Cartilage Canals Research Articles

Cartilage matrix changes in the developing epiphysis: Early events on the pathway to equine osteochondrosis?

The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

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.

Micro-computed tomography of early lesions of osteochondrosis in the tarsus of foals

IntroductionOsteochondrosis (OC) is an important developmental orthopedic disease of human and equine patients. The disease is defined as a focal disturbance in enchondral ossification. In horses, the disturbance can occur secondary to failure of the blood supply to growth cartilage. Diagnosis of the early, subclinical stages that can clarify the etiology is currently confined to cross-sectional histological examination. The potential for micro-computed tomography (micro-CT) with angiography to detect early lesions of OC has not yet been investigated. Materials and methodsNine Standardbred foals bred from parents with OC of the tarso-crural joint were sacrificed at weekly intervals from birth to 7 weeks of age. Permanent barium angiograms were created within one hind limb post mortem, and samples collected from two predilection sites for OC within the tarso-crural joint of the perfused hind limb. The resulting 18 sample blocks were scanned with a custom-built micro-CT equipment set-up, and analyzed as 2D slices and 3D volume rendered models before sectioning for conventional histological examination. ResultsHistological examination identified eight early lesions in seven locations within six joints from the nine foals. Micro-CT with angiography was able to detect seven lesions in the same sites as histological examination. Lesions consisted of non-perfused foci within growth cartilage. No perfused vessels exited from subchondral bone deep to any lesion. Six of the seven lesions were associated with focal defects in the subchondral bone plate. Evidence of ongoing ossification was seen in three out of the seven lesions and included one separate center of ossification. ConclusionMicro-CT was a useful technique for examination of early lesions of OC. The results of micro-CT were compatible with failure of cartilage canal vessels at the point where they cross the ossification front. Resultant areas of ischemic chondronecrosis were associated with focal delay in enchondral ossification as visualized in 3D volume rendered models. Micro-CT combined with histology clarified the role of different forms of ossification in the secondary repair responses to lesions.

Perlecan, the “jack of all trades” proteoglycan of cartilaginous weight‐bearing connective tissues

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.

Epiphyseal cartilage canal blood supply to the tarsus of foals and relationship to osteochondrosis

Pathological changes in the blood supply to growth cartilage have been implicated in the pathogenesis of osteochondrosis (OC) in horses, but have not been reported using vascular perfusion techniques. To describe the developmental pattern of cartilage canal vessels in the distal tibial epiphysis and talar growth cartilage of foals. Nine foals bred from parents with OC were sacrificed between the ages of 0 and 7 weeks to undergo a barium perfusion procedure. The distal end of the tibia and the entire talus were cleared in methyl salicylate and perfused vessels studied in the intact bones. Slabs with a thickness of 4-5 mm from 3 predilection sites for OC were examined in the stereomicroscope and with light microscopy. Cartilage canals were present for a limited period of growth. Perfused vessels initially entered canals from the perichondrium. Vessels in the proximal portion of canals retained their perichondrial arterial source throughout. With time, the ossification front advanced to incorporate the mid-portion of canals; and anastomoses formed between canal vessels and subchondral vessels. A shift occurred and vessels in the distal terminus of canals came to use subchondral vessels as their arterial source. Twelve histological lesions were found in 7 foals. All contained necrotic vessels surrounded by necrotic growth cartilage and 3 caused macroscopically visible delay in endochondral ossification. Lesions were located where vessels traversed the ossification front to enter the distal terminus of canals. Cartilage canal vessels are particularly susceptible to failure at the point where they cross the ossification front, with consequences for the viability of those chondrocytes that depend on them. A better understanding of how lesions of OC arise may improve the ability to identify, monitor, prevent and treat this disorder. Involvement of cartilage canals in the pathogenesis of equine tarsal OC plausibly explains several clinical features of this disease.

Endochondral Ossification of Chick Embryonic Femora in vitro and on Chorioallantoic Membrane

We observed angiogenesis and endochondral ossification of the femora of chick embryos in vitro, on chorioallantoic membrane (CAM), and in vivo to clarify the cellular processes of avian endochondral ossification. We found that the inside of the in vitro femora was still filled with chondrocytes, despite being cultured for 10 days, while calcification of the diaphysis was not observed. As well, only the cartilage tissue of the epiphysis was enlarged. By contrast, blood vessels invaded into the diaphysis and the marrow cavity was formed in CAM-cultured femora. Cartilage canals extended from the marrow cavity and reached to the resting chondrocyte zone, with normal endochondral ossification occurring as in vivo. This study demonstrates that endochondral ossification occurs in femora in CAM culture similar to that in in vivo femora, but not in in vitro femora. The ossification is dependent on vascular invasion into the embryonic femora. In conclusion, for the endochondral ossification of long bones it is essential to supply the embryo with blood vessels. CAM culture system was found to be a superior endochondral ossification model of the embryonic femur.

Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

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.

Cartilage canals in the medial coronoid process of young Golden Retrievers

The aim of the study was to establish the location of cartilage canals in the medial coronoid process (MCP) of the ulna of young Golden Retrievers, a breed that is predisposed to fractures of the medial coronoid process (FMCP). To determine whether the presence of cartilage canals could be associated with the predilection site of FMCP, the right elbows of nine young Golden Retrievers (aged 4, 6, 8, 10, 13, 16, 18, 22 and 24 weeks) were dissected and, with no prior decalcification, the formaldehyde-fixed MCPs were embedded in methylmethacrylate. The entire MCPs were serially sectioned in the frontal plane from cranial to caudal and the sections (5 μm) were routinely stained. Between the ages of 5 and 10 weeks, three main cartilage canals were visible – medial, central and lateral. All originated from the periosteum of the distal parts of the MCP and ended proximally under the articular cartilage. Branches of the main canals were seen more cranially and caudally. At the age of 13 weeks, the central canal was absent, and the remaining canals showed a smaller diameter. From 16 weeks onwards, no cartilage canals were seen. No direct relationship could be established between the predilection site of FMCP (lateral part of the MCP) and the presence/absence of cartilage canals, since both medial and lateral canals disappeared at the same age. Further research is needed to elucidate the pathogenesis of FMCP.

Early lesions of osteochondrosis in the distal tibia of foals

Material available for research into osteochondrosis (OC) in humans tends to represent chronic lesions. Comparative studies of early lesions in young animals are, therefore, important in clarifying the pathogenesis of OC in humans. Recent studies in pigs provide strong evidence that lesions of articular OC are associated with a focal failure in the cartilage canal vascular supply to epiphyseal growth cartilage (articular-epiphyseal cartilage complex excluding the articular cartilage). The purpose of the present study was to examine histological sections from a specific predilection site for articular OC in the distal tibia of a large number of young foals to determine if the same is true in horses. Material from the distal tibiae of 100 foals aged from 191 days of gestation to 153 days old was collected from routine submissions of fetuses and foals for post mortem examination. The tibiae were band-sawed into slabs, and selected slabs were processed for histology, stained with hematoxylin and eosin, and examined using light microscopy. Early subclinical developmental stages of OC were found in the most common site for clinical OC lesions of horses in nine of 100 foals aged 12 to 122 days old. All lesions contained areas of chondrocyte necrosis that were associated with cartilage canal necrosis in five of nine foals. Five of these foals also had focal disruption of enchondral ossification at the chondro-osseous junction in the same site. Early lesions purported to play a role in the initial stages of articular OC in the distal tibia of horses were characterized by chondrocyte necrosis and likely occurred secondary to a failure of cartilage canal vascular supply to epiphyseal growth cartilage. The similarities in appearance between early lesions of piglets and foals suggest that information gained in one species may be transferable to others, including humans.

Effect of in ovo immobilization on development of chick hind‐limb articular cartilage: An evaluation using micro‐MRI measurement of delayed gadolinium uptake

To examine the effect of immobilization on the development of articular cartilage, we assessed glycosaminoglycan (GAG) content in the chick articular surface by delayed gadolinium-enhanced MRI of cartilage (dGEMRIC). Chick embryos were paralyzed by decamethonium bromide (DMB) from day 10 to either day 13 or day 16. The GAG content of the chick knee was compared with that of nonparalyzed chick embryos. Histologic analysis was unable to quantify GAG content; however, dGEMRIC demonstrated that GAG content was higher in the femoral condyles of the nonparalyzed embryos on day 13, and on day 16 the GAG content was lower in both the femoral condyles and the tibial plateaus of the nonparalyzed embryos. These results suggest that paralysis delays embryonic hind-limb development. Osteoblastic activity at the cartilage canal, as demonstrated by staining for alkaline phosphatase (ALP), was present only in the nonparalyzed chick embryos on day 16. The GAG content of the cartilage decreased when the cartilage canals began to form on day 16. The effect of immobilization on hind-limb development was indicated by the differences in the GAG content of the cartilage anlage measured by dGEMRIC in the developing knee joint of paralyzed and nonparalyzed embryonic chicks.

Matrix Metalloproteinase-9 Induces the Formation of Cartilage Canals in the Chondroepiphysis of the Neonatal Rabbit

An investigation of matrix metalloproteinase-9 (MMP-9) and its influence on vascular invasion in the secondary ossification center at the chondroepiphysis of developing long bones was undertaken. The effect of MMP-9 was compared with that of basic fibroblast growth factor (b-FGF), a potent angiogenic factor, and we assessed the chorioallantoic membrane (CAM) culture as a model for angiogenesis in osteochondral tissue. Seventy-two femoral and seventy-two humeral heads of thirty-six four-day postnatal rabbits were dissected immediately after each animal was killed. Solutions of MMP-9, b-FGF, and phosphate-buffered saline solution were applied, and the femoral and humeral chondroepiphyseal explants were incubated for ten days in CAM culture. This was used as an in vivo model to investigate the growth of blood vessels into the femoral and humeral heads of the neonatal rabbit. The explants were harvested from the CAM culture and analyzed histologically. A three-day incubation was also performed to look for early signs of vascular ingrowth into the cartilage matrix. One hundred and twenty epiphyses from thirty rabbits were placed onto CAM culture successfully; of these, two were harvested at three days to assess early changes and 118 were harvested at ten days. Forty of the 118 cultures were still viable when harvested after ten days, giving a 33% yield. Both MMP-9 and b-FGF caused an increased vascular invasion into the chondroepiphysis. New blood vessels derived from the chorioallantoic membrane within cartilage canals were more numerous in MMP-9 treated epiphyses, and larger canals were more commonly seen when compared with a control group. These findings confirmed that b-FGF is angiogenic at the chondroepiphysis. Matrix metalloproteinase-9 appears to be implicated in vascular invasion and induces the formation of new cartilage canals at the chondroepiphysis. The CAM culture model was a useful model for investigating angiogenesis in osteochondral tissue. This study adds to the understanding of the complex biochemical interaction that occurs in cartilage when the advancing vasculature begins growing into the chondroepiphysis. A better knowledge of this angiogenic process will enable a better understanding of the pathological failure or disturbance of vasculogenesis, which results in dysplastic growth disorders and osteonecrosis.

Identification and location of bone‐forming cells within cartilage canals on their course into the secondary ossification centre

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.

Computerized Three‐Dimensional Reconstruction of Cartilage Canals in Chick Tibial Chondroepiphysis

Cartilage canals have been extensively investigated, in particular as to their mode of formation, morphologic distribution, function and fate. We studied the morphological pattern of the cartilage canals of the right upper chondroepiphysis of the tibia of chick embryos, Hamburger-Hamilton stages 35-42, in serial sagittal wax sections and in reconstructions made with AutoCAD software. The spatial arrangement of the canals is presented in a series of drawings made according to computerized images. The canals were penetrated from three distinct surfaces: the anterior and superior surfaces of the tubercle and the posterior surfaces of the medial and lateral condyles. Immediately after entering, nearly all the canals were extended toward the medial or lateral aspects of the chondroepiphysis, from which no canals took their origin. All of the cartilage canals connected with the perichondrium, and their branching did not follow a specific pattern. The condylar canals did not unite with the tubercular ones.

The Cartilage Canals of the Epiphyseal Growth Cartilage and their Role in the Formation of Lesions of Osteochondrosis

Cartilage canals are temporary vascular structures within the growth cartilage. They were described as early as 1736 and the first perfusion study performed already in 1743. In its most complex form, the vascular structure consists of an arteriole, originating in the perichondrial plexus and branching out into an anastomosing network of capillaries. The capillaries circle back along the arteriole and rejoin into a venule that follows the course of the arteriole back to the plexus. The canals regress with age in a process designated as chondrification, where the content of the canals is replaced by cartilage. The process of chondrification is considered physiological; however, premature regression has been associated with the formation of lesions of osteochondrosis. The purpose of the present study was to gain further insight into the nature of chondrification and the initial steps in the pathogenesis of osteochondrosis and the relationship between these events and age, growth rate, weight and femoral shape of the individual animals. This involved studies of the distribution and pattern of regression of the cartilage canals. The articular‐epiphyseal cartilage complexes of the distal femur of 48 half‐siblings of the Norwegian Landrace pig breed were studied, combining techniques of perfusion and tissue clearing with histological and immunohistological methods. The results of this study indicate that the physiological regression of the cartilage canals is an orderly and time‐dependent process in which the endothelial cells at the termination of the vessels die by apoptosis, while perivascular cells differentiate into chondrocytes that fill in the cartilage canal with matrix. However, as the ossification front advances, the deepest portion of some of the canals form anastomoses with vessels of the subchondral bone, giving the axial portion of the growth cartilage a new source of blood supply. Lesions of osteochondrosis, typically occurring in the axial growth cartilage, are characterized by necrosis of the portion of the affected cartilage canal distal to a point of interruption, with subsequent necrosis of adjacent resting zone growth cartilage. This interruption occurs at the junction between cartilage and bone where anastomoses are formed between cartilage canal vessels and subchondral vessels.

Juvenile kyphosis in pigs

The development of kyphotic lesions in pigs with different weights from herds with a high frequency of the lesion was analysed pathologically, radiologically, and for alkaline phosphatase, calcium, magnesium, and phosphate in blood samples. The development of kyphosis was caused by the formation of ventral hemivertebrae due to the absence of ventral vertebral epiphyseal centres of ossification. Within the ventral parts of affected vertebral epiphyses, the retained chondroid tissue was dysplastic and the contents of cartilage canals with vessels often clotted with fibrin were reduced. As lesions such as fractures and inflammation that may be a secondary cause of kyphosis in pigs were absent and all blood parameters were within normal range, secondary causes of the condition, including metabolic disturbances, were excluded. It can be seen that the present type of porcine kyphosis does not develop until later in life as the ossification centres within the epiphyses of vertebrae have a retarded appearance. As the present porcine type of kyphosis pathomorphologically is comparable with Scheuermann's kyphosis in man, it constitutes a spontaneous model for this common cause of structural kyphosis of the thoracic or thoracolumbar spine.

Development of sensory innervation in rat tibia: co‐localization of CGRP and substance P with growth‐associated protein 43 (GAP‐43)

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.

Chondrocytes are released as viable cells during cartilage resorption associated with the formation of intrachondral canals in the rat tibial epiphysis

The development of cartilage canals is the first event of the ossification of the epiphyses in mammals. Canal formation differs from vascular invasion during primary ossification, since the former involves resorption of resting cartilage and is uncoupled from bone deposition. To learn more about the fate of resorbed chondrocytes during this process, we have carried out structural, cell proliferation, and in situ hybridization studies during the first stages of ossification of the rat tibial proximal epiphysis. Results concerning the formation of the cartilage canals implied the release of resting chondrocytes from the cartilage matrix to the canal cavity. Released chondrocytes had a well-preserved structure, expressed type-II collagen, and maintained the capacity to divide. All these data suggested that chondrocytes released into the canals remained viable for a specific time. Analysis of the proliferative activity at different regions of the cartilage canals showed that the percentage of proliferative chondrocytes at areas of active cartilage resorption was significantly higher than that in zones of low resorption. These results are consistent with the hypothesis that resting chondrocytes surrounding canals have a role in supplying cells for the development of the secondary ossification center. Since released chondrocytes are at an early stage of differentiation greatly preceding their entry into the apoptotic pathway and are exposed to a specific matrix, cellular, and humoral microenvironment, they might differentiate to other cell types and contribute to the ossification of the epiphysis.

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.

Immunohistochemical localization of calcitonin gene-related peptide and substance P in the rat knee cartilage at birth.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have been found in the perichondrium and within the cartilage canals. It is still unknown whether they exert a direct effect on chondrocytes during joint development. We processed 28 knees of newborn Wistar rats in 7 different fashions to perform histology and immunohistochemistry studies. Positive immunoreactivity against CGRP and SP was found in the inner aspect of the perichondrium in a close contact with chondrocytes. The presence of CGRP and SP indicates the presence of nerves fibers, and precedes the development of cartilage canals. Nerve fibers may play a role in the development of synovial joints before and during the presence of cartilage canals. The presence of CGRP and SP in the cartilage at birth may be involved in the early postnatal maturation of synovial joints. It remains to be determined whether autonomic innervation is later involved in age-related degenerative joint disease.