossal bone is 46.1 +/- 1.1 mm, and on the mandible--25 +/- 9 mm. The width of fixation of the musculus mylohyoideus on the mandible is 52.6 +/- 1.2 mm. The angles between the masseter muscles, the mandibular body and the occlusive plane have also been determined.     

Hypertrophic Chondrocytes in the Rabbit Growth Plate Can Proliferate and Differentiate into Osteogenic Cells when Capillary Invasion Is Interposed by a Membrane Filter

The fate of hypertrophic chondrocytes during endochondral ossification remains controversial. It has long been thought that the calcified cartilage is invaded by blood vessels and that new bone is deposited on the surface of the eroded cartilage by newly arrived cells. The present study was designed to determine whether hypertrophic chondrocytes were destined to die or could survive to participate in new bone formation. In a rabbit experiment, a membrane filter with a pore size of 1 µm was inserted in the middle of the hypertrophic zone of the distal growth plate of ulna. In 33 of 37 animals, vascular invasion was successfully interposed by the membrane filter. During 8 days, the cartilage growth plate was enlarged, making the thickness 3-fold greater than that of the nonoperated control side. Histological examination demonstrated that the hypertrophic zone was exclusively elongated. At the terminal end of the growth plate, hypertrophic chondrocytes extruded from their territorial matrix into the open cavity on the surface of the membrane filter. The progenies of hypertrophic chondrocytes (PHCs) were PCNA positive and caspase-3 negative. In situ hybridization studies demonstrated that PHCs did not express cartilage matrix proteins anymore but expressed bone matrix proteins. Immunohistochemical studies also demonstrated that the new matrix produced by PHCs contained type I collagen, osteonectin, and osteocalcin. Based on these results, we concluded that hypertrophic chondrocytes switched into bone-forming cells after vascular invasion was interposed in the normal growth plate.     

Long-term results of deep defects in articular cartilage. A scanning electron microscope study.

Core defects produced in the medial femoral condyle of the rabbit were studied by scanning electron microscopy and light microscopy over a period of 2 years. In some cases the defect was filled by hyaline articular cartilage with a fairly smooth surface, but in others the tissue was markedly fibrillated and resembled fibrous tissue and fibrocartilage. Appearances suggesting disintegration of the newly formed cartilage were seen in some cases. It would appear that a continuation of this process can lead to the exposure of subchondral bone. In one instance no repair tissue or new cartilage could be identified but the surrounding old cartilage had formed a shelf over the defect. The cartilage surrounding the defect was either normal or showed superficial fibrillation, and/or flow formation, and/or fissures.     

Chondroid bone and secondary cartilage contribute to apical dentary growth in juvenile Atlantic salmon

The microstructure and tissue composition of the dentary bone in Atlantic salmon salmo salar parr were examined using a variety of histological and whole‐mount techniques. Proximally, the dentary is composed of typical cellular lamellar bone with Sharpey's fibres extending dorsally, proximally and ventrally. Meckel's cartilage is located medially through the entire length of the dentary, and degrades distally resulting in a short transitional zone between hyaline cartilage and connective tissue. At the distal tip of the dentary, isogenic clusters of chondrocytes of periosteal origin were observed secreting small amounts of pericellular cartilage matrix within the bone matrix. These characteristics are highly indicative of secondary chondrogenesis, and suggest that the apical part of the dentary bone in Atlantic salmon does not grow via 'pure' intramembranous ossification, but rather via a modified mode of periosteal ossification involving secondary cartilage and chondroid bone. Furthermore, the unusual mode of gender‐related dentary growth (kype formation) in adult male Atlantic salmon could be the continuation of a general mode of salmonid apical dentary growth.     

Das coronoid der paulchoffatiidae (multituberculata; ober-jura)

For the first time the presence of a rudimentary coronoid bone in the mandible can be proved, among the Kimmeridgian Paulchoffatiidae of the Guimarota coal mine near Leiria/Portugal. In 3 specimens remains of the bone in situ are present; in others only its sutural surface is preserved. The coronoid is a small bone placed immediately posteriorly to the second molar, variable in outline. In shape and position it is more similar to the coronoid of the contemporaneous Docodonta —Haldanodon — than to that of the also contemporaneous Eupantotheria, in which it is larger and situated higher on the coronoid process. Other accessorial bones, the sulcus mylohyoideus and the Meckelian cartilage are not present. With that it is proved that in all Mammalian orders known from the Guimarota coal mine — Docodonta, Eupantotheria, Multituberculata — accessorial bones in the lower jaw persist in a more or less degree, and it seems probable that in Kimmeridgian time the presence of such reptilian bones is the normal condition in mammals.     

Ultrastructure of the deltoid muscle in wrestlers with habitual shoulder dislocation and during rehabilitation after surgical treatment

Electron microscopical investigation of the musculus deltoideus bioptates has been performed in wrestlers with a habitual shoulder-slip (7 persons). In the same persons contralateral muscle of the healthy arm has been studied (4 persons) and state of the muscle after operative treatment of the shoulder-slip (4 persons) has been analysed. At repeated shoulder-slips signs of the muscle atrophy are noted; this is clear from destructive changes of myofibrils, appearance of a great number of necrotized fibers, outgrowth of the connective tissue. In two cases ectopic formation of the bone in the muscle tissue has been observed. Increasing number of myosatellitocytes, appearance of newly formed fibers after the operative treatment contributes to restoration of the atrophied muscles. During rehabilitation period after the operative eradication of the shoulder-slip, when the program of the restorative loading is working out, it is necessary to take into consideration the severity of the trauma and duration of the disease, in order to avoid lesions of the weakend muscles.     

The epiphyseal cartilage and growth of long bones in Rana catesbeiana.

The structure of the epiphyseal cartilage of the bullfrog Rana catesbeiana and its role in the growth of long bones were examined. The epiphyseal cartilage was inserted into the end of a tubular bone shaft, defining three regions: articular cartilage, lateral articular cartilage and growth cartilage. Joining the lateral cartilage to the bone was a fibrous layer of periosteum, rich in blood vessels. Osteoblasts with alkaline phosphatase activity were found on the surface of the periosteal bone, which presented a fibrous non-mineralised tip. The growth cartilage was inside the bone. The proliferative chondrocytes presented perpendicular separation of daughter cells and there was no columnar arrangement of the cells. Furthermore, chondrocyte hypertrophy was not associated with either calcification or endochondral ossification, in apparent contrast to the avian and mammalian models. Finally, there was no reinforcement system capable of directing cell volume increase into longitudinal growth. Since bone extension depends on the intramembranous ossification of the periosteum, the growth cartilage is inside and not at the end of the bone and the cells in the growth cartilage show no columnar arrangement and separate in a direction perpendicular to the long bone axis, we conclude that the growth cartilage mainly contributes to the radial expansion of the bone.     

Structure of skeletal muscles in leghorn type chicken from conservative and parent flocks

Intensive selection conducted within closed populations has led to the creation of specialized chicken strains that differ significantly in meat yield and reproduction performance. The effect of the selection conducted on the birds is differentiation identified not only on the molecular but also on the cellular level, among other things in the skeletal muscles. The aim of this study was to compare the structure of chosen homological skeletal muscles from Leghorn chickens (LSL), originating from parent flock, intensively selected for reproductive traits and from conservative flock (G99), unselected for many generations. The structure of musculus pectoralis superficialis and musculus biceps femoris (the thickness of the muscle fibres and the share of the fibre types in the bundle) in 8 and 20 week old chickens was compared. A significant impact of the origin on all examined slaughter parameters was recorded. Body weight before slaughter, carcass weight and the weight of breast and leg muscles in 8 weeks old LSL chicken made up from 60% to about 85% of the respective values in the G99 Leghorn. Lack of red fibres in the breast muscles of all the individuals from the parental flock (LSL) was noted, whereas in 12 individuals (among 24) from the conservative flock (G99), red fibres were observed in this muscle from 2.75% up to 7.09%. White fibres in 8 week old chicks were always thicker, both in pectoralis superficialis and biceps femoris muscle in birds with higher body weight as well as higher weight of breast and legs muscles, i.e. in chicks from conservative flock (G99), P<0.01. However, in 20 week old birds, the diameters of the white fibres were similar in both groups. Also the diameters of the red fibres in musculus biceps femoris in 8 week old chickens were higher in cockerels and pullets from conservative flock (G99).     

Epithelial-mesenchymal interactions in the development of chick facial primordia and the target of retinoid action

The development of the chick face involves outgrowth of buds of tissue, accompanied by the differentiation of cartilage and bone in spatially defined patterns. To investigate the role of epithelial-mesenchymal interactions in facial morphogenesis, small fragments of facial tissue have been grafted to host chick wing buds to continue their development in isolation. Fragments of the frontonasal mass give rise to typical upper-beak-like structures: a long central rod of cartilage, the prenasal cartilage and an egg tooth. Meckel's cartilage, characteristic of the lower beak, develops from fragments of the mandible. Removal of the ectoderm prior to grafting leads to truncated development. In fragments of frontonasal mass mesenchyme only a small spur of cartilage differentiates and there is no outgrowth. The mandible is less affected; a rod of cartilage still forms but the amount of outgrowth is reduced. Retinoid treatment of chick embryos specifically affects the development of the upper beak and outgrowth and cartilage differentiation in the frontonasal mass are inhibited. The mandibles, however, are unaffected and develop normally. In order to investigate whether the epithelium or the mesenchyme of the frontonasal mass is the target of retinoid action, recombinations of retinoid-treated and untreated facial tissue have been grafted to host wing buds. Recombinations of retinoid-treated frontonasal mass ectoderm with untreated mesenchyme develop normally whereas recombinations of untreated ectoderm with retinoid-treated mesenchyme lead to truncations. The amount of outgrowth in fragments of mandibular tissue is slightly reduced when either the ectoderm or the mesenchyme has been treated with retinoids. These recombination experiments demonstrate that the mesenchyme of the frontonasal mass is the target of retinoid action. This suggests that retinoids interfere with the reciprocal epithelial-mesenchymal interactions necessary for outgrowth and normal upper beak development.     

Mode of formation of the flight muscles in a butterfly Pieris brassicae

The formation and development of the dorsal longitudinal flight muscles of the butterfly Pieris brassicae L have been studied by electron and light microscopy. These imaginal muscles arise from two symmetrical pairs of mesothoracic larval muscles, which are morphologically indistinguishable from the other wall muscles at the beginning of the 5th larval instar. However, 2 days before the end of this instar an accumulation of myoblasts is observed at the median region of these muscle fibres. The muscle fibres are penetrated by the myoblasts and broken into fragments. Progressive dedifferentiation of the larval fibrillar material in each of the muscle fragments is observed during the first days of the pupal development. The myoblasts within the basal lamina of the original larval muscle fibres remain associated with the muscle fragments. Myoblasts then fuse with the larval muscle fragments, which simultaneously fuse with each other. This results in the formation of rudimentary imaginal muscle fibres. The development of these fibres, particularly myofibrillogenesis, is studied until the emergence of the imago.     

Soft-tissue bone interface: How do attachments of muscles,tendons, and ligaments change during growth? A light microscopic study

This study addressed the problem of how soft structures maintain approximately the same relative positional relationships during long bone growth. Attachments of the popliteus muscle, semitendinosus tendon, medial collateral knee ligament, and extensor retinaculum were examined histologically in rabbits, aged 2-60 days, to determine the manner in which soft structures attached to long bones during growth. Soft structures inserted principally into fibrous periosteum or perichondrium in the age range studied. However, an extensive collagen fiber framework within the cellular periosteum and perichondrium, present by at least 2 days of age, linked the fibrous periosteum or perichondrium to subjacent bone or cartilage. Maturation of soft tissue-bone interfaces was viewed from two related perspectives. The first stressed temporal patterning of cartilage and bone differentiation. The second emphasized incorporation of attachments of soft structures into bone and cartilage matrices during growth and remodeling. Differentiation and remodeling of bone and cartilage varied not only with age, but also between regions of attachment of single muscles and ligaments. Insertion regions were characterized by the presence of coarse-fibered periosteal bone and chondroid bone, both morphologically intermediate between fibrocartilage and lamellar bone. These results provide evidence that periosteal attachments, characterizing the soft-tissue bone interface, are a necessary structural prerequisite for compensatory movement and invariance of the relative positions of muscles, tendons, and ligaments during long bone growth.     

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.