Annexin VIII is differentially expressed by chondrocytes in the mammalian growth plate during endochondral ossification and in osteoarthritic cartilage.

Endochondral ossification is the developmental process that leads to the formation and coordinated longitudinal growth of the majority of the vertebrate skeleton. Central to this process is chondrocyte differentiation occurring in the growth plate that lies at the junction between the epiphyseal cartilage and the bone. To identify novel factors involved in this differentiation process, suppression subtractive hybridization was performed to amplify preferentially cDNAs uniquely expressed in fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes. The subtracted product was used to screen a fetal bovine chondrocyte cDNA library. One of the cDNA clones identified encoded the bovine orthologue of annexin VIII, a protein not previously described in the growth plate. Northern and Western blotting confirmed that annexin VIII was expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. Immunofluorescence localized annexin VIII largely to the chondrocyte cell membrane. In a preliminary study, we examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. Thus annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification and may act as a marker for cells undergoing inappropriate differentiation in OA.     

Cartilage abnormalities are associated with abnormal Phex expression and with altered matrix protein and MMP-9 localization in Hyp mice

X-linked hypophosphatemic rickets (HYP) in humans is caused by mutations in the PHEX gene. This gene mutation is also found in Hyp mice, the murine homologue of the human disease. At present, it is unknown why loss of Phex function leads to cartilage abnormalities in Hyp mice. In the present study, we compared in wild-type and Hyp mice Phex protein localization in cartilage of developing long bone as well as localization of skeletal matrix proteins and matrix metalloproteinase-9 (MMP-9). Also compared were chondrocyte apoptosis in the growth plate, mineralization and cartilage remnant retention in the metaphysis, and chondroclast/osteoclast characteristics in the primary spongiosa. Phex protein was detected in proliferating and hypertrophic chondrocytes in growth plate cartilage of wild-type mice, but not in Hyp mice. Hyp mice exhibited a widened and irregular hypertrophic zone in growth plate cartilage showing hypomineralization, increased cartilage remnants from the growth plate in both metaphyseal trabecular and cortical bone, and fewer and smaller chondroclasts/osteoclasts in the primary spongiosa. Increased link protein and C-propeptide of type II procollagen of Hyp mice reflected the increase in chondrocytes and matrix in the cartilaginous growth plate and in bone. In addition, growth plate osteocalcin and bone sialoprotein levels were decreased, while osteonectin was increased, in hypertrophic chondrocytes and cartilage matrix in Hyp mice. MMP-9 in hypertrophic chondrocytes was also reduced in Hyp mice and fewer apoptotic hypertrophic chondrocytes were detected. These findings suggest that Phex may control mineralization and removal of hypertrophic chondrocytes and cartilage matrix in growth plate by regulating the synthesis and deposition of certain bone matrix proteins and proteases such as MMP-9.     

Human cartilage glycoprotein 39 (HC gp-39) mRNA expression in adult and fetal chondrocytes, osteoblasts and osteocytes by in-situ hybridization

OBJECTIVE: To examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in human cartilage and bone. DESIGN: In-situ hybridization analysis was used to examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in adult human osteoarthritic articular cartilage from various stages of disease, as well as in human osteophytic tissue and in human fetal bone. RESULTS: In cartilage from patients with mild osteoarthritic cartilage degeneration, HC gp-39 was expressed at moderate to high levels only in chondrocytes of the superficial zone. In advanced OA cartilage, cloning chondrocytes of the superficial zone expressed high levels of HC gp-39 and chondrocytes of the mid- and deep zones were also positive. HC gp-39 was undetectable in the chondrocytes of normal articular cartilage. In osteophytic tissue, the expression of HC gp-39 mRNA was intense in flattened, end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation. Proliferating osteoblasts expressed low to moderate levels. Notably, mature osteocytes were negative for HC gp-39 expression. Chondrocytes in the secondary ossification center of developing fetal cartilage demonstrated high expression while growth plate and mineralized cartilage chondrocytes had lower expression. Osteoblasts at sites of endochondral and intramembranous bone formation were positive for expression of HC gp-39. CONCLUSIONS: The stage-specific expression of HC gp-39 in fetal development and adult remodelling bone and cartilage provides evidence for a specific functional or structural role for HC gp-39 in bone and cartilage tissue. HC gp-39 is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue, and its distribution within the tissue changes as disease progresses. OA is characterized not only by cartilage degeneration, but by increased subchondral bone formation and osteophytosis. The results from this study indicate that the increased HC gp-39 expression in OA serum and synovial fluid may reflect not only cartilage degeneration but increased osteogenesis.     

Immunohistological analysis of transglutaminase factor XIIIA expression in mouse embryonic growth plate.

Previously we demonstrated the expression of Factor XIIIA (FXIIIA), a coagulation transglutaminase, in avian embryonic growth plate. To explore whether FXIIIA is also expressed by chondrocytes of the mammalian cartilage anlagen of bones, we analyzed the mouse embryonic growth plate by immunostaining using anti-FXIIIA antibodies developed against human and chicken proteins. We revealed the expression of FXIIIA in the epiphyseal growth plate, where FXIIIA appears first intracellularly in the zone of proliferation/maturation, and remains intra- and extracellularly throughout the hypertrophic zone. Externalization of FXIIIA occurs before mineralization. Transglutaminase activity was assayed in organ cultures using rhodamine-labeled synthetic substrate Pro-Val-Lys-Gly. Enzymatic activity shows a restricted distribution in cartilage and correlates with FXIIIA expression pattern, suggesting that cartilagenous transglutaminase activity is due, at least partially, to the FXIIIA isoform. We conclude, that coagulation factor FXIIIA is expressed by chondrocytes of embryonic mouse long bone cartilages in a strictly regulated pattern, which correlates with chondrocyte differentiation and matrix mineralization.     

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase‐Dependent Cell Death and Leads to Mild Growth Retardation

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome‐dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage‐specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage‐specific collagen type 2 promoter–driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3‐methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase‐3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase‐3 cleavage. This was associated with activation of caspase‐9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase‐dependent death of chondrocytes. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

S-100 protein in human cartilage lesions

S-100 protein is an acidic calcium-binding protein that was originally isolated from the mammalian central nervous system in 1965. Initially, S-100 protein was thought to be specific to neuroectodermal tissues, but its presence in chondrocytes was recently reported. This study is an analysis of the distribution of S-100 protein in lesions of human cartilage and its possible significance. Several cartilaginous tumors, both benign and malignant, as well as normal epiphyseal growth plates, were examined for S-100 protein by the immunoperoxidase technique. Each cartilaginous lesion that was examined showed immunoreactivity for S-100 protein. The staining product was noted only intracellularly. The highest intensity of staining was seen in the hypertrophic chondrocytes of the zone of provisional calcification in the growth plate and in the large chondrocytes located adjacent to areas of matrix mineralization in cartilaginous tumors. In normal epiphyseal growth plates, the intensity of staining increased in chondrocyte cytoplasm as one moved from the proliferating columnar chondrocytes through the zone of hypertrophic chondrocytes to the hypertrophic, degenerating chondrocytes in the zone of provisional calcification. In cartilaginous tumors, the cells of enchondroma and of the cartilaginous cap of osteochondroma were more immunoreactive than those of chondromyxoid fibroma. In benign chondroblastoma, the chondroblasts were less reactive than the chondrocytes in areas of chondroid matrix production. The latter areas of chondroblastomas showed stronger immunoreactivity in the matrix-enclosed cells adjacent to areas of mineral deposition. Among conventional chondrosarcomas, grade-I tumors showed greater immunoreactivity of the chondrocyte cytoplasm than did those of a higher grade, in which chondroid matrix production was less abundant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagen metabolism is markedly altered in the hypertrophic cartilage of growth plates from rats with growth impairment secondary to chronic renal failure.

Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase-3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham-operated rats fed ad libitum (SAL) and sham-operated rats pair-fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase-3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia.     

Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.

Skeletal growth depends on endochondral ossification in growth plate cartilage, where proliferation of chondrocytes, matrix synthesis, and increases in chondrocyte size all contribute to the final length of a bone. To learn more about the potential role of matrix synthesis/degradation dynamics in the determination of bone growth rate, we investigated the expression of matrix collagens and collagenase 3 in tibial growth plates in three age groups of rats (21, 35, and 80 days after birth), each characterized by specific growth rates. By combining stereological and in situ hybridization techniques, it was found that the expression of matrix collagens and collagenase 3 was specifically turned on or off at specific stages of the chondrocyte-differentiation cycle, and these changes occurred as a temporal sequence that varied depending of animal growth rate. Furthermore, the expression of these matrix proteins by a growth plate chondrocyte was found to be sped up or slowed down depending of the growth rate. In addition to expression of types II and X collagen, collagenase-3 expression was found to constitute a constant event in the series of changes in gene expression that takes place during the chondrocyte-differentiation process. Collagenase-3 expression was found to show a biphasic pattern: it was intermittently expressed at the proliferative phase and uniformly expressed at the hypertrophic stage. An intimate relationship between morphological and kinetic changes associated with chondrocyte hypertrophy and changes in the expression pattern of matrix collagens and collagenase 3 was observed. Present data prove that the matrix synthesis/degradation dynamics of the growth plate cartilage varied depending on growth rate; these results support the hypothesis that changes in matrix degradation and synthesis are a critical link in the sequence of tightly regulated events that lead to chondrocytic differentiation.     

Reimplantation of growth plate chondrocytes into growth plate defects in sheep

Defects in growth plates due to trauma, infection, or genetic causes can result in bone formation across the defect, bridging the epiphysis and metaphysis, resulting in growth arrest and limb deformation. We have investigated the capacity of implanted chondrocyte cultures to prevent this process. Sheep growth plate chondrocytes were isolated, and after culture at high density produced easily manipulated cartilaginous discs. The tissue was implanted into growth plate defects produced in lambs and the response was assessed histologically. Following implantation, cultures continued to proliferate and maintain a cartilage-like matrix. After 8 to 12 weeks, hypertrophic maturation chondrocyte columnation, and associated endochondral calcification were observed. Culture implantation was always associated with local immune inflammatory reaction, which continued throughout the course of investigation. Cellular survival was variable and resulted in the presence of viable implants as well as residual cartilage matrix devoid of chondrocytes; however, implanted chondrocyte discs always prevented bone bridge formation. These findings encourage the expectation that cultured chondrocytes may provide a useful replacement for the inert interpositional materials currently used in the treatment of growth arrest. The potential of this technique for growth plate replacement, however, requires a more predictable rate of implant survival. The likely reasons for implant loss are discussed.     

Molecular basis of achondroplasia, hypochondroplasia, and thanatophoric dysplasia]

Fibroblast growth factor 2 (FGF2) inhibits proliferation and hypertrophy of chondrocytes in the growth plate, synthesis of cartilage matrix, terminal differentiation of hypertrophic chondrocytes and matrix calcification. Recent studies have found that mutations in the receptor for fibroblast growth factor 3 (FGFR3) cause achondroplasia, hypochondroplasia and thanatophoric dysplasia. These mutations evoke uncontrolled stimulation of the receptor, leading to inhibition of bone growth. Inactivation of the receptor in experimental animals causes excessive chondrocyte proliferation and abnormal bone length. Chondrocyte stem cells proliferate in the ossification groove of Ranvier and contribute to both peripheral and longitudinal growth of the growth plate. They express FGFR3, have a potential to differentiate into chondrocytes and are therefore considered adequate for healing cartilage defects in the articular surface. It is at present unknown what happens to the chondrocyte precursor cells in the ossification groove of patients with FGFR3 mutation.     

A poptosis of hypertrophic chondrocytes in rat cartilaginous growth plate

Calcifying cartilages undergo endochondral ossification, a process in which cartilage is replaced by bone. These tissues contain chondrocytes that proliferate, leading, to differentiation and hypertrophy. Recent histological and biochemical studies suggest that hypertrophic chondrocytes undergo apoptosis. We investigated the process of this cell death to determine when fragmentation of DNA, a hallmark of apoptosis, occurs during cellular commitment to hypertrophy, and to test the hypothesis that the chondrocytes are intrinsically programmed to undergo apoptosis. End-labeling of fragmented DNA of rat proximal tibiae revealed that a majority of hypertrophic cells bore fragmented DNA, indicating that apoptosis was in progress in this zone. In pelleted chondrocyte cultures isolated from, rat rib growth plates and employed in an in vitro model of a growth plate, hypertrophic cells were also positive for end-labeling. Gel electrophoresis of DNA isolated from the chondrocyte cultures at 1–3 weeks yielded the ladder formation characteristic of apoptosis. We conclude that the chondrocytes in the growth plate are programmed to self-annihilate by apoptosis and that the apoptotic process is closely associated with the commitment to hypertrophy.     

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

Characterization of growth plate mitochondria

The growth plate chondrocyte plays a central role in growth plate function. The purpose of this study was to characterize the respiratory and calcium transport properties of isolated mammalian growth plate chondrocytes and mitochondria obtained from these cells and to quantitate the mitochondrial weight and volume fraction in each zone of the growth plate. A new method was developed for isolation of mitochondria from chondrocyte suspensions. Isolated chondrocyte mitochondria demonstrated an eightfold increase in oxygen consumption in response to calcium and a two- to threefold increase in oxygen consumption in response to adenosine diphosphate. Similar responses were observed in chondrocytes treated with digitonin. The mitochondrial protein content of the growth plate and hyaline cartilage chondrocytes is significantly less than hepatocytes. Conversely, the chondrocyte mitochondrial cytochrome aa3 content is similar to mitochondria from a wide variety of sources. A zonal analysis of the growth plate demonstrates an increase in the mitochondrial weight (protein) fraction from the reserve to the hypertrophic zone whereas the mitochondrial volume fraction decreases from the reserve to the hypertrophic zone. The findings of this study emphasize the dependence of chondrocytes on glycolysis as a prime energy source and support the concept that chondrocyte mitochondria have become specialized in the process of matrix calcification.     

PTH Receptor Signaling in Osteoblasts Regulates Endochondral Vascularization in Maintenance of Postnatal Growth Plate

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone. © 2014 American Society for Bone and Mineral Research.     

Bone vitamin D-dependent calcium-binding protein is localized in chondrocytes of growth-plate cartilage

Summary The cellular distribution of vitamin D-dependent calcium-binding protein (CaBP) was examined in rat and chicken bone by immunocytochemical methods using an antiserum raised against purified chicken intestinal CaBP. In EDTA-decalcified, Vibratome sections of growing rat long bones, specific CaBP immunostaining was observed in cytoplasm of chondrocytes of the growth plate, particularly in regions of calcification. In undecalcified, frozen sections from neonatal rat, positive staining was seen in chondrocytes of tibial growth plate and also in chondrocytes of the long bones of the skull. No specific immunostaining was observed in osteoblasts, osteocytes or osteoclasts in mineralized bone. In frozen sections of tibias from 19-day-old chick embryos specific immunostaining was again confined to dividing chondrocytes of the growth plate and was much less intense in “resting” cartilage. The finding of CaBP in chondrocytes, cells known to possess specific receptors for 1,25-dihydroxyvitamin D₃ and to respond to the hormone, suggests a possible functional role for CaBP in chondrocyte maturation, differentiation and/or cartilage calcification.     

Localization of type X collagen in canine growth plate and adult canine articular cartilage

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.     

Integrins and extracellular matrix proteins in the human childhood and adolescent growth plate

Interaction of chondrocytes with the surrounding matrix significantly influences differentiation and growth. These processes involve cell surface proteins, particularly integrins. The aim of this study was to compare the expression of integrins (alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta5 subunits) together with matching binding proteins in human childhood and adolescent growth plate cartilage using immunohistochemistry. Integrin beta1 was detected in all chondrocytes of the growth plate cartilage, beta3 only in osteoclasts of the opening zone, and beta5 in hypertrophic chondrocytes and osteoblasts. Integrin alpha1, alpha2, and alpha5 subunits were expressed by chondrocytes in the proliferative and hypertrophic zone as well as in osteoblasts and osteoclasts. Integrin av and alpha6 subunits were present in chondrocytes of all zones, alpha3 only in osteoclasts. Collagen type II and fibronectin were seen throughout the growth plate, collagen type X in the hypertrophic zone, collagen type I in the ossifying trabecules. Laminin was expressed by chondrocytes in the resting zone and more weakly in the proliferative zone, collagen VI was present in the pericellular and interterritorial matrix in all zones of the growth plate. These results differ from previous reports on the distribution of integrins in the fetal growth plate. However, there was no difference in integrin expression in children before and during puberty. Our results indicate that integrin expression is not influenced by endocrine factors during sexual maturation and suggest that the process of skeletal maturation is not regulated via altered integrin expression.