Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Human long bone development in vivo: analysis of the distal femoral epimetaphysis on MR images of fetuses.

PURPOSE: To investigate human long bone development in vivo by analyzing distal femoral epimetaphyseal structures and bone morphometrics on magnetic resonance (MR) images of fetuses. MATERIALS AND METHODS: An institutional review board approved this retrospective study, and informed consent was waived. Included were 272 MR imaging examinations (April 2004-July 2011) in 253 fetuses with a mean gestational age (GA) of 26 weeks 6 days (range, 19 weeks 2 days to 35 weeks 6 days) without known musculoskeletal abnormalities. Two independent readers qualitatively analyzed epiphyseal and metaphyseal shape, secondary ossification, and the perichondrium on 1.5-T echo-planar MR images and correlated the results with the GA that was derived from previous fetal ultrasonography (US). Diaphyseal and epiphyseal morphometric measurements were correlated with GA by means of the Pearson correlation and linear regression. MR imaging measurements of diaphyseal length and US normative values were compared graphically. Interreader agreement analysis was performed with weighted κ statistics and the intraclass correlation coefficient. RESULTS: With advancing GA, the epiphyseal shape changed from spherical (r(2) = 0.664) to hemispherical with a notch (r(2) = 0.804), and the metaphyseal shape changed from flat (r(2) = 0.766) to clearly undulated (r(2) = 0.669). Secondary ossification (r(2) = 0.777) was not observed until 25 weeks 3 days. The perichondrium decreased (r(2) = 0.684) from 20 weeks onward. Correlation coefficients were 0.897 for diaphyseal length, 0.738 for epiphyseal length, and 0.801 for epiphyseal width with respect to GA. The range of measurements of diaphyseal length was larger than that of the reported US normative values. Interreader agreement was good for bone morphometrics (intraclass correlation coefficient, 0.906-0.976), and moderate for bone characteristics (weighted κ, 0.448-0.848). CONCLUSION: Prenatal MR imaging allows visualization of human bone development in vivo by means of epimetaphyseal characteristics and bone morphometrics. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13112441/-/DC1.

Foetal and postnatal equine articular cartilage development: magnetic resonance imaging and polarised light microscopy.

Adult articular cartilage (AC) has a well described multizonal collagen structure. Knowledge of foetal AC organisation and development may provide a prototype for cartilage repair strategies, and improve understanding of structural changes in developmental diseases such as osteochondrosis (OC). The objective of this study was to describe normal development of the spatial architecture of the collagen network of equine AC using 1.5 T magnetic resonance imaging (MRI) and polarised light microscopy (PLM), at sites employed for cartilage repair studies or susceptible to OC. T2-weighted fast-spin echo (FSE) sequences and PLM assessment were performed on distal femoral epiphyses of equine foetuses, foals and adults. Both MRI and PLM revealed an early progressive collagen network zonal organisation of the femoral epiphyses, beginning at 4 months of gestation. PLM revealed that the collagen network of equine foetal AC prior to birth was already organised into an evident anisotropic layered structure that included the appearance of a dense tangential zone in the superficial AC in the youngest specimens, with the progressive development of an underlying transitional zone. A third, increasingly birefringent, radial layer developed in the AC from 6 months of gestation. Four laminae were observed on the MR images in the last third of gestation. These included not only the AC but also the superficial growth plate of the epiphysis. These findings provide novel data on normal equine foetal cartilage collagen development, and may serve as a template for cartilage repair studies in this species or a model for developmental studies of OC.

Distal femoral epiphyses ossification center diameter and third trimester gestational age in Iranian population.

OBJECTIVE: The epiphyses ossification centers appear late in gestation, when traditional biometric measurements are the least accurate, and they can be useful in determining third trimesters gestational age. To evaluate fetal distal femoral epiphysis (DFE) size in various ages of gestation and establish a reference chart for Iranian population. MATERIALS & METHODS: DFE diameter was measured in 1300 normal singleton pregnancies, between 28 and 40 weeks. Mean diameter in each week of gestation was evaluated. RESULTS: The DFE is not visualized in 28 weeks' gestation. It appeared in a small proportion of the fetuses (5%) as early as the 29th week. DFE was detectable by ultrasonography increased dramatically to 56% at 33 weeks' reaching 94% at 36 weeks and 100% at 37 weeks gestation. CONCLUSION: Ultrasonographic visualization of the distal femoral epiphyses ossification center is a useful marker of fetal third trimesters gestational age.

[Histogenesis of Japanese quail bone and cartilage tissues at the final stages of embryonic development in microgravity].

The article reports the results of comparative histological studies of skeleton development in 14- and 16-day Japanese quail embryos grown in space flight and in the 1-g gravity. The investigation revealed retardation of cartilage replacement by bone in the femur and tibia in space embryos as compared with their controls. Perichondral ossification metaphysis was reached by day 14 both in the space and control embryos. Destruction of cartilaginous diaphysis advanced onto the proximal and distal parts including the periphery. Tibia and femur cartilaginous cores in space embryos were destructed worse than in the controls in consequence of insufficient minerals supply. Perichondral ossification in the 16-day space and control embryos was alike close to completion spreading as far as the epiphysis. Long bones metaphysis was abundant in monomorphic cells as a growth bank existing during and 7 days post hatching. However, absence, in contrast to the controls, of osteogensis sites in long bones epiphysis, suggested retardation of chondrocytes calcification in these areas, as well as of ossification in space embryos.

Maturation of collagen Ketoimine cross-links by an alternative mechanism to pyridinoline formation in cartilage.

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH(4). On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis.

Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Here we report that Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone. Coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt factors show specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Moreover, Wif-1 was able to block Wnt3a-mediated activation of the canonical Wnt signalling pathway. Consequently, Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells. These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage biology.

Early lesions of articular osteochondrosis in the distal femur of foals.

Failure of the cartilage canal blood supply to epiphyseal growth cartilage has been implicated in the pathogenesis of articular osteochondrosis in horses and other animal species. In a previous study of the developmental pattern of the blood supply in the tarsus of foals, early lesions of osteochondrosis were consistently found in regions where the cartilage canal vessels traversed the chondro-osseous junction. The developmental pattern of blood vessels has also been described in the distal femoral epiphysis; however, the group of foals examined in that study did not have lesions of osteochondrosis in this location. Therefore, the relationship between the occurrence of early lesions of osteochondrosis and the developmental pattern of the blood supply to epiphyseal growth cartilage in this site in foals has not been examined. Distal femora were collected from 30 fetuses and foals (up to 11 months old) submitted for postmortem examination. Sections from the lateral trochlear ridge and medial femoral condyle of both hind limbs were examined histologically. Sixteen cartilage lesions were found in 7 of the 30 fetuses and foals. All lesions contained evidence of cartilage canal necrosis and ischemic chondronecrosis. The lesions were located in regions where cartilage canal vessels traversed the chondro-osseous junction, as previously observed in the tarsus. The location and morphology of lesions indicated that a subclinical stage of ischemic chondronecrosis existed that preceded and predisposed to the development of osteochondrosis dissecans and subchondral bone cysts.

Common skeletal growth retardation disorders resulting from abnormalities within the mesenchymal stem cells reservoirs in the epiphyseal organs pertaining to the long bones.

Among the objectives in writing the current chapter were the curiosity and the interest in allocating the sites and routes of migration of the reservoirs of the mesenchymal precartilaginous stem cells of the developing limbs in health and in disease. We chose to emphasize the events believed to initiate in these regions of stem cells, which may lead to growth retardation disorders. Thus, this narrow niche touches an enlarged scope of developmental biology angles and fields. The enclosed coverage sheds light on part of the musculoskeletal system, skeletogenesis, organogenesis of mobile structures and organs, the limbs, joints and digits (arthrology). It appears that the key role of the cartilage-bone regions is their responsibility to replenish the physis with committed chondrocytes, during the developmental, maturation and puberty periods. We shall start by outlining the framework of normal limb formation, the modalities, signals and the agents participating in this biological creation and regulation, illustrating potential sites that might deviate from normal development during the growth periods.

Development of the mandibular condylar cartilage in human specimens of 10-15 weeks' gestation.

This study analyses some morphological and histological aspects that could have a role in the development of the condylar cartilage (CC). The specimens used were serial sections from 49 human fetuses aged 10-15 weeks. In addition, 3D reconstructions of the mandibular ramus and the CC were made from four specimens. During weeks 10-11 of development, the vascular canals (VC) appear in the CC and the intramembranous ossification process begins. At the same time, in the medial region of the CC, chondroclasts appear adjacent to the vascular invasion and to the cartilage destruction. During weeks 12-13 of development, the deepest portion of the posterolateral vascular canal is completely surrounded by the hypertrophic chondrocytes. The latter emerge with an irregular layout. During week 15 of development, the endochondral ossification of the CC begins. Our results suggest that the situation of the chondroclasts, the posterolateral vascular canal and the irregular arrangement of the hypertrophic chondrocytes may play a notable role in the development of the CC.

Lysozyme in epiphyseal cartilage. II. The effect of egg white lysozyme on mouse embryonic femurs in organ cultures.

Embryonic mouse femoral cartilage, like the epiphyseal cartilage of the calf scapula, contains large amounts of lysozyme. The addition of egg white lysozyme to organ cultures of embryonic mouse femurs induces unique alterations in the gross and microscopic morphology of the femurs. The sites of these alterations are precisely related to the natural distribution of lysozyme in calf scapula. If the exogenous lysozyme is withdrawn from the culture, the morphological changes disappear, accompanied by a resumption or derepression of growth. The effect on growth is evident only in 17-day embryos. These observations support the idea that lysozyme has a physiological role in cartilage, perhaps related to a regulatory mechanism in bone formation.

Prenatal growth map of the mouse knee joint by means of deformable registration technique.

Joint morphogenesis is the process during which distinct and functional joint shapes emerge during pre- and post-natal joint development. In this study, a repeatable semi-automatic protocol capable of providing a 3D realistic developmental map of the prenatal mouse knee joint was designed by combining Optical Projection Tomography imaging (OPT) and a deformable registration algorithm (Sheffield Image Registration toolkit, ShIRT). Eleven left limbs of healthy murine embryos were scanned with OPT (voxel size: 14.63µm) at two different stages of development: Theiler stage (TS) 23 (approximately 14.5 embryonic days) and 24 (approximately 15.5 embryonic days). One TS23 limb was used to evaluate the precision of the displacement predictions for this specific case. The remaining limbs were then used to estimate Developmental Tibia and Femur Maps. Acceptable uncertainties of the displacement predictions computed from repeated images were found for both epiphyses (between 1.3µm and 1.4µm for the proximal tibia and between 0.7µm and 1.0µm for the femur, along all directions). The protocol was found to be reproducible with maximum Modified Housdorff Distance (MHD) differences equal to 1.9 µm and 1.5 µm for the tibial and femoral epiphyses respectively. The effect of the initial shape of the rudiment affected the developmental maps with MHD of 21.7 µm and 21.9 µm for the tibial and femoral epiphyses respectively, which correspond to 1.4 and 1.5 times the voxel size. To conclude, this study proposes a repeatable semi-automatic protocol capable of providing mean 3D realistic developmental map of a developing rudiment allowing researchers to study how growth and adaptation are directed by biological and mechanobiological factors.

Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.

[Changes of the femoral bone microstructure in the reindeer during the fetal period of ontogenesis].

The diaphysis of the femoral bone was studied morphometrically in 30 reindeer fetuses aged 2-7.5 months. The formation of the diaphyseal cavity of the femoral bone took place from 3 till 7.5 months, rapidly progressing during all the fetal period. The thickness of the periosteum in the epiphysis and the diaphysis of the bone increased from 2 to 6 months inclusive, whereupon it decreased by the time of birth. The cartilaginous tissue in the epiphyses was present from 2 till 7.5 months; its growth was registered up to 3 months in a proximal epiphysis, while it continued till 4 months in a distal epiphysis. Later on, the thinning of the cartilage was noted till the birth. The thickness of spongy substance of bone epiphyses increased with the fetal age. The osteons in the diaphysis of the femoral bone were formed in 2-month-old fetus, their numbers were found to increase with age. The compact substance of the diaphysis of the femoral bone increased in thickness till 5 months, whereupon the process of thinning of tissue till the time of birth, was noted.

Expression patterns of matrix metalloproteinases and vascular endothelial growth factor during epiphyseal ossification.

UNLABELLED: In situ hybridization studies allowed for the localization of three MMPs and the angiogenic factor VEGF during secondary ossification. MMPs were widely expressed during ossification of the secondary center, whereas expression of VEGF was restricted to later stages. INTRODUCTION: The spatiotemporal expression patterns of the matrix metalloproteinases gelatinase-B (MMP-9), collagenase-3 (MMP-13), and membrane-type 1 metalloproteinase (MMP-14) and the angiogenic peptide vascular endothelial growth factor (VEGF) were studied during development of the proximal epiphysis of the rat tibia. MATERIALS AND METHODS: Cell expression was analyzed by in situ hybridization. Studies on osteoclastic activity, matrix mineralization, cell proliferation, and vascular progression were also performed. RESULTS: MMP-9, MMP-13, and MMP-14 were expressed in discrete perichondrial cells that gave way to sites of intrachondral canal formation. High expression levels for the three MMPs were found at the blind ends of advancing intrachondral canals and at the expanding borders of the marrow space. Signals for MMP-9 and MMP-13 were in close proximity but did not overlap, whereas MMP-14 was expressed in both MMP-9+ and MMP-13+ cells. VEGF was not expressed during formation of intrachondral vascular canals but was observed in hypertrophic chondrocytes during formation of the bone marrow cavity. CONCLUSIONS: Expression of MMPs and VEGF are constant events during development of the secondary ossification center. We propose that MMPs are involved in targeting proteolytic activity during epiphyseal development. VEGF is not expressed during early formation of vascular canals, but it may have a role in the formation of the bone marrow cavity.

Zebrafish evx1 is dynamically expressed during embryogenesis in subsets of interneurones, posterior gut and urogenital system.

The even-skipped-related homeobox genes (evx) are widely distributed through animal kingdom and are thought to play key role in posterior body patterning and neurogenesis. We have cloned and analyzed the expression of evx1 in zebrafish (see also Borday et al. (Dev. Dyn. 220 (2001) in press) which displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurones prior to their axonal outgrowth, identified as primary interneurones and a subset of Pax2.1(+) commissural interneurones. In the hindbrain, evx1 is expressed in reticulospinal interneurones of rhombomeres 5 and 6 as well as in rhombomere 7 interneurones. The latest emerging evx1(+) interneurones in the hindbrain correspond to commissural interneurones. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the uro-genital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast other vertebrates, evx1displays no early nor caudal expression in zebrafish.

Use of a novel joint-simulating culture system to grow organized ex-vivo three-dimensional cartilage-like constructs from embryonic epiphyseal cells.

A method for growth and maintenance of vital cartilaginous tissue is necessary for cartilage repair by in-vitro produced biologic implants. A previously tested perfusion system simulating joint activity was used. Whole epiphyses collected from thirty 11-day-old chick embryos were divided into two groups. One group was grown in a tissue culture dish for 10 days. The other group was placed in a perfusion system termed a joint-simulating device (JSD). After a period of 10 days, histology and immunohistochemistry were performed on five epiphyses from each group. Histologically, epiphyses grown in the device coalesced into a homogenous three-dimensional mass. The bridging tissue between individual epiphyses was highly cellular (PCNA staining positive) and was composed of mesenchymal stem cells as shown by expression of FGF receptor 3. No such tissue formed between epiphyses in the tissue culture dish and the epiphyseal cores were shown to be necrotic. The rest of the epiphyses were evaluated for radioactive sulfate incorporation into glycosaminoglycans (GAGs). A tenfold increase in sulfate incorporation occurred in epiphyses grown within the JSD as compared to the traditional culture method. In conclusion, embryonic epiphyses could be a suitable source for the ex-vivo growth of tissue-engineered cartilage constructs that might later be used as an in-vivo cartilage implant. The joint simulating device effectively maintains cartilage viability and bioactivity for as long as 10 days.

Growth plate stress distribution implications during bone development: a simple framework computational approach.

Mechanical stimuli play a significant role in the process of long bone development as evidenced by clinical observations and in vivo studies. Up to now approaches to understand stimuli characteristics have been limited to the first stages of epiphyseal development. Furthermore, growth plate mechanical behavior has not been widely studied. In order to better understand mechanical influences on bone growth, we used Carter and Wong biomechanical approximation to analyze growth plate mechanical behavior, and explore stress patterns for different morphological stages of the growth plate. To the best of our knowledge this work is the first attempt to study stress distribution on growth plate during different possible stages of bone development, from gestation to adolescence. Stress distribution analysis on the epiphysis and growth plate was performed using axisymmetric (3D) finite element analysis in a simplified generic epiphyseal geometry using a linear elastic model as the first approximation. We took into account different growth plate locations, morphologies and widths, as well as different epiphyseal developmental stages. We found stress distribution during bone development established osteogenic index patterns that seem to influence locally epiphyseal structures growth and coincide with growth plate histological arrangement.

Initiation of the bony epiphysis in long bones: chronology of interactions between the vascular system and the chondrocytes.

Many events occur concurrently during the initiation of the secondary ossification center in the cartilaginous epiphyses of long bones. We have investigated the chronology of interactions between the vascular system and epiphyseal chondrocytes by culturing explanted heads of femurs and humeri from pre- and neonatal rabbits on the chorioallantoic membrane (CAM) of growing chick embryos. We confirmed that, on the whole, the epiphyseal cartilage was resistant to vascular invasion, whereas the physeal growth plate was resorbed. However, new CAM-derived cartilage canals occasionally penetrated through the articular surface. This caused death of those chondrocytes in the immediate vicinity of the canal but no further reaction. If explants already contained a bony epiphysis and were halved prior to culture, CAM-derived vessels were attracted to the spongiosa. From there they pushed into the uncalcified cartilage, indicating that calcification was not a prerequisite for vascular invasion. Where at least two vessels were in apposition, a new pseudo-ossification center was initiated: chondrocytes became hypertrophic and the matrix calcified. This suggests that cumulative release of diffusible factors from more than one vessel was the trigger for chondrocyte hypertrophy, which, in turn, led to the initiation of the bony epiphysis. CAM cultures thus provide an experimental model for both the quiescent angiogenesis of cartilage canal formation and the reactionary angiogenesis associated with chondrocyte hypertrophy. By exploiting the different anatomy of CAM-derived vascularity, events that occur concurrently in vivo can be specially separated in CAM culture.