Legg-Calvé-Perthes disease. Histochemical and ultrastructural observations of the epiphyseal cartilage and physis

Biopsy specimens of the lateral aspect of the femoral head and neck were obtained from five children with Legg-Calvé-Perthes disease and were studied using histochemistry and electron microscopy. Beneath the normal articular cartilage there was a thick zone of hyaline (epiphyseal) cartilage containing sharply demarcated areas of hypercellular and fibrillated cartilage with prominent blood vessels. The fibrillated cartilage was strongly positive to alcian blue, weakly positive to periodic acid-Schiff, and positive to aniline blue. The interterritorial matrix in the hypercellular areas was weakly positive to both alcian blue and periodic acid-Schiff. Ultrastructural examination of these areas revealed many irregularly oriented large collagen fibrils and variable amounts of proteoglycan granules. These results suggest that in the fibrillar areas there are: (1) a high proteoglycan content, (2) a decrease in structural glycoproteins, and (3) a different size of collagen fibrils from that of normal epiphyseal cartilage. The hypercellular areas had a decrease in proteoglycans, glycoproteins, and collagen. The lateral physeal margin was often irregular, with a marked reduction of collagen and proteoglycan granules, and contained numerous large lipid inclusions. CLINICAL RELEVANCE: The abnormal areas in the epiphyseal cartilage of patients with Legg-Calvé-Perthes disease have different histochemical and structural properties from normal cartilage and from fibrocartilage. This suggests that the disease could be a localized expression of a generalized, transient disorder of epiphyseal cartilage that is responsible for delayed skeletal maturation. The cartilage lesions are similar to those seen in the vertebral plates in patients with juvenile kyphosis. Whether the epiphyseal cartilage abnormalities are primary or are secondary to ischemia remains uncertain; however, it appears that the collapse and necrosis of the femoral head could result from the breakdown and disorganization of the matrix of the epiphyseal cartilage, followed by abnormal ossification.     

Elastin fibers display a versatile microfibril network in articular cartilage depending on the mechanical microenvironments

Elastin fibers are major extracellular matrix macromolecules that are critical in maintaining the elasticity and resilience of tissues such as blood vessels, lungs and skins. However, the role of elastin in articular cartilage is poorly defined. The present study investigated the organization of elastin fiber in articular cartilage, its relationship to collagen fibers and the architecture of elastin fibers from different mechanical environments by using a kangaroo model. Five morphologies of elastin fibers were identified: Straight fiber, straight fiber with branches, branching fibers directly associated with chondrocyte, wave fiber and fine elastin. The architecture of the elastin network varied significantly with cartilage depth. In the most superficial layer of tibial plateau articular cartilage, dense elastin fibers formed a distinctive cobweb‐like meshwork which was parallel to the cartilage surface. In the superficial zone, elastin fibers were well organized in a preferred orientation which was parallel to collagen fibers. In the deep zone, no detectable elastin fiber was found. Moreover, differences in the organization of elastin fibers were also observed between articular cartilage from the tibial plateau, femoral condyle, and distal humerus. This study unravels the detailed microarchitecture of elastin fibers which display a well‐organized three‐dimensional versatile network in articular cartilage. Our findings imply that elastin fibers may play a crucial role in maintaining the integrity, elasticity, and the mechanical properties of articular cartilage, and that the local mechanical environment affects the architectural development of elastin fibers. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1345–1353, 2013     

Organization and cellular biology of the perichondrial ossification groove of ranvier: a morphological study in rabbits.

The perichondrial ossification groove of Ranvier, a circumferential groove in the periphery of the epiphyseal cartilage, was studied in rabbits whose ages ranged from one week to eight months using light and electron microscopy, autoradiography after labeling with 3H-thymidine, 3H-proline, and 3H-glucosamine, and histochemical staining for proteoglycans and alkaline phosphatase. By these methods, three groups of cells were identified within the groove: 1. A group of densely packed cells deep in the groove, which are the progenitor cells for the osteoblasts that form the bone bark, a cuff of bone surrounding the epiphyseal growth-plate region and the adjacent part of the metaphysis. 2. A group of more widely dispersed, relatively undifferentiated mesenchymal cells and fibroblasts, some of which are chondroblast precursors that probably contribute to appositional chondrogenesis and growth in width of the epiphyseal cartilage. 3. Fibroblasts and fibrocytes among sheets of highly oriented and organized collagen fibers which form a fibrous layer that is continuous with the outer fibrous layer of the periosteum and with the perichondrium. This layer also sends fibers into the epiphyseal cartilage and anchors the periosteum firmly to the epiphyses as bone growth proceeds.     

Ultrastructural Modifications of the Extracellular Matrix Upon Calcification of Growth Plate Cartilage as Revealed by Quick-Freeze Deep Etching Technique

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils. Received: 19 November 1996 / Accepted: 15 October 1997     

Expression of type I,type II,and type X collagen genes during altered endochondral ossification in the femoral epiphysis of osteosclerotic (oc/oc) mice

The osteosclerotic (oc/oc) mouse, a genetically distinct murine mutation that has a functional defect in its osteoclasts, also has rickets and shows an altered endochondral ossification in the epiphyseal growth plate. The disorder is morphologically characterized by an abnormal extension of hypertrophic cartilage at 10 days after birth, which is later (21 days after birth) incorporated into the metaphyseal woven bone without breakdown of the cartilage matrix following vascular invasion of chondrocyte lacunae. In situ hybridization revealed that the extending hypertrophic chondrocytes expressed type I and type II collagen mRNA, as well as that of type X collagen and that the osteoblasts in the metaphysis expressed type II and type X collagen mRNA, in addition to type I collagen mRNA. The topographic distribution of the signals suggests a possible co-expression of each collagen gene in the individual cells. Immunohistochemically, an overlapping deposition of type I, type II, and type X collagen was observed in both the extending cartilage and metaphyseal bony trabeculae. Such aberrant gene expression and synthesis of collagen indicate that pathologic ossification takes place in the epiphyseal/metaphyseal junction of oc/oc mouse femur in different way than in normal endochondral ossification. This abnormality is probably not due to a developmental disorder in the epiphyseal plate but to the failure in conversion of cartilage into bone, since the epiphyseal plate otherwise appeared normal, showing orderly stratified zones with a proper expression of cartilage-specific genes.     

The effect of detachment of the articular cartilage from its calcified zone on the cartilage microstructure, assessed by 2H-spectroscopic double quantum filtered MRI.

Most studies on articular cartilage properties have been conducted after detachment of the cartilage from the bone. In the present work we investigated the effect of detachment on collagen fiber architecture. We used one-dimensional (2)H double quantum filtered MRI on cartilage bone plugs equilibrated in deuterated saline. The quadrupolar splittings observed in the different zones were related to the degree of order and the density of the collagen fibers. The method is non-destructive, allowing for measurements on the same plug without the need for fixation, dehydration, sectioning and decalcification. Detachment of the radial from the calcified zone resulted in swelling of the cartilage plug in physiological saline and a concomitant decrease in the quadrupolar splitting. The effect of mechanical pressure on the (2)H quadrupolar splittings for the detached cartilage and for the calcified zone-bone plugs were compared with those of the same zones in the intact cartilage-bone plug. The splitting in the radial zone of the detached cartilage collapsed at much smaller loads compared to the intact cartilage-bone plug. The effect of the load on the size of the cartilage was also greater for the detached plug. These results indicate that anchoring of the cartilage to the bone through the calcified zone plays an important role in retaining the order of the collagen fibers. The water (2)H quadrupolar splitting in intact and proteoglycan-depleted cartilage was the same, indicating that the proteoglycans do not contribute to the ordering of the collagen fibers.     

Three-dimensional collagen architecture in bovine articular cartilage

The three-dimensional architecture of bovine articular cartilage collagen and its relationship to split lines has been studied with scanning electron microscopy. In the middle and superficial zones, collagen was organised in a layered or leaf-like manner. The orientation was vertical in the intermediate zone, curving to become horizontal and parallel to the articular surface in the superficial zone. Each leaf consisted of a fine network of collagen fibrils. Adjacent leaves merged or were closely linked by bridging fibrils and were arranged according to the split-line pattern. The surface layer (lamina splendens) was morphologically distinct. Although ordered, the overall collagen structure was different in each plane (anisotropic) a property described in previous morphological and biophysical studies. As all components of the articular cartilage matrix interact closely, the three-dimensional organisation of collagen is important when considering cartilage function and the processes of cartilage growth, injury and repair.     

The cartilage-bone interface

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.     

The isolation of two types of collagen from embryonic bovine epiphyseal cartilage

The nature of the collagen in embryonic and postnatal bovine epiphyseal cartilage was studied by amino acid analysis, extraction in denaturants, and extraction in denaturants after prior digestion of the cartilage with pepsin. Components of both Type I collagen and Type II collagen were isolated by molecular sieve filtration from the gelatin extracted from 4–5 month old embryonic bovine epiphyseal cartilage, and characterized by their behavior during ion exchange chromatography, amino acid composition and their content of hydroxylysine and glycosydically substituted hydroxylysine residues. Type I collagen accounted for about one-third of the total collagen content of epiphyseal cartilage. It was preferentially extracted in denaturant salts, whilst Type II collagen, essentially insoluble in denaturants, could be extracted from the cartilage only after prior digestion of the cartilage with pepsin. The marked preponderance of α components compared with higher molecular weight polymers in the extracted Type I gelatin suggested a paucity of intramolecular crosslinkages, and the lability of the intermolecular crosslinks to denaturants at neutral pH, suggests that the intermolecular, aldehyde-derived crosslinks of Type, I collagen in cartilage are similar to those in the Type I collagen of bone rather than those present in the Type I collagen of skin and other non-mineralized connective tissues. The marked differences in the solubility properties of Type I and Type II collagens of cartilage suggests that the stability and possibly the chemical nature of the intermolecular crosslinkages in Type II collagen, as well as the number of intramolecular crosslinkages, differs in the two types of collagen present in cartilage.     

骺板软骨细胞复合三维支架体外构建组织工程软骨的研究

目的探讨将骺板软骨细胞复合三维支架经体外培养,构建组织工程软骨的效果及其生物学特点. 方法将3周龄幼兔第1代骺板软骨细胞与液态的生物凝胶混合,接种于聚磷酸钙纤维/L-聚乳酸(CPPF/PLLA)三维支架材料,构建组织工程软骨组织块,连续培养4周.行大体、倒置显微镜及组织学、Ⅰ型和Ⅱ型胶原免疫组织化学光镜观察,定量检测硫酸糖胺多糖(GAG)含量. 结果构建的组织工程软骨块在培养过程中能保持其初始外形,种子细胞呈稳定的三维均相分布,外观逐渐呈乳白色、半透明,硬度亦不断增加.培养1周有软骨细胞陷窝形成,2周后形成富含Ⅱ型胶原和蛋白聚糖、具有典型软骨组织结构的工程化软骨,且Ⅰ型胶原逐渐转为阴性.4周时构建软骨的组织结构与天然骺板软骨相类似,硫酸GAG含量平均为天然骺板软骨的34%以上. 结论骺板软骨细胞复合三维支架体外培养可生成典型软骨,且可形成类似天然骺板软骨的组织结构,能满足修复骺板缺损的基本要求.体外培养1～2周可能是植入体内修复骺板缺损的较佳时机.     

Altered postnatal expression of insulin-like growth factor-I (IGF-I) and type X collagen preceding the Perthes' disease-like lesion of a rat model.

The spontaneously hypertensive rat (SHR) is a widely used animal model for the study of hypertension. It also exhibits an osteonecrosis of the femoral epiphysis that resembles the clinical features of Perthes' disease in humans. In this rat model, occlusion of the epiphyseal vessels occurs as a result of a breakdown of the mechanically vulnerable epiphysis. The postnatal development of the epiphysis recapitulates the serial events of the endochondral ossification (i.e., cartilage formation), chondrocyte hypertrophy, cartilage mineralization, vascularization, and introduction of osteoblasts that form the secondary ossification center within the epiphysis. In the present study, a detailed radiographic and histological analysis demonstrates that the osteonecrosis is preceded by a disturbance of the cartilage mineralization and a disturbance of the ossification, despite a normal hypertrophy of the epiphyseal cartilage. These observations suggest that abnormal development of the femoral epiphysis occurs much earlier than manifestation of the osteonecrosis. They lead us to a hypothesis that yet-unclarified transitional events between the cartilage hypertrophy and the cartilage mineralization may be affected in SHRs. Type X collagen is a developmentally regulated matrix molecule that is implicated in the mineralization of the hypertrophied chondrocytes. We show that the expression of type X collagen during epiphyseal ossification is delayed in SHRs (vs. normal controls), suggesting disturbed growth and/or differentiation of the epiphyseal chondrocytes. Postnatal growth and differentiation of the chondrocytes at least partly depend on insulin-like growth factor-I (IGF-I), which is produced by the chondrocytes in response to the pituitary growth hormone and stimulates cartilage growth in situ. The present study demonstrates an altered IGF-I expression during early postnatal life in SHRs and suggests that the altered IGF-I expression as well as the following delay in upregulation of type X collagen may cause the mechanical vulnerability of the femoral epiphysis in SHRs.     

Experimental ischemia of porcine growth cartilage produces lesions of osteochondrosis.

Osteochondrosis is a disorder of growth cartilage in which a focal failure of blood supply has been proposed as an important initiating factor. In the present study we investigated the effect on epiphyseal growth cartilage of experimentally interrupting the blood supply to a limited area of the distal femur of growing pigs. In 12 pigs, a thin full-thickness cartilage slab was removed from the abaxial margin of the medial condyle, thereby transecting a limited number of cartilage canals. The pigs were culled 1, 2, 3, 7, 14, 21 and 29 days post-surgery. The condylar cartilage was studied histologically, immunohistologically and by use of the TUNEL method. The transection induced cellular death of cartilage canal elements followed by cellular death of chondrocytes within the deep layers of the resting zone of the epiphyseal growth cartilage. However, in the superficial layers of the resting zone, chondrocytes appeared to proliferate into and subsequently chondrify some of the necrotic cartilage canals. The dying and dead cells were TUNEL-positive, but active caspase 3-negative. The loss of vascular supply induced increased VEGF-immunostaining in chondrocytes surrounding the affected area. We conclude that transection of cartilage canals produces chondronecrosis in the deep resting zone of the epiphyseal growth cartilage similar to that observed in spontaneously occurring osteochondrosis.     

Functional anatomy of articular cartilage under compressive loading Quantitative aspects of global, local and zonal reactions of the collagenous network with respect to the surface integrity

OBJECTIVE: To assess the influence of local compressive loading on the arrangement of the collagenous fibers in intact articular cartilage. To quantitate the zonal deformation of intact cartilage under load. To analyse the influence of removal of the tangential zone on the load-induced changes. MATERIALS AND METHODS: 380 cylinder shaped cartilage-on-bone samples (d=7 mm) were harvested from 20 bovine femoral heads. In 120 of them the tangential zone was removed. All samples were loaded for 20 min by 0.42 MPa or 0.98 MPa. After proteoglycan extraction, fixation in 4% formalin, dehydration by increasing concentrations of acetone, critical point drying, freeze-fracturing and gold-coating the samples were analysed by scanning-electron-microscopy. RESULTS: Fiber bulging away from the center of load occurred in an area larger than the directly loaded one and its extent increased parallel to loading (P< 0.01). Crimp was seen only under the indenter and spread with increasing load from the intermediate zone into the tangential zone and radial zone. The absolute height of tangential zone and intermediate zone together remained constant under all loading situations at the costs of the radial zone. All changes due to loading were fully reversible. Removal of the tangential zone reduced the area of bulging (P< 0.01) but markedly increased the amount of crimp. Overall radial strain was not altered, but overall superficial tangential strain was increased by up to 20% (P< 0.01) and high peaks in the local distribution of superficial tensile strain developed. CONCLUSIONS: The collagenous architecture is a dynamic property of the articular cartilage adapting to its respective loading situation. Crimp reflects local compressive strain. Under compressive loading larger portions of cartilage than the directly loaded areas are functionally included in the process of load transmission. During this process the tangential zone and the intermediate zone form a common functional unit providing a high degree of fiber cross-linkage as a possible mechanism to increase zonal compressive stiffness. Removal of the tangential zone seems to impair distribution of a locally applied compressive load sideways and leads to a reduced cartilage volume included in the process of load transmission. An intact tangential zone contributes to prevent peaks of surface tensile strain.     

Variation of collagen fiber alignment in a joint surface: a scanning electron microscope study of the tibial plateau in dog, rabbit, and man

To determine if articular cartilage collagen fiber organization differs with location on the tibial plateau, specimens from dogs, humans, and rabbits were studied by scanning electron microscopy. Joint surfaces were fixed, dehydrated, and fractured radially so that the periphery could be compared with the center on single specimens. Generally, fibers were more tightly packed in the lateral side than in the medial and the periphery as compared with the center, where the cartilage was consistently thicker and the radial zone was dominant and composed of straight vertical fibers. In the periphery, the tangential and transitional zones were better developed and contributed up to 50% of the cartilage depth in comparison to only 5% centrally. The soft, dull, malacic appearance of the center results from lack of a true surface layer of tangential collagen fibers.     

Development of the attachment zones in the rat anterior cruciate ligament: changes in the distributions of proliferating cells and fibrillar collagens during postnatal growth.

The development of the attachment zones of the anterior cruciate ligament (ACL) is an important consideration when examining the structural properties. The aim of this study was to elucidate the morphological changes and the distribution of proliferating cells and collagen types I, II and III at the attachment zones of the rat ACL during postnatal growth. The majority of proliferating cell nuclear antigen (PCNA) immunostained cells were noted near the ligament insertion, especially at the tibial site, and these cells gradually changed to fibrochondrocyte-like cells but still produced collagen types I and III at birth until one month old when rapid longitudinal growth of the ACL took place. After one month when the rate of the ligament growth decreased to one thirtieth of that during the first month and the epiphyseal cartilage at the attachment zone had been replaced by bone, these fibrochondrocyte-like cells began to produce collagen type II and reveal safranin O staining. The immunolabelling pattern to collagen type III was similar to that of PCNA immunostaining during the growth phase. Our findings show that the fibrochondrocytes at the attachment zone may develop from the ligament cells and act as a growth zone for the ligament during the period of ligament growth, and that subsequently, these cells begin to synthesis collagen type II and proteoglycans after epiphyseal ossification. These observations mainly occurred at the tibial attachment zone.     

Morphometric Changes in the Epiphyseal Plate of the Growing and Young Adult Male Rat After Long-Term Salmon Calcitonin Administration

The function of the epiphyseal plate is related to the differentiation and maturation of the chondrocytes, especially of the hypertrophic zone. Salmon calcitonin exerts a positive effect on chondrocytes of different types of cartilage, e.g., articular cartilage, osteochondral callus formation, and the epiphyseal plate. In the present study, the effect of long-term daily salmon calcitonin treatment upon epiphyseal plate function was examined in 80 male Wistar rats aged 12 weeks at the beginning of the experiment. A daily dose of 6 IU of salmon calcitonin enhanced the number of the chondrocytes of the hypertrophic zone of the upper tibial epiphyseal plate, increased the mean thickness of the epiphyseal plate, and accelerated the longitudinal growth of long bones. It was found that the peripheral growth of the epiphyseal plate was delayed after calcitonin treatment in comparison with the placebo-treated animals. The most effective period for calcitonin treatment on epiphyseal plate function seems to be the late accelerated period of growth, i.e., puberty. In conclusion, long-term salmon calcitonin treatment has a beneficial effect on longitudinal skeletal growth and this effect remains throughout the adult life of the animal. Salmon calcitonin does not enlarge the surface of the epiphyseal plate.     

Pathogenesis of osteochondrosis juvenilis Scheuermann

In osteochondrosis juvenilis Scheuermann, foci of various sizes in the cartilaginous end plates of the vertebral bodies display a loosening or complete interruption of the collagen fibers. These findings, together with an alteration and occasional absence of the growth zone, may result in the typical deformation of the vertebral bodies. Electron micrographs of the areas with optically absent collagen fibers reveal collagen fibrils. They are arranged in an irregular pattern. We conclude that a disturbance of collagen or ground substance biosynthesis is of importance in the pathogenesis of juvenile osteochondrosis.     

Resistant proteoglycans in epiphyseal plate cartilage. Variations in their distribution in relationship to age and species

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphyseal plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.     

Matrix Vesicles Mediate Mineralization of Human Thyroid Cartilage

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca²⁺-P_i complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca²⁺ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca²⁺ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage. Received: 21 July 1999 / Accepted: 2 November 1999