Hypertrophic Growth Plate Chondrocytes Research Articles

Role of interleukin‐8 in PiT‐1 expression and CXCR1‐mediated inorganic phosphate uptake in chondrocytes

The proinflammatory chemokine interleukin-8 (IL-8) induces chondrocyte hypertrophy. Moreover, chondrocyte hypertrophy develops in situ in osteoarthritic (OA) articular cartilage and promotes dysregulated matrix repair and calcification. Growth plate chondrocyte hypertrophy is associated with expression of the type III sodium-dependent inorganic phosphate (Pi) cotransporter phosphate transporter/retrovirus receptor 1 (PiT-1). This study was undertaken to test the hypothesis that IL-8 promotes chondrocyte hypertrophy by modulating chondrocyte PiT-1 expression and sodium-dependent Pi uptake, and to assess differential roles in this activity. The selective IL-8 receptor CXCR1 and the promiscuous chemokine receptor CXCR2 were used. Human knee OA cartilage, cultured normal bovine knee chondrocytes, and immortalized human articular chondrocytic CH-8 cells were transfected with CXCR1/CXCR2 chimeric receptors in which the 40-amino acid C-terminal cytosolic tail domains were swapped and site mutants of a CXCR1-specific region were generated. Up-regulated PiT-1 expression was detected in OA cartilage. IL-8, but not IL-1 or the CXCR2 ligand growth-related oncogene alpha, induced PiT-1 expression and increased sodium-dependent Pi uptake by >40% in chondrocytes. The sodium/phosphate cotransport inhibitor phosphonoformic acid blocked IL-8-induced chondrocyte hypertrophic differentiation. Signaling mediated by kinase Pyk-2 was essential for IL-8 induction of PitT-1 expression and Pi uptake. Signaling through the TSYT(346-349) region of the CXCR1 cytosolic tail, a region divergent from the CXCR2 cytosolic tail, was essential for IL-8 to induce Pi uptake. Our results link low-grade IL-8-mediated cartilaginous inflammation in OA to altered chondrocyte differentiation and disease progression through PiT-1 expression and sodium-dependent Pi uptake mediated by CXCR1 signaling.

Histochemical visualization of the cartilage hyaladherins using a biotinylated hyaluronan oligosaccharide bioaffinity probe.

Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.

Thyroxine stimulates transglutaminase activity in articular chondrocytes

Objectives: Thyroid hormones induce features of the hypertrophic phenotype in mature articular chondrocytes as well as in growth plate chondrocytes. Hypertrophic chondrocytes are responsible for extracellular matrix mineralization, with formation of bone mineral in growth plate cartilage and pathologic calcium crystals in aging articular cartilage. Elevated activity levels of the two transglutaminase (Tgase) enzymes (type II Tgase and Factor XIIIA (FXIIIA)) have recently been described as additional features of hypertrophic growth plate chondrocytes. Because Tgases may participate in pathologic mineralization in aging cartilage, we explored the effects of thyroid hormones on Tgase activity in articular chondrocytes. Methods: Adult porcine articular chondrocytes were incubated with or without 250–750 nM l-thyroxine (T4) or 10–100 nM 3,3′,5-tri-iodothyronine (T3). Tgase activity was measured with a standard radiometric assay. The effects of thyroid hormones on protein and mRNA levels of type II Tgase and FXIIIA were determined. As Tgase activity can be stimulated by proteases, endoproteinase levels were also measured. The mechanisms of these effects were explored. Results: T4 (750 nM) or T3 (100 nM) stimulated Tgase activity by twofold in articular chondrocytes at 4 h and increased the percentage of Tgase activity in the extracellular matrix. Chondrocytes rapidly converted T4 to T3, but the time course suggests similar mechanisms for T4 and T3. T4-induced Tgase activity was suppressed with cycloheximide and protein kinase C inhibitors. The effects of T4 on type II Tgase and FXIIIA levels were modest, but T4 strongly induced endoproteinase activity in chondrocytes. Conclusions: We report in this study that thyroid hormones increase Tgase activity in articular chondrocytes via a non-genomic mechanism, which may involve increased endoproteinase secretion.

Expression of VEGF121 and VEGF165 in hypertrophic chondrocytes of the human growth plate and epiphyseal cartilage.

Vascular endothelial growth factor (VEGF) plays an important role during endochondral bone formation in hypertrophic cartilage remodelling. We examined VEGF and VEGF receptor expression in tibiae from fetuses, newborns and children immunohistochemically. Expression of mRNA for the different VEGF splice forms and for VEGF receptors KDR and FLT-1 was analysed by reverse transcription-polymerase chain reaction (RT-PCR). VEGF could be immunolocalized intracellularly in the hypertrophic chondrocytes of the growth plate and in the chondrocytes around cartilage canals of the epiphysis, respectively. The resting zone and the proliferative zone of the growth plate were VEGF-negative. In cartilage samples of all growth plates analysed, VEGF121 and VEGF165 were identified as the only VEGF splice forms expressed. RT-PCR for VEGF mRNA of normal hyaline cartilage was negative. At vessels growing into the hypertrophic cartilage FLT-1 (VEGFR-1) and KDR (VGEFR-2) could be visualized. Reverse transcription-polymerase chain reaction (RT-PCR) substantiated the results regarding FLT-1 and KDR expression. The results of our study suggest that the splice forms VEGF121 and VEGF165 and the receptors KDR and FLT-1 of the known angiogenetic peptide VEGF play a role in process of endochondral ossification.

Adenomatous polyposis coli (APC), beta-catenin, and cadherin are expressed in human bone and cartilage.

Members of the cadherin and catenin families are involved in chondrogenesis and catenin gene mutations have been detected in malignant tumours of bone. This study was undertaken to assess in detail expression of cadherin, beta-catenin and the associated tumour suppressor gene product APC in bone and cartilage at different stages of human skeletal maturity and in non-neoplastic and neoplastic osteoarticular disease. Immunohistochemical staining of formalin-fixed paraffin-embedded normal and osteoarthritic adult articular cartilage, fetal growth plate and a series of tumours of bone and cartilage was undertaken with a panel of antibodies against APC, beta-catenin, and pan-cadherin. This study demonstrated expression of APC, beta-catenin and cadherin in normal and diseased bone and cartilage. APC was present both in osteoblasts and osteoclasts but not in osteocytes. Although only weak APC staining of occasional growth plate hypertrophic chondrocytes and normal articular chondrocytes was seen, APC staining was increased in osteoarthritic articular cartilage. beta-catenin and pan-cadherin staining was strongly positive in osteoclasts and osteoblasts, with expression being lost when bone cells differentiated into osteocytes. Expression of APC, beta-catenin and pan-cadherin in bone tumours was similar to that of non-neoplastic adult tissues. These findings suggest previously unrecognized roles for APC in regulation of function of chondrocytes, osteoblasts and osteoclasts and support the view that catenin-cadherin interactions are important in regulation of bone cell activity. Abnormalities of expression or function of these molecules may be important in formation of bone tumours and their clinical behaviour.

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate.

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.

Regulatory roles of zinc in matrix vesicle-mediated mineralization of growth plate cartilage.

Zinc (Zn2+) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn2+ action are not well understood. In this study we investigated the effects of Zn2+ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn2+ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn2+-treated cultures showed similar APase activity, but vesicles from RA/Zn2+-treated cultures contained significantly less Ca2+ and Pi. MVs isolated from RA-treated cultures were able to take up Ca2+ and mineralize in vitro, whereas vesicles isolated from RA/Zn2+-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca2+-Pi-phospholipid complexes, did not restore the Ca2+ uptake abilities of MVs isolated from RA/Zn2+-treated cultures, suggesting that vesicles from RA/Zn2+-treated cultures did not contain functional Ca2+-Pi-phospholipid complexes. Zn2+ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca2+-dependent, EDTA-reversible binding of these molecules to the membrane surface. However, Zn2+ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn2+ interferes with the assembly of annexins in the MV membrane. In addition, Zn2+ inhibited annexin II-, V-, and VI-mediated Ca2+ influx into liposomes. In conclusion, Zn2+ inhibits the mineralizing competence of intravesicular Ca2+-Pi-phospholipid complexes and function of annexin channels, thereby controlling Ca2+ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization.

Stability of Alternatively Spliced IGF-I mRNA in Growth Plate Chondrocytes

Insulin-like growth factor-I (IGF-I) enhances the proliferation and hypertrophy of growth plate chondrocytes leading to increased growth plate width thus promoting bone elongation. Differing quantities of the multiple IGF-I transcripts within the growth plate suggest differential regulation of IGF-I. To assess this, the relative stabilities of the 1Ea, 1Eb, and 2Ea IGF-I mRNA classes in 2-6 week old rat growth plates were evaluated. The mean estimated half-life of class lEa was 3.7 ± 0.05 h, while classes 1Eb and 2Ea decayed gradually over the course of the experiment. Estimated half-lives for each IGF-I mRNA species were unchanged at all ages examined. Incubation with Act D enhanced the transcription of class 1Ea, 1Eb, and 2Ea mRNAs to varying degrees. This implies that the differential stability of alternative IGF-I mRNA classes may be an inherent regulatory component that is not influenced by an animal's developmental state, and the differential abundance of alternative IGF-I mRNA species in the growth plate throughout development is due to transcriptional differences.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

Hypertrophy of growth plate chondrocytes in vivo is accompanied by modulations in the activity state and surface area of their cytoplasmic organelles.

The rate of longitudinal bone growth is regulated primarily by modulations in the activity of epiphyseal plate hypertrophic chondrocytes, these being manifested as changes in cell and matrix volume. It was the purpose of this study to ascertain whether the cytoplasmic organelles representing the cellular production apparatus, i.e. rough endoplasmic reticulum, Golgi apparatus and mitochondria, contribute to these changes by modulating their rate of activity or by increasing/decreasing the surface area and/or volume of their membranes. Using rats at different stages of growth, the surface areas and volumes of the three organellar systems were quantified in epiphyseal plate chondrocytes at the onset and termination of hypertrophy by ultrastructural stereology. Matrix synthesis during the same span was assessed by monitoring the production of its principal components, namely, fibrillar collagen (ultrastructural morphometry) and glycosaminoglycans (quantitative (35)S-autoradiography). Each organelle adapts to increases (21- to 35-day-old rats) and decreases (35- to 80-day-old rats) in growth rate by its own individual combination of the two alternative mechanisms, but modulations in the level of activity predominate over alterations in the surface area or volume of their membranes. These findings point to the danger of relying solely on data gleaned from a quantitative ultrastructural analysis of organellar parameters and emphasise the necessity of conducting functional assays in parallel, as performed here.

IGF-I expression is downregulated in hypertrophic growth-plate chondrocytes in vivo but not in vitro

IGF-I expression is downregulated in hypertrophic growth-plate chondrocytes in vivo but not in vitro

The domain of hypertrophic chondrocytes in growth plates growing at different rates.

In this study, we tested the hypotheses that (a) both the domain volume (volume of the cell and the matrix it has formed) and matrix volume of juxtametaphyseal hypertrophic chondrocytes in the growth plate is tightly controlled, and that (b) the domain volume of juxtametaphyseal hypertrophic chondrocytes is a strong determinant of the rate of bone length growth. We analyzed the rate of bone length growth (oxytetracycline labeling techniques) and nine stereologic and kinetic parameters related to the juxtametaphyseal chondrocytic domain in the proximal and distal radial and tibial growth plates of 21- and 35-day-old rats. The domain volume increased with increasing growth rates, independent of the location of the growth plate and the age of the animal. Within age groups, the matrix volume per cell increased with increasing growth rates, but an identical growth plate had the same matrix volume per cell in 21- and 35-day-old rats. The most suitable regression model (R2 = 0.992) to describe the rate of bone length growth included the mean volume of juxtametaphyseal hypertrophic chondrocytes and the mean rate of cell loss/cell proliferation. This relationship was independent of the location of the growth plate and the age of the animal. The data suggest that the domain volume of juxtametaphyseal hypertrophic chondrocytes, as well as the matrix volume produced per cell, may be tightly regulated. In addition, the volume of juxtametaphyseal hypertrophic chondrocytes and the rate of cell loss/rate of cell proliferation may play the most important role in the determination of the rate of bone length growth.

P-glycoprotein is expressed in the mineralizing regions of the skeleton.

Using oligonucleotide primers specific for the human MDR 1 gene, we were able to identify a specific amplicon using RT-PCR from total bovine growth plate chondrocyte RNA. The identification of MDR mRNA in growth plate chondrocytes led us to examine the precise distribution of MDR P-glycoprotein in bone and cartilage. We applied two monoclonal antibodies (C219 and C494) to human fetal, neonatal, and childhood growth plates and bone. In growth plates, P-glycoprotein was detected at high levels in a perilacunar distribution in the calcifying zone and at lower levels in hypertrophic, but not proliferative or reserve zone, chondrocytes. P-glycoprotein was also observed in perichondrial chondrocytes, in perivascular chondrocytes and matrix in the fetal cartilage anlage, and in osteoblasts and the surface osteoid matrix of newly formed bone trabeculae in the primary spongiosa. The recently described chloride channel of P-glycoprotein suggests a potential role of P-glycoprotein in growth plate chondrocyte hypertrophy.

Defective bone formation in Krox-20 mutant mice.

Endochondral ossification is the prevalent mode of vertebrate skeleton formation; it starts during embryogenesis when cartilage models of long bones develop central regions of hypertrophy which are replaced by bony trabeculae and bone marrow. Although several transcription factors have been implicated in pattern formation in the limbs and axial skeleton, little is known about the transcriptional regulations involved in bone formation. We have created a null allele in the mouse Krox-20 gene, which encodes a zinc finger transcription factor, by in frame insertion of the E. coli lacZ gene and shown that hindbrain segmentation and peripheral nerve myelination are affected in Krox-20-/- embryos. We report here that Krox-20 is also activated in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that its disruption severely affects endochondral ossification. Krox-20-/- mice develop skeletal abnormalities including a reduced length and thickness of newly formed bones, a drastic reduction of calcified trabeculae and severe porosity. The periosteal component to bone formation and calcification does not appear to be affected in the homozygous mutant suggesting that the major role for Krox-20 is to be found in the control of the hypertrophic chondrocyte-osteoblast interactions leading to endosteal bone formation.

THE EXPRESSION OF TYPE X COLLAGEN mRNA BUT NOT ALKALINE PHOSPHATASE mRNA INCREASES AS GROWTH PLATE CHONDROCYTES HYPERTROPHY

In order to study the regulation of the process of growth plate chondrocyte hypertrophy, freshly isolated newborn calf rib growth plate chondrocytes were separated using Percoll density gradient centrifugation. Five subpopulations of chondrocytes of different size were obtained. Alkaline phosphatase activity/ugDNA was lowest in the smallest cells and increased with increasing cell size. mRNA isolated from the five subpopulations was analyzed by northern blot using radiolabeled cDNAs encoding the genes for type II and X collagen, alkaline phosphatase and G-6-PD. The type II collagen gene was highly expressed in all subpopulations. The expression of the type X collagen gene was lowest in the smallest cells and relative expression increased with increasing cell size. By contrast, the expression of the alkaline phosphatase gene did not vary with cell size. These data suggest that while type X collagen synthesis in maturing growth plate chondrocytes may be regulated at the gene level, the increase in alkaline phosphatase activity observed in maturing growth plate chondroctyes may be post-transcriptionally regulated.

Polymerase chain reaction — amplification of the coding sequence of the type X collagen gene from genomic DNA and identification of a polymorphism that changes GLY to ARG at position 545 by single-strand conformation polymorphism analysis

Type X collagen is a specific product of hypertrophic growth plate chondrocytes and it has been suggested that mutations in the corresponding gene (COL1OA1) may be responsible for certain heritable disorders affecting growth plate cartilage such as the epiphyseal dysplasias. We have amplified the coding region of COL1OA1 employing polymerase chain reaction (PCR) of genomic DNA. Single-strand conformation polymorphism (SSCP) analysis of PCR products followed by direct sequencing identified a G to C transition that results in a Gly to Arg substitution at position 545 of the polypeptide chain. The sequence variation was confirmed by restriction enzyme analysis with BsaJ 1. Analysis of a family with multiple epiphyseal dysplasia ruled out this sequence change as a cause of the disease. This is the first report showing application of SSCP for detection of a sequence variant in COL1OA1.

Binding of insulin-like growth factor-I (IGF-I) to primary cultures of chondrocytes from rat rib growth cartilage

Binding of insulin-like growth factor I (IGF-I) to cultured resting, proliferative and hypertrophic growth plate chondrocytes was investigated. The optimal binding conditions and the extent of degradation of the 125I-IGF-I at 20 °C were analyzed in a time-course study. The maximal binding without noticeable degradation was observed after 3 h. The binding of IGF-I to the proliferative cells was 2-fold higher than to the resting and the hypertrophic cells. On the proliferative chondrocytes two classes of receptors with different affinities were found. 125I-IGF-I could be displaced from the proliferative cells by unlabelled IGF-I, IGF-II and insulin, respectively. Half maximal binding was observed at 0.3 nmol/l (= 2.2 μg/l) of IGF-I, 4.3 nmol/l (= 32 μg/l) of IGF-II and 350 nmol/l (= 2000 μg/l) of insulin. No specific binding of human growth hormone (hGH) could be demonstrated. When binding of epidermal growth factor (EGF) to the proliferative cells was assessed, little, but specific binding was observed.

The Fate of Hypertrophic Chondrocytes in Growth Plates Transplanted Intramuscularly in the Rabbit

Sixteen implants of fetal growth cartilage of New Zealand white rabbits of both sexes were implanted intramuscularly into an adult animal of the opposite sex. Endochondral ossification was recognized in 11 of the 16 implants. The sex of the osteoblasts and chondrocytes was determined by counting the sex chromatin body in five areas of abundant endochondral ossification. The bone grown adjacent to the implant was predominantly of recipient type in all of the specimens (p less than 0.01). Most of the chondrocytes gradually lost stainability to Feulgen-Schiff stain as they matured, and only a few cells were stained in the calcified zone, indicating the deterioration of nuclear DNA. Thus, the majority of the growth plate chondrocytes are considered to die and be replaced by osteoblasts of a different origin.

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture.

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.