Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

Ontogenetic explanation for tegmen tympani dehiscence and superior semicircular canal dehiscence association. / Explicación ontogénica para la asociación entre dehiscencia del tegmen tympani y dehiscencia del canal semicircular superior.

OBJECTIVES: To analyze the ontogeny of the superior semicircular canal and tegmen tympani and determine if there are common embryological factors explaining both associated dehiscence. METHODS: We analyzed 77 human embryological series aged between 6 weeks and newborn. Preparations were serially cut and stained with Masson's trichrome technique. RESULTS: The tegmental prolongation of tegmen tympani and superior semicircular canal originate from the same structure, the otic capsule, and have the same type of endochondral ossification; while the extension of the squamous prolongation of tegmen tympani runs from the temporal squama and ossification is directly of intramembranous type. The nuclei of ossification of the superior and external semicircular canals and accessory of tegmen collaborate in the ossification of the tegmental extension and by growth extend to the tegmental prolongation. This fact plus the fact that both structures share a common layer of external periosteum could explain the coexistence of lack of bone coverage in tegmen and superior semicircular canal. CONCLUSION: The development of the semicircular canal and tegmen tympani could explain the causes of the association of both dehiscences.

Distribution of matrix proteins in perichondrium and periosteum during the incorporation of Meckel's cartilage into ossifying mandible in midterm human fetuses: an immunohistochemical study.

Immunohistochemical localization of versican and tenascin-C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin-C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin-C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin-C immunoreactivity in the primary bone marrow was also weak, but tenascin-C positive areas did not overlap with versican-positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican-positive perichondrium and tenascin-C-positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin-C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage.

[Early bone and cartilage histogenesis in embryonic Japanese quails in the conditions of microgravity].

The article presents the results of a comparative histological investigation of skeletal bones genesis in Japanese quail embryos developed in the spaceflight microgravity (space group) and laboratory (control group). Total preparations of 4-day-old embryos from both groups demonstrated clearly that the cartilaginous anlage of the femoral bone had central, dyaphisial, 2 epiphysial and 2 proliferation zones. By day 7 of embryogenesis, cartilaginous anlages had grown in size in both groups due to intensive chondrocytes multiplication and gain in the intercellular substance mass. Tibial cuff in space embryos measured half and femoral cuff was 2.3 times smaller in comparison with these parameters in the control group. In addition, intensity of chondrocyte multiplication was reduced Histological profiles of the femur and tibia in 10-day old embryos of the control pointed to enhancement of osteogenesis. The metaphysis zone contained distinct mitosis figures on different stages of division. Bone deposition could be seen below the peristoma. The osteogenesis cuff spread up to the femoral anlage metaphysis; cartilage was calcined. Space embryos display retard osteogenesis. There were ingrown blood vessels in the region of cartilage destruction; however, vessels grown in the periosteum were less in number as compared with the laboratory control. Also, the perichondral ossification layer was considerably thinner, whereas the osseous cuff was 1.3 and 1.45 times shorter in the femur and tibia, respectively. To sum up, the histological investigation of bones from 4-, 7- and 10- day old Japanese quail embryos demonstrated retardation of osteogenesis in the conditions of microgravity.

Intracellular tension in periosteum/perichondrium cells regulates long bone growth.

Perichondrium/periosteum is involved in regulating long bone growth. Long bones grow faster after removal or circumferential division of periosteum. This can be countered by culturing them in conditioned medium from perichondrium/periosteum cells. Because both complete removal and circumferential division are effective, we hypothesized that perichondrium/periosteum cells require an intact environment to release the appropriate soluble factors. More specifically, we propose that this release depends on their ability to generate intracellular tension. This hypothesis was explored by modulating the ability of perichondrium/periosteum cells to generate intracellular tension and monitoring the effect thereof on long bone growth. Perichondrium/periosteum cells were cultured on substrates with different stiffness. The medium produced by these cultures was added to embryonic chick tibiotarsi from which perichondrium/periosteum was either stripped or left intact. After 3 culture days, long bone growth was proportionally related to the stiffness of the substrate on which perichondrium/periosteum cells were grown while they produced conditioned medium. A second set of experiments demonstrated that the effect occurred through expression of a growth-inhibiting factor, rather than through the reduction of a stimulatory factor. Finally, evidence for the importance of intracellular tension was obtained by showing that the inhibitory effect was abolished when perichondrium/periosteum cells were treated with cytochalasin D, which disrupts the actin microfilaments. Thus, we concluded that modulation of long bone growth occurs through release of soluble inhibitors by perichondrium/periosteum cells, and that the ability of cells to develop intracellular tension through their actin microfilaments is at the base of this mechano-regulated control pathway.

European Society of Biomechanics S.M. Perren Award 2010: an adaptation mechanism for fibrous tissue to sustained shortening.

The mechanism by which fibrous tissues adapt upon alterations in their mechanical environment remains unresolved. Here, we determine that periosteum in chick embryos resides in an identical mechanical state, irrespective of the developmental stage. This state is characterized by a residual tissue strain that corresponds to the strain in between the pliant and stiffer region of the force-strain curve. We demonstrate that periosteum is able to regain that mechanical equilibrium state in vitro, within three days upon perturbation of that equilibrium state. This adaptation process is not dependent on protein synthesis, because the addition of cycloheximide did not affect the response. However, a functional actin filament network is required, as is illustrated by a lack of adaptation in the presence of cytochalasin D. This led us to hypothesize that cells actively reduce collagen fiber crimp after tissue shortening, i.e. that in time the number of recruited fibers is increased via cell contraction. Support for this mechanism is found by visualization of fiber crimp with multiphoton microscopy before the perturbation and at different time points during the adaptive response.

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.

Residual periosteum tension is insufficient to directly modulate bone growth.

Periosteal incision is one of the less severe interventions used to correct mild long bone growth pathologies. The mechanism responsible for this growth modulation is still unclear. A generally adopted hypothesis is that incision releases compressive force created by tensioned periosteum. We set out to evaluate the feasibility of this hypothesis by quantifying the stress level imposed on cartilage by periosteum tension in the rapid growth phase of chick embryos and evaluating if tension release could be responsible for modulating growth. Residual force in embryonic periosteum was measured in a tensile tester. A finite element model was developed, based on geometry determined using optical projection tomography in combination with histology. This model was then used to calculate the stress-distribution throughout the cartilage imposed by the periosteum force and to evaluate its possible contribution in modulating growth. Residual periosteal force in e17 chick tibiotarsi resulted in compressive stresses of 6 kPa in the proliferative zone and tensile stresses up to 9 kPa in the epiphyseal cartilage. Based on the literature, these compressive stresses are estimated to reduce growth rates by 1.1% and calculated tensile stresses increase growth rates by 1.7%. However, growth rate modulations between 8% and 28% are reported in the literature upon periosteum release. We therefore conclude that the increased growth, initiated by periosteal incision, is unlikely to be predominantly the result of mechanical release of cartilage compression by periosteum tension. However, increased epiphyseal growth rates due to periosteal tension, may contribute to bone morphogenesis by widening the epiphysis.

Periosteal biaxial residual strains correlate with bone specific growth rates in chick embryos.

It has been proposed that periosteal residual tensile strains influence periosteal bone apposition and endochondral ossification. The role of bone growth rates on the development of residual strains is not well known. This study examined the relationships between specific growth rate and residual strains in chick tibiotarsi. We measured length and circumference during embryonic days 11-20 using microCT. Bones grew faster in length, with longitudinal and circumferential specific growth rates decreasing from 17 to 9% and 14 to 8% per day, respectively. To calculate residual strains, opening dimensions of incisions through the periosteum were analysed using finite element techniques. Results indicate that Poisson's ratio for an isotropic material model is between 0 and 0.04. For the model with Poisson's ratio 0.03, longitudinal and circumferential residual strains decreased from 46.2 to 29.3% and 10.6 to 3.9%, respectively, during embryonic days 14-20. Specific growth rates and residual strains were positively correlated (p<0.05).

Perichondrial-mediated TGF-beta regulation of cartilage growth in avian long bone development.

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.

Identification and characterization of a previously undetected region between the perichondrium and periosteum of the developing avian limb.

In developing long bones, the growing cartilage and bone are surrounded by the fibrous perichondrium (PC) and periosteum (PO), respectively, which provide cells for the appositional growth (i.e., growth in diameter) of these tissues. Also during the longitudinal growth of a bone, the cartilage is continuously replaced by bony tissue, giving rise to the widely held assumption that the PC concomitantly gives rise to the PO. Except for this morphological correlate, however, no evidence exists for a direct conversion of PC cells to PO cells, and our observations presented here question this assumption. Instead, we have obtained evidence suggesting that a previously undescribed region exists between the PC and PO. This region, termed the border region (BR), has several unique characteristics which distinguish it from either the PC or PO, including (1) its lack of being determined to differentiate as either cartilage or bone, (2) its ability to preferentially elicit the invasion of blood vessels, and (3) its ability to undergo preferential growth.

[Expressions of Cbfal and osterix in osteoblasts on human acellular amniotic membrane].

OBJECTIVE: To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane (HAAM). METHODS: To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and 10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The real-time PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simply-cultured osteoblasts were used as the control group (n=20). RESULTS: The expression of Cbfal was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day (P < 0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results (P < 0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference (P > 0.05). CONCLUSION: Cbfal and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.

A light and electron microscopic study of the limb long bones perichondral ossification in the quail embryo (Coturnix coturnix japonica).

The perichondral ossification of the limb long bones in the quail embryo is investigated, in this study, by means of light and electron microscopy. Longitudinal sections of the humerus, radius, ulna, femur, tibia and fibula stained with haematoxylin-eosin were examined by the light microscope. Ultrathin cross sections were selected for the electron microscope as well. Light microscopic analysis showed that the ossification began at the same time in the long bones of the wing and leg. At the embryonic day 6, all the cartilaginous rudiments consisted of three zones. The central zone composed of hypertrophic chondrocytes, a second zone on either side of the central zone, which consisted of flattened cells and a third zone, which represented the epiphyseal region. A thin sheath of osteoid and a bi-layered perichondrium-periosteum surrounded the central zone of the cartilaginous rudiments of the long bones. The perichondrium consisted of a layer of osteoblasts, in contact with the cartilage, and a layer of fibroblasts. At the embryonic day 7, the thickness of the calcified osteoid ring increased and a vasculature appeared between the layer of osteoblasts and the layer of fibroblasts. At the embryonic day 8, a second sheath of periosteal bone began to be formed. Concurrently, vascular and perivascular elements began to invade the cartilage. The ossification spread towards the distal ends of both the diaphysis. At the electron microscopic level, the osteoblasts of the perichondium showed cytoplasmatic characteristics of cells involved in protein synthesis. The perichondral ossification is the first hallmark of the osteogenesis in the long bones. The observations reported above, are in accordance with previous studies in the chick embryo.

Positive regulation of endochondral cartilage growth by perichondrial and periosteal calcitonin.

Our previous studies showed that during the embryonic development of avian long bones, growth of the cartilaginous component is regulated by multiple factors secreted by the surrounding perichondrium (PC) and periosteum (PO). The activities of these factors--which include both positive and negative regulators--can be detected in conditioned media from PC and PO cell cultures. In the present study, we have obtained evidence suggesting that a positive regulator is the peptide hormone calcitonin (CT). By mass spectrometry of conditioned media, one of the components has a molecular mass of 3.4 kDa, the size of chicken CT. By RT-PCR the tissue and cell cultures contain mRNA for CT, and by immunohistochemistry the cells contain the protein. That the protein is normally secreted is suggested by further immunohistochemical analyses, which show that cells treated with monensin, a compound that blocks exocytosis, contain elevated intracellular CT. Functionally, the addition of CT to organ cultures of long bone rudiments effects increased growth in a manner similar to that of the PC- and PO-conditioned media. Taken together, these data suggest that secretion of CT by the PC and PO effects, in a paracrine manner, positive stimulation of growth in the underlying cartilage.

Multiple mechanisms of perichondrial regulation of cartilage growth.

We previously observed that the perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO-conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO-free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor-beta1 [TGF-beta1]) act in a manner consistent with this PC/PO-mediated regulation. The results suggest that RA and TGF-beta1, per se, are not the regulators in the PC/PO-conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO-free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF-beta1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not "precise" in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial-derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF-beta1, when combined with our previous results showing similar--yet clearly different--"precise" regulation by the PC/PO-conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements.

Expression of slit-2 and slit-3 during chick development.

The Drosophila and vertebrate slit proteins have been characterized as secreted chemorepellents recognized by the robo receptor proteins that function principally for the guidance of neuronal axons and neurons. slit genes are also expressed in the limb. To provide a basis for the determination of slit functions in the limb we have isolated and characterized the expression of chick slit-2 and slit-3 in the developing limb and other tissues of the chick embryo. Both genes share similar expression profiles in the chick embryo when compared to that of their mammalian homologues, particularly in the neural tube. In the limb, their expression patterns suggest their involvement in many aspects of limb development. In the early limb bud, slit-2 is expressed in the peripheral mesenchyme and invading muscle precursors, while slit-3 expression is restricted to the future chondrogenic core of the limb bud. At later stages, both slit genes are expressed in interdigital mesenchyme, in inner periosteal cells, and in mesenchyme immediately radial to the periosteum and under the epidermis. slit-3 is also expressed in proliferating chondrocytes during cartilage development, while slit-2 is expressed in later muscle masses and peripherally to joints in the autopod.

Regulation of endochondral cartilage growth in the developing avian limb: cooperative involvement of perichondrium and periosteum.

The perichondrium and periosteum have recently been suggested to be involved in the regulation of limb growth, serving as potential sources of signaling molecules that are involved in chondrocyte proliferation, maturation, and hypertrophy. Previously, we observed that removal of the perichondrium and periosteum from tibiotarsi in organ culture resulted in an overall increase in longitudinal cartilage growth, suggesting negative regulation originating from these tissues. To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. The requirement for regulatory factors from both the perichondrium and periosteum suggests a novel mechanism of regulation. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes.

Deer antlerogenic periosteum: a piece of postnatally retained embryonic tissue?

This article reviews the research findings on the piece of periosteum overlying the lateral crest of prepubertal deer frontal bone, known as antlerogenic periosteum (AP). AP was initially discovered by Hartwig and Schrudde in 1974 when searching for the tissue that gives rise to antlers. In their experiment, when AP was transplanted elsewhere on the deer body it formed ectopic antlers. This clearly shows that AP possesses full self-differentiating ability, an attribute that can only be paralleled by embryonic tissue in mammals, like lateral plate mesoderm (LPM). Studies along this line by Goss in the 1980s further demonstrated that AP also holds the patterning information for antler formation. In the 1990s, our group carried out a series of studies on this unique tissue. The results showed that some of the critical features of AP resemble those of embryonic tissues, such as the astonishing growth potential in vivo and in vitro, and rich glycogen content. Histological observations and cell lineage tracing using a genetic marker convincingly demonstrate that pedicles and antlers are the derivatives of AP. Based on these findings, we advanced a hypothesis that AP is a piece of postnatally retained embryonic tissue. Morphological and histological examinations on the presumptive antler growth regions in deer prenatal life showed that the growth of primordial pedicles is initiated in the early pregnant stage (about 55 days) but then ceases (about 100 days) and is subsequently repressed at the late stage of pregnancy. The epidermis overlying the primordial pedicles resembles the apical ectoderm ridge (multicellular layer). These results strongly support our hypothesis. The results from the specific comparison between deer antler formation (from AP in postnatal) and mammalian limb development (from LPM in prenatal) showed that the ontogeny of antlers and limbs are comparable, and that deer antler has the same level of regulative properties as mammalian limbs. We believe that revealing the mechanism underlying the retention of embryonic tissue properties by AP until deer postnatal life will have important implications in biomedical research. Antler formation from AP offers an ideal model to work with in investigating how a self-differentiating system functions.

Osteogenesis from cultured chick periostea has a specific requirement for chloride.

Bone development, like embryonic development in general, depends on a particular internal electrical milieu. Ions are the carriers of currents that maintain this internal environment. In embryonic bone, chloride is a major carrier of such current. To explore the role chloride plays in embryonic bone development we performed several ion-removal experiments, using the chick periosteal osteogenesis (CPO) system as our model. We found that if chloride is reduced in the medium and replaced with a nontoxic anion, alkaline phosphatase (ALP) activity does not rise, nor does osteogenic development occur. However, acid phosphatase (AP) activity is not affected by level of chloride. Experiments using metabolic inhibitors showed that explants cultured in low chloride medium remain viable. Dose-response studies revealed that the response of ALP activity to chloride concentration is sigmoidal, with a [Cl-]0.5 of 45.9 mM. Reciprocal transfers of explants between complete and low chloride medium show that the rise in ALP activity depends on the length of time explants are cultured with chloride. In contrast, such transfer experiments show that osteogenesis requires chloride only during days 2-3 of culture.

Extent of ossification at the amputation plane is correlated with the decline of blastema formation and regeneration in Xenopus laevis hindlimbs.

Xenopus laevis larvae gradually lose the ability to regenerate lost hindlimb structures as they progress through metamorphosis. Previous studies have suggested that this loss of regenerative capacity occurs in a proximal-to-distal fashion. We assessed the quality of overall regeneration and early bud blastema formation in order to evaluate previous explanations for this loss of regenerative ability in Xenopus. We further examined the extent to which epidermis, basement membrane, dermis, cartilage, bone, periosteum, and accumulated mesenchyme within the blastema are involved in the decline of regenerative abilities during mid-metamorphic stages of development. Each tissue was scored based on its contributions to the regeneration blastema, in accordance with previously reported blastemal descriptions. Tadpoles amputated at the ankle and tarsal-metatarsal joints scored objectively higher within the overall regeneration and blastema quality rating systems. Both joint sites met more criteria associated with regeneration-capable blastemas than tadpoles amputated through the middle of the tarsus, especially at later stages of metamorphosis. The three amputation sites studied began to vary in their ability to regenerate skeletal elements and to generate productive blastemas during the same stages at which we initially observed ossification of the tarsus. These results suggest that the decline of Xenopus hindlimb regeneration does not occur in a strictly proximal-to-distal fashion but rather is dependent at later stages on the state of ossification of the structure through which amputation occurs. Our morphological and cellular observations reveal specific times and places during Xenopus hindlimb development at which further investigations into tissue-specific molecular events during early regeneration should be focused.