Stefano Longato

Structure, formation and role of cartilage canals in the developing bone

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes?

We investigated the development of cartilage canals to clarify their function in the process of bone formation. Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three‐dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation. We conclude that cartilage canals contribute to both perichondral and endochondral bone formation and that osteoblasts have the same origin in both processes.

Epithelial and Muscular Regionalization of the Human Developing Anorectum

In the past, interpretations of anorectal development were mainly based on analysis of serially sectioned embryos of various nonhuman species as well as some human specimens. A four‐dimensional view of the developmental situation in the human has never been established nor connected to recent findings obtained from newer molecular techniques. We, therefore, investigated human embryonic and fetal pelves by means of immunohistochemistry and in situ hybridization to elucidate differentiation and interaction of epithelial and mesenchymal layers of the anorectum. To emphasize spatial as well as sequential morphological development, we produced three‐dimensional reconstructions of the specimens at hand. Research conducted proved that the decisive steps of epithelial and muscular differentiation occur between the 7th and 9th week after conception. This study elucidates a biphasic epithelial “closure” in the anal canal and interactions between epithelium, smooth musculature, and skeletal musculature. Based on the results presented here, it is possible to describe the pathogenesis of two anorectal malformations: the imperforate anal membrane and the anal membrane stenosis. This study will now provide the basis for further research into developmental processes occurring before the ones examined. Anat Rec, 1449‐1458, 2007.

Bone development in the femoral epiphysis of mice: the role of cartilage canals and the fate of resting chondrocytes.

In mammals, the exact role of cartilage canals is still under discussion. Therefore, we studied their development in the distal femoral epiphysis of mice to define the importance of these canals. Various approaches were performed to examine the histological, cellular, and molecular events leading to bone formation. Cartilage canals started off as invaginations of the perichondrium at day (D) 5 after birth. At D 10, several small ossification nuclei originated around the canal branched endings. Finally, these nuclei coalesced and at D 18 a large secondary ossification centre (SOC) occupied the whole epiphysis. Cartilage canal cells expressed type I collagen, a major bone‐relevant protein. During canal formation, several resting chondrocytes immediately around the canals were active caspase 3 positive but others were freed into the canal cavity and appeared to remain viable. We suggest that cartilage canal cells belong to the bone lineage and, hence, they contribute to the formation of the bony epiphysis. Several resting chondrocytes are assigned to die but others, after freeing into the canal cavity, may differentiate into osteoblasts. Developmental Dynamics 236:2077–2088, 2007.

Localization of tartrate‐resistant acid phosphatase (TRAP), membrane type‐1 matrix metalloproteinases (MT1‐MMP) and macrophages during early endochondral bone formation

Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate‐resistant acid phosphatase (TRAP) and membrane type‐1 matrix metalloproteinases (MT1‐MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP‐positive chondroclasts resorbed the cartilage matrix. However, occasionally co‐expression of TRAP and MT1‐MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1‐MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1‐MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone‐lining cells. Considering the well‐known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1‐MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.

VEGF and its role in the early development of the long bone epiphysis

In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio‐temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.

Computer-Assisted Surgery in the Edentulous Jaw Based on 3 Fixed Intraoral Reference Points

PURPOSE In computer-assisted implantation surgery, the transfer of prosthodontic-guided planning to the operative site is usually based on a registration template. The precise repositioning of the registration template is crucial for high accuracy and is compromised in edentulous jaws. The purpose was to determine the in vitro registration and targeting accuracy for surgical navigation in the edentulous jaw based on 3 fixed intraoral reference points. MATERIALS AND METHODS Edentulous maxilla and mandible cadaver specimens were provided with 3 fixed reference-point screws. A resin template with matrices for the fixed reference-point screws was produced and connected to a Vogele-Bale-Hohner registration mouthpiece and external registration frame with a snap-lock system. Surgical implants were planned on computed tomographic data and the corresponding dental stone casts were drilled under guidance of an optical navigation system. For evaluation of the registration accuracy, fiducial registration error was recorded and application accuracy was evaluated by fusion of postsurgical computed tomographic scans of the drilled dental stone casts with the presurgical planning computed tomogram. RESULTS In 9 maxillas and 5 mandibles, 14 registrations and 104 stone cast drillings were performed. The mean fiducial registration error was 0.49 +/- 0.14 mm (0.37 to 0.9 mm). The mean total error at the tip of the borehole was 0.88 +/- 0.65 mm (0.0 to 4.24 mm). The mean lateral errors were 0.51 +/- 0.49 mm (0.0 to 2.80 mm) at the base and 0.46 +/- 0.34 mm (0.0 to 1.5 mm) at the tip of the borehole, respectively. The mean angular error was 0.83 +/- 0.60 degrees (0.0 to 2.5 degrees ). CONCLUSION Three fixed intraoral reference points successfully support a registration mouthpiece and provide in vitro registration and targeting accuracy that is comparable to tooth-supported registration templates or bone marker registration.

Growth curves of the fetal prostate based on three-dimensional reconstructions: a correlation with gestational age and maternal testosterone levels

There are two papers in this section this month. The first, by authors from Austria, concerns growth curves of the fetal prostate, correlating gestational age and maternal testosterone using three‐dimensional reconstructions. The second, from the UK, investigated testicular microlithiasis, and found no convincing evidence that alone it is a premalignant condition.

Extraordinary branching pattern of the aortic arch.

A CT‐scan of a 75‐year‐old patient showed an aneurysm of the descending aorta with a maximum diameter of 4.8 cm involving the left subclavian artery. Due to the fact that the patient had several comorbidities including a severe chronic obstructive pulmonary disease he was treated only conservatively. However, there were several interesting findings on the CT‐scan: the branching pattern of the aortic arch revealed a left carotid artery arising as first side branch of the distal part of the ascending aorta. This vessel crosses the midline right in front of the trachea. Apart from that the patient did not have a brachiocephalic trunk: the right carotid artery arose as the first branch from the aortic arch and crossed the right subclavian artery anteriorly. Furthermore, the left subclavian seemed to arise from the descending aorta and not from the aortic arch. Clin. Anat. 26:1006–1007, 2013.

Dysplasia, architectural derangement, extracellular matrix degradation and regulation of Tgf-ß3 within the interureteric muscle in children with vesicoureteral reflux

Purpose The interureteric muscle represents an essential part of an adequate antireflux mechanism. Biopsies from children operated on for vesicoureteric reflux were investigated using morphological and immunohistochemical methods to evaluate smooth muscle configuration, potential muscle dysplasia and connective tissue changes in that area. Material and methods Biopsies of the interureteric muscle were obtained from 22 ureterorenal units undergoing reflux surgery. Mean age of patients was 32 months, reflux grades III to V, respectively. Routine histological paraffin embedded sections were stained -actin to evaluate the presence,α with haematoxylin, eosin and smooth muscle allocation and morphological integrity of the interurerteric smooth muscle architecture. Indirect immunohistochemical methods were used to assess the extracellular matrix for collagen composition. Extracellular matrix involvement was assessed estimating transforming growth factor- s III (TGF-s3) expression. Results All biopsies showed different grades of muscle atrophy and degradation. In these smooth muscle tissue lacking regions, disordered fibre arrangement associated with a 2 to 3-fold increase in endomysial and perimysial connective tissue in particular collagen type III was observed. Increased mesenchymal cell proliferation in the degenerated tissue areas was associated with a significant TGF-s3 expression of the surrounding muscle cells and correlated with organized collagen deposition. Conclusions Extracellular matrix remodelling with interstitial fibrosis of the longitudinal muscle sheath of the ureterovesical junction may be mediated through upregulation of TGF-s3. Diminution or even complete loss of contractile elements of the interureteric muscle in children with congenital vesicoureteral reflux is obviously associated with an inadequate antireflux mechanism of the vesicoureteral junction.