Reviewer's comments on the paper entitled: “Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: A possible role of the terminal webs in ameloblast movement during secretion,” by S. Nishikawa

The ultrastructural distribution of two noncollagenous proteins, osteopontin (OPN) and osteocalcin (OC), originally extracted from bone matrix and proposed to play an important role in bone formation, was examined in the matrices of bone and cartilage from embryonic and postnatal chicken tibial growth plates by high-resolution immunocytochemistry using the colloidal gold technique. In bone, immunolabeling patterns using polyclonal antibodies against chicken OPN and OC were generally similar in that both showed an intense, but regionally variable, labeling of mineralized bone matrix and small mineralization loci dispersed throughout the osteoid and containing prominent condensed organic material. Unmineralized osteoid showed weak-to-moderate labeling. In the mineralized bone matrix proper, labeling was predominantly associated with amorphous, electron-dense patches of organic material among the collagen fibrils. In growth plate cartilage, both proteins first appeared related to calcified cartilage in the hypertrophic zone, although the labeling patterns were somewhat different. For OPN, gold particles were mostly associated with an organic lamina limitans-like density containing condensed, filamentous organic matrix at the periphery of small nodules and large masses of calcified cartilage, with additional moderate labeling throughout the interior of the calcified cartilage. For OC, labeling was observed over filamentous structures throughout the calcified cartilage matrix, with some, but less, labeling at the periphery. In the lowermost zones of the growth plate, the major reaction using both antibodies was found over a layer of dense, amorphous organic material at the periphery of the calcified cartilage at the future bone/calcified cartilage interface, a labeling pattern that persisted following bone deposition at these sites. OPN and to a lesser extent OC were also concentrated in cement (resting, reversal) lines. Throughout the bone and cartilage of the tibia, cells of both the osteoblastic and the osteoclastic lineages were found directly apposed to labeled surfaces and lamina limitans of organic matrix containing OPN and OC. In summary, it is concluded form the immunocytochemical data presented here that the association of OPN and OC with mineralized regions of the extracellular matrices of bone and cartilage and the accumulation of these proteins at tissue surfaces and interfaces are consistent with the hypotheses that they play a role in the extracellular mineralization process per se and/or that they may mediate cell adhesion and dynamics.© Willey-Liss, Inc.     

High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia.

The ultrastructural distribution of two noncollagenous proteins, osteopontin (OPN) and osteocalcin (OC), originally extracted from bone matrix and proposed to play an important role in bone formation, was examined in the matrices of bone and cartilage from embryonic and postnatal chicken tibial growth plates by high-resolution immunocytochemistry using the colloidal gold technique. In bone, immunolabeling patterns using polyclonal antibodies against chicken OPN and OC were generally similar in that both showed an intense, but regionally variable, labeling of mineralized bone matrix and small mineralization loci dispersed throughout the osteoid and containing prominent condensed organic material. Unmineralized osteoid showed weak-to-moderate labeling. In the mineralized bone matrix proper, labeling was predominantly associated with amorphous, electron-dense patches of organic material among the collagen fibrils. In growth plate cartilage, both proteins first appeared related to calcified cartilage in the hypertrophic zone, although the labeling patterns were somewhat different. For OPN, gold particles were mostly associated with an organic lamina limitans-like density containing condensed, filamentous organic matrix at the periphery of small nodules and large masses of calcified cartilage, with additional moderate labeling throughout the interior of the calcified cartilage. For OC, labeling was observed over filamentous structures throughout the calcified cartilage matrix, with some, but less, labeling at the periphery. In the lowermost zones of the growth plate, the major reaction using both antibodies was found over a layer of dense, amorphous organic material at the periphery of the calcified cartilage at the future bone/calcified cartilage interface, a labeling pattern that persisted following bone deposition at these sites. OPN and to a lesser extent OC were also concentrated in cement (resting, reversal) lines. Throughout the bone and cartilage of the tibia, cells of both the osteoblastic and the osteoclastic lineages were found directly apposed to labeled surfaces and lamina limitans of organic matrix containing OPN and OC. In summary, it is concluded from the immunocytochemical data presented here that the association of OPN and OC with mineralized regions of the extracellular matrices of bone and cartilage and the accumulation of these proteins at tissue surfaces and interfaces are consistent with the hypotheses that they play a role in the extracellular mineralization process per se and/or that they may mediate cell adhesion and dynamics.     

Expression of Collagen Types I,II, IX,and X in the Mineralizing Turkey Gastrocnemius Tendon

The turkey gastrocnemius tendon mineralizes by intramembranous ossification with a transient chondrogenic phase. The mineralizing zone has hypertrophic chondrocytes similar to endochondral bone formation. These similarities prompted the evaluation of this tendon for the presence of type X collagen in the mineralizing zone. Tendons were removed, radiographed, decalcified, and embedded for frozen sections. Seral sections were H&E stained and immunostained individually with antibodies specific collagens (types I, II, IX, and X). Type I collagen was distributed widely throughout the mineralized tendon extracellular matrix. Types II and IX collagen were at the mineralized/non-mineralized junction. Type X collagen was in the pericellular matrix of hypertrophic chondrocytes and in some calcified matrix. These data support the theory that the gastrocnemius tendon has fibrocartilage characteristics and that type X collagen has a role in the tissue's mineralization. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.     

Immunolocalization of keratan sulfate proteoglycan in rat calvaria

We investigate, by the immunogold method, the localization of keratan sulfate (KS) proteoglycan in rat calvaria in order to clarify the detailed process of intramembranous ossification. KS was localized in bone nodules corresponding to calcified nodules, close to the saggital suture of calvaria. The immunoreactivity decreased in fully calcified regions distant from the suture. Electron microscopic observation revealed that KS was distributed in and around matrix vesicles, among collagen fibrils at the initial crystal deposition stage, and then concentrated in bone nodules. According to the progress of mineralization, KS tended to be localized in the peripheral region of the nodules. In addition, these nodules came in contact with collagen fibrils which also showed KS-positive reactivity. In cell organelles of osteoblasts, KS was detected in the Golgi apparatus. These findings suggest that osteoblasts in intramembranous ossification sites actively synthesize KS. KS in the calcified nodules, as well as other glycosaminoglycans in osteoid, may play an important role in additional and/or collagenous calcification by trapping calcium ions through its negative charge.     

Osteopontin distribution in the canine skeleton during growth and structural maturation

The distribution of osteopontin (OPN) was studied immunohistochemically in cells and extracellular matrix in the humerus, scapula, and lumbar vertebrae of growing (age: 6 weeks, 12 weeks, 4.5 months) and adult dogs. OPN was expressed in hypertrophic chondrocytes of epiphyseal cartilage and in chondrocytes of the deep zone of mature articular cartilage, where extracellular matrix was also stained. OPN expression was strongest in 4.5-month-old puppies in cells of the osteoblastic lineage. It also varied with microlocation and was pronounced in areas prone to resorption due to modelling and remodelling activities. Osteoclasts were always strongly labelled with OPN. OPN deposition in extracellular bone matrix was detected particularly as a delineation of cartilage cores within secondary trabeculae and as a lining of the trabecular surfaces in resorption microlocations. The OPN distribution pattern is discussed here for each cell population with regard to its functional implications.     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

Gender-specific distribution of glycosaminoglycans during cartilage mineralization of human thyroid cartilage

The role of glycosaminoglycans (GAG) in the process of cartilage mineralization, especially in the hypertrophic zone of growth plates, is not yet fully understood. Human thyroid cartilage can serve as a model to observe matrix changes associated with cartilage mineralization because the processes follow a distinct route, progress very slowly and show sexual differences. Histochemical staining for low sulphated GAG (chondroitin-4- and -6-sulphates) was decreased in the interterritorial matrix of thyroid cartilage starting at the beginning of the fifth decade, but not in the pericellular or territorial matrix of chondrocytes. Because cartilage mineralization progressed in the interterritorial matrix it seems likely that a decreasing content of chondroitin-4- and -6-sulphates is involved in the mineralization process. This hypothesis is supported by the observation that immunostaining for chondroitin-4- and -6-sulphates was weaker in mineralized cartilage areas than in unmineralized areas, whereas there was no difference in staining for keratan sulphate. In all life decades, female thyroid cartilages contained more chondrocytes with a territorial rim of chondroitin-4- and -6-sulphates probably preventing cartilage mineralization compared with age-matched male specimens. Taken together, the characteristic distribution pattern of chondroitin-4- and -6-sulphates being more concentrated in female than in male thyroid cartilages provided evidence that these macromolecules decrease in cartilage mineralization.     

Morphological observations concerning the pattern of mineralization of the normal and the rachitic chick growth cartilage

Summary The entire calcified layer of the chick growth cartilage is penetrated by canals that contains blood vessel complexes: some of these canals pass through all the layers of the cartilage from the resorptive zone at the metaphysis, through the mineralizing, hypertrophic, proliferative and resting regions. This study aimed to provide more details of the 3-D microanatomy of this region and to establish whether there are differences in the process and progress of mineralization compared with the established mammalian model.Proximal tibial heads from 6 to 8 weeks old normal and vitamin D deficient chickens were rapidly frozen and prepared for scanning electron microscopy using freezefracture, freeze-drying, plasma ashing, and chemical deproteinization techniques. Cartilage samples were also embedded in PMMA and polished for BSE imaging. Other samples were prepared for light microscopy.Zones of (mineralized) cartilage several cells thick separate adjacent canals. At the mineralizing front, calcification of the matrix is most advanced close to the canals, but the matrix adjacent to the canal lumens does not calcify. Mineralisation of the cartilage matrix is incomplete and small fenestrae of unmineralized matrix connect chondrocyte lacunae. These discontinuities in matrix calcification could serve as a route for diffusion of nutrients, metabolites and dissolved gases.The calcified cartilage is more mineralized than the contiguous developing bone. Osteoblasts surrounded by bone were seen to occupy the lacunae of hypertrophic chondrocytes. We tentatively suggest that some osteoblasts represent a terminal stage in the differentiation of hypertrophic chondrocytes.The rachitic cartilage was disorganised. It was penetrated by irrugular vascular canals and exhibited a greatly expanded hypertrophic zone. The matrix was mineralized and mineral particles and clusters were spread throughout the matrix. However, these centres did not become continuous with adjacent or contiguous mineral. The results indicate that an absence of vitamin D affects crystal growth rather than initiation.     

Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation. These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.     

Association of the C-Propeptide of Type II Collagen with Mineralization of Embryonic Chick Long Bone and Sternal Development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation.

These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.     

Osteoclastic invasion and mineral resorption of fetal mouse long bone rudiments are inhibited by culture under intermittent compressive force

To study the effect of low-magnitude mechanical stimuli on mineralized matrix metabolism, fetal mouse long bone rudiments were cultured for 5d in the absence or presence of intermittent (0.3 Hz) compressive force (ICF) of 132 g/cm2. ICF treatment stimulated mineralization of the diaphyseal bone collar as well as hypertrophic cartilage, but inhibited the release of 45Ca from prelabeled rudiments. ICF also inhibited the migration of osteoclasts and their precursors from the periosteum into the diaphysis and the subsequent excavation of a primitive marrow cavity. These data suggest that osteoclasts are sensitive to mechanical stimuli. Mechanical stimulation seems to protect the bone rudiment against osteoclastic attack and has a strong anabolic effect on mineral metabolism.     

Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice

The epiphyseal growth plate, where chondrocytes proliferate and differentiate, is the major site for longitudinal bone growth, matrix synthesis and mineralization. Glucose is an important energy source for the metabolism and growth of chondrocytes. The family of facilitative glucose transporters (GLUTs) mediates glucose transport across the plasma membrane in mammalian cells. We used immunocytochemical methods with anti-GLUT antibodies to investigate the localization of GLUTs in chondrocytes of the epiphyseal growth plate in 3 age groups of rats (3, 7, and 28 days after birth). Intense immunoreactivity of GLUT isoforms 1-5 was detected in chondrocytes of 3-day and 7-day old rats, and all GLUTs were localized in the maturation zone of the hypertrophic zone. On postnatal day 28, chondrocytes in the maturation zone showed intense GLUT1, 4 and 5 immunoreactivity, and weak GLUT2 and 3 immunoreactivity. In addition to chondrocytes in the maturation zone, those in the degenerative zone and in the zone of provisional calcification showed strong GLUT4 and 5 immunoreactivity. Autoradiography of bone sections from 4-week old mice injected with 14C-2-deoxyglucose showed high silver grain density within matrix tissue in the reserve and proliferative zones but not around chondrocytes. However, in the hypertrophic zone, silver grain density was high in matrix and chondrocytes. These data indicate that chondrocytes in the hypertrophic zones use glucose as energy source. High levels of GLUT4 expression imply that glucose use in chondrocytes is regulated by insulin. Expression of GLUT5 in chondrocytes suggests that fructose is also used as an energy source.     

Osteoclastic Invasion and Mineral Resorption of Fetal Mouse Long Bone Rudiments are Inhibited by Culture Under Intermittent Compressive Force

To study the effect of low-magnitude mechanical stimuli on mineralized matrix metabolism, fetal mouse long bone rudiments were cultured for 5d in the absence or presence of intermittent (0.3 Hz) compressive force (ICF) of 132 g/cm². ICF treatment stimulated mineralization of the diaphyseal bone collar as well as hypertrophic cartilage, but inhibited the release of ⁴⁵Ca from prelabeled rudiments. ICF also inhibited the migration of osteoclasts and their precursors from the periosteum into the diaphysis and the subsequent excavation of a primitive marrow cavity.

These data suggest that osteoclasts are sensitive to mechanical stimuli. Mechanical stimulation seems to protect the bone rudiment against osteoclastic attack and has a strong anabolic effect on mineral metabolism.     

Topographical and histological examination of osteophytes taken from arthrotic femoral heads.

Until now it is not known whether osteophytes of the femoral head develop because of pathological joint alterations or arise from normal remodeling processes secondary to osteoarthrosis. Firstly, we analysed the topographical localization of osteophytes. We then compared the extracellular matrix components of macroscopically normal cartilage from the margin of osteophytes with osteophytic cartilage from weight bearing and non-weight bearing zones by histochemical staining of low and heavily sulfated glycosaminoglycans. For examination 65 femoral heads were taken during endoprosthetic hip surgery. Osteophytes from different locations and macroscopically normal cartilage from the margin of osteophytes were excised, decalcified and embedded in paraplast. A lateral or medial localization of osteophytes (47 cases) was more common than a ventral or dorsal position (18 cases). Histochemical staining for low and heavily sulfated glycosaminoglycans from normal cartilage at the rim of osteophytes was stronger in the unmineralized cartilaginous zones compared to the mineralized cartilaginous zone. Weight bearing zones of osteophytic cartilage, on the other hand, showed an even distribution of the two differently sulfated glycosaminoglycans. Surprisingly, non-weight bearing zones of osteophytic cartilage showed a weaker staining for low and especially for heavily sulfated glycosaminoglycans in the superficial cartilage layer than in the deep cartilage layer. Altogether, osteophytic cartilage can be regarded as a reparative phenomenon for two reasons: Firstly, osteophytes arise very often at the weight bearing lateral and medial femoral head. Secondly, despite local differences in osteophytic cartilage, the same types of glycosaminoglycans are synthesized as in normal cartilage at the margin of osteophytes.     

Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification

We examined the spatio-temporal pattern of type X collagen mRNA and its protein in the embryonic chick vertebrae undergoing ossification by in situ hybridization and immunohistochemistry. Hypertrophic chondrocytes, producing type X collagen, were developed as islands of cells in a few vertebral body segments of stage 36 embryos. These cells were increased in number at stages 37 and 38 and they expressed high levels of type X collagen mRNA and deposited its protein in the matrix. Blood vessels entered from the perichondrium at stage 37 and invaded deeply into hypertrophic cartilage at stage 38. As the vertebrae grew further at stage 40, the leading front of active hypertrophic chondrocytes with high levels of type X mRNA shifted from the midvertebral perivascular area towards intervertebral borders, while the perivascular area retained a number of inactive hypertrophic chondrocytes with low levels of type X mRNA. Type X collagen was found in large amounts throughout the matrix areas containing both active and inactive hypertrophic chondrocytes. Calcium was detected by von Kossa's technique in hypertrophic cartilage matrix in a small amount at stage 37, in parts of the matrix with type X collagen deposition in succeeding stages, and finally in almost the entire area of type X collagen deposition at stage 45. The vertebral segments of stage 45 embryos also showed a clearly reversed pattern of expression between type X collagen mRNA and types II and IX collagen mRNAs. The results demonstrate that the production of type X collagen by hypertrophic chondrocytes precedes both vascular invasion and mineralization of the matrix, suggesting that hypertrophic chondrocytes have an important role in regulating these events.     

Origin-associated features of chondrocytes in mouse Meckel''s cartilage and costal cartilage: an in vitro study

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.     

Ultrastructural changes associated with the mineralization of deer antler cartilage

The maturation and mineralization of deer antler cartilage were investigated ultrastructurally by using enzymatic digestions and subsequent staining with ruthenium red (RR) or phosphotungstic acid (PTA). RR staining of matrix granules was observed in the immature prechondroblastic matrix and became more intense as the cartilage matured into a mineralized tissue. The granules got larger and more numerically dense in the mature matrix. There were matrix granules that coalesced around matrix vesicles or remnants of such in the mineralized zone. These granules were observed after demineralization, and they were RR and acidic PTA-positive (they were not susceptible to hyaluronidase nor trypsin digestion, however). It appears that the granules were modified such that the matrix vesicle formed a centralized nidus for mineralization. The growth of hydroxyapatite crystals along matrix granules (which in this zone may or may not represent proteoglycan monomers) may have caused the coalescence. Microfibrils associated with matrix granules probably represented the hyaluronic acid core of the large proteoglycan complexes because of their susceptibility to hyaluronidase digestion.     

Cartilage canals in human thyroid cartilage characterized by immunolocalization of collagen types I, II, pro-III, IV and X

In this study the collagenous composition of cartilage canals in human thyroid cartilage, which are perichondral invaginations of blood vessels and connective tissue, and the surrounding cartilage matrix were investigated by immunolabelling with specific antibodies against type I, II, pro-III, IV and X collagen. During childhood and early adolescence no cartilage canals were detected in thyroid cartilage, and immunolabelling for type IV collagen was restricted to basal lamina components of blood vessels in the perichondrium. First immunolabelling for type IV collagen, belonging to blood vessels in cartilage canals, in both sexes was detected about the end of the second decade; it was localized in the dorsal part of the thyroid cartilage plate. At this time thyroid cartilage has already reached its final form and size. As revealed by von Kossa staining, vascularization preceded mineralization and ossification. In contrast to the male thyroid cartilage plate, no immunostaining for type IV collagen and no ossification was detected in the ventral half of female thyroid cartilage even in advanced age. The extracellular matrix of cells in cartilage canals showed positive immunostaining for collagen types I and pro-III as well as for collagen type II, indicating that the cells in the canal possess fibroblastic and chondrogenic properties. The extracellular matrix of hypertrophic chondrocytes adjacent to cartilage canals showed strong immunoreactivity for type X collagen. First mineralization was detected close to cartilage canals, suggesting that mineralization in human thyroid cartilage starts in the extracellular matrix adjacent to cartilage canals.