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Ultrastructural organization of type XI collagen in fetal bovine epiphyseal cartilage.

Type XI collagen was localized with polyclonal antibodies specific for alpha 1 (XI) and alpha 2 (XI) chains in the resting zone of epiphyseal cartilage from calf fetuses. The immunofluorescence technique was used on sections of cartilage, and the immunogold labelling technique for electron microscopy on fibrils isolated from cartilage and, for the first time, in situ on blocks of cartilage fractured in liquid nitrogen. Immunofluorescence showed that without pepsin treatment the staining of type XI collagen was restricted to the pericellular zones; after pepsin treatment, the staining was co-distributed with that of type II collagen. Immunoelectron microscopy performed on isolated fibrils and on cartilage blocks showed that after disruption of fibrils with pepsin, type XI collagen was labelled on small filaments on the fibrils. When the fibrils were not disrupted, labelling was observed in situ only at the ends of the fibrils or on cross-sections of some fibrils. These results indicate that type XI collagen is located inside type II collagen fibrils in fetal bovine epiphyseal cartilage, as already postulated for embryonic chicken sterna.

Animals↗

Effects of the calcium channel blocker nifedipine on epiphyseal growth plate and bone turnover: a study in rabbit.

The potential effects of a calcium channel blocker (nifedipine) on epiphyseal growth plate and bone remodeling have been investigated in growing rabbits. The treated group received 6 mg/kg/day nifedipine twice daily by gavage for 10 weeks. An untreated group was used as control; with this dose, neither toxic effects nor decrease in the body weight have been observed. No modifications of blood phosphocalcic parameters have been found. In the treated group there is a significant lower cancellous bone volume, lower osteogenesis, shorter labeled perimeters, and lower mineral apposition rate than in the control group. Epiphyseal growth plate thickness is lower than in the untreated animals and considerable morphological changes are observed in the growth zone compared with the control group. A decrease in the growth of humerus length was found. In conclusion, nifedipine affects bone physiology, especially with consequences on bone growth. These effects appear to be quantitatively important, and there is the possibility of bone side effects on therapeutic use in humans, especially in young subjects.

Animals↗

Zonal analysis of metabolic profiles of articular-epiphyseal cartilage chondrocytes: a histochemical study.

Articular-epiphyseal cartilage from the femur of New Zealand rabbits was subjected to histochemistry for determination of the presence of metabolic enzymes along its zonal stratification. Glycolytic enzymes were strongly reactive in all of the zones. Krebs cycle enzymes, enzymes of the hexose monophosphate shunt and the respiratory chain enzymes showed a progressive increase in reactivity from the tangential zone through the top half of the epiphyseal zone. Indicators of lipid metabolism were fairly high in all regions of the cartilage.

Animals↗

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate.

In a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 mum. The half maximal transport rate occurred at 34 mum Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of the ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.

Animals↗

[Behavior of artificially produced defects in the epiphyseal plate of rabbits after transplantation of autologous and homologous rib cartilage].

In the first part of this study one could demonstrate that a defined mechanical traumatization of the distal epiphyseal plate of femur produced a constant development of bone fossilization which united the epiphysis to the metaphysis as a histopathological substract. In the second part--a correspondingly set-up defect--on the opposite side of the distal epiphyseal plate femur was filled up either by an autogeneous or homogeneous cartilage from the rib of a rabbit. The autogeneous rib transplantations were in this connection in a good position to prevent a partial bone-bridge-development between the epiphysis and metaphysis. The homogeneous transplantations showed some bone fossilizations, on the whole though satisfactory results with regard to the prevention of a partial obstruction could be obtained.

Animals↗

Action of estradiol on epiphyseal growth plate chondrocytes.

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17beta-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10(-13) M to 10(-7) M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10(-7) M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.

Alkaline Phosphatase↗

Anaplastic large cell lymphoma presenting as an epiphyseal lytic lesion--a case report with clinico-pathologic correlation.

Anaplastic large cell lymphoma (ALCL) is predominantly a systemic disease with nodal involvement, but extranodal involvement can occur either as the primary presentation or during the disease course. Primary epiphyseal involvement is extremely rare with lymphomas. This case report illustrates an 8-year old boy who first presented with pain over the right upper extremity, which was initially treated as epiphyseal osteomyelitis. A few weeks later, he presented with abdominal pain and an abdominal wall mass, which on biopsy proved to be an anaplastic large-cell lymphoma.

Bone Neoplasms↗

Previous synovectomy or epiphyseal stapling and the influence on knee replacement in juvenile chronic arthritis.

Seventy-seven anatomically graduated components (AGC) total knee arthroplasties (TKA) were performed on 52 patients with juvenile chronic arthritis. According to the nature of previous surgery on the knee, the patients were subdivided into three groups. The mean age at onset of disease in 23 patients with previous synovectomy of the knee was 11 (1.5-16) years, the mean age at the time of synovectomy was 20 (4-42) and the mean age when arthroplasty was performed was 31 (18-45) years. In nine patients with previous epiphyseal stapling, the mean age at disease onset was 4 (1.5-8) years, at stapling 8 (4-16) years, and at arthroplasty 23 (18-30) years. In patients with no previous surgery, the mean age at disease onset in this group was 7 (1.5-16) years and the mean age at arthroplasty 34 (16-64) years. Patients with need for epiphyseal arrest had an early disease onset and knee replacement in early adulthood. The mean age at knee replacements was highest in the group with no prior surgery.

Adolescent↗

Studies on the time frame for ossification of the medial clavicular epiphyseal cartilage in conventional radiography.

Radiological assessment of the degree of ossification of the medial clavicular epiphyseal cartilage plays a vital part in forensic age diagnosis of living adolescents and young adults. A total of 873 plain chest radiographs requested by the staff medical officer for members of staff aged 16-30 at the University Hospital Charité were evaluated retrospectively. Of these X-rays 699 permitted an assessment of ossification of at least 1 side of the clavicle. In addition to the customary stages (1: non-ossified epiphysis, 2: discernible ossification centre, 3: partial fusion, 4: total fusion) a stage 5 was also defined, characterised by the disappearance of the epiphyseal scar following total fusion. The earliest age at which stage 3 was detected in either gender was 16 years. Stage 4 was first observed in women at 20 years and in men at 21 years. In both genders, the earliest observation of stage 5 was at 26 years. It was concluded that plain chest radiographs can essentially be used to assess clavicular ossification. In practice, if overlap in posterior-anterior views impedes evaluation, a lateral view should also be taken to facilitate age estimation. In forensic practice the reference values of the present paper should be applied.

Adolescent↗

Concomitant double epiphyseal injuries of the tibia with vascular compromise: a case report.

A rare case of double epiphyseal injuries in the same tibia is reported. A 4-year-old child was struck by a vehicle and presented with the above fractures and vascular compromise to the extremity. Following localization of the vascular occlusion level by arteriography, popliteal vessel anastomosis was attempted. The vascular repair failed, resulting in disarticulation through the knee. Simultaneous displaced proximal and distal epiphyseal injury in the same tibia has not been previously reported in the literature. The most serious complication of injury to the proximal tibia is vascular compromise. A delay in recognition or intervention in such cases can be devastating.

Child, Preschool↗

Cyclic AMP-dependent inhibition of collagen synthesis in avian epiphyseal cartilage cells: effect of chicken and human parathyroid hormone and parathyroid hormone-related peptide.

Avian cartilage cells derived from epiphyseal growth-plate and avian skin fibroblasts were cultured in vitro. Production of cAMP by cartilage cells was stimulated by the synthetic fragments (1-34) of chicken (cPTH), human (hPTH) parathyroid hormone and by parathyroid hormone-related peptide (PTHrP). The enhancement of cAMP production by any of the peptides could be blocked by the parathyroid hormone analogue (3-34)PTH, suggesting interaction with PTH specific receptors. When incubated with [3H]proline, both cell types released radiolabelled collagenase-digestible and non-digestible proteins into the medium. cPTH, hPTH, PTHrP, forskolin, prostaglandin E2 (PGE2) and 8-bromo cAMP inhibited collagen production in cartilage cells with only minor effects on non-collagenase digestible proteins. No effect of cAMP on collagen production by fibroblasts was observed. The present results provide additional evidence that avian growth-plate cartilage cells are targets for PTH, and are first to demonstrate the response of a non-mammalian system to mammalian PTHrP. The data suggest that collagen production by epiphyseal growth-plate cartilage cells is inhibited by PTH and that this inhibition is mediated by cAMP.

Animals↗

Nucleoside triphosphate pyrophosphatase of rabbit matrix vesicles, a mechanism for the generation of inorganic pyrophosphate in epiphyseal cartilage.

Inorganic pyrophosphate (PPi) may be important in the regulation of mineralisation but its origin in epiphyseal cartilage is ill-defined. Nucleoside triphosphate pyrophosphatase is one potential source, as this enzyme catalyses the formation of PPi from nucleoside triphosphates. This enzyme has been identified in matrix vesicles derived from rabbit epiphyseal cartilage and a method developed to measure the activity using ATP as substrate in intact matrix vesicles under relatively physiological conditions. The enzyme had a high affinity for ATP (Km less than 10 microM) and was also active towards GTP, CTP and UTP. Disruption of the matrix vesicle membrane by sonication failed to alter the activity. Treatment of sonicated matrix vesicles with Triton X-100 increased the activity which may indicate a direct effect of the detergent on the enzyme. Activity towards ATP was inhibited substantially by ADP and AMP and by another potential substrate beta,gamma-methyleneadenosine 5'-triphosphate. Dichloromethylene bisphosphonate, an analogue of the product PPi, inhibited the activity to a lesser extent. Two other potential substrates, NADP+ and thymidine 5'-monophosphate p-nitrophenyl ester were only weakly inhibitory as was 1-hydroxyethylidene 1,1-bisphosphonate. These results imply that nucleoside triphosphates are the substrates in vivo and the inhibitory effects of ADP and AMP suggest mechanisms whereby this activity could be regulated.

Animals↗

Ultrastructural localization of alpha-parvalbumin in the epiphyseal plate cartilage and bone of growing rats.

The distribution of the calcium-binding protein, alpha-parvalbumin, in the epiphyseal plate cartilage and bone of growing rats was examined by electron microscope immunocytochemistry of undecalcified samples. Parvalbumin immunoreactivity, as revealed by gold particles, increased with maturation of chondrocytes and was maximal in the zone of calcification. It was found in the cytoplasm of chondrocytes, osteoblasts and osteocytes, corroborating light microscope observations. The immunolabeling was associated with amorphous electron-dense material in the cytoplasm and not bound to membranes. There was moderate parvalbumin immunolabeling over the dense chromatin in the nuclei of chondrocytes and bone cells, but none in the cell processes of mature and hypertrophic chondrocytes, in the matrix vesicles themselves, or in the cell processes of osteoblasts. However, there was parvalbumin immunoreactivity in the cell processes of the osteocytes of compact cortical bone. The uncalcified and calcified matrix of the epiphyseal cartilage, the osteoid, and the fully mineralized cortical bone were devoid of parvalbumin immunoreactivity. Thus, immunoreactive parvalbumin is confined to the cell bodies of chondrocytes and osteoblasts, and is unlikely to be directly involved in mineral deposition. The maximal parvalbumin immunoreactivity in the last terminal chondrocytes of the zone of calcification suggests that the protein is involved in buffering intracellular Ca2+, preventing the stimulation of degenerative processes by high intracellular calcium. The parvalbumin immunoreactivity in the cell processes of osteocytes of compact cortical bone seems to indicate that this calcium-binding protein may be involved in the regulation of Ca2+ fluxes and hence in calcium homeostasis in bone.

Animals↗

Impairment and activity limitation associated with epiphyseal dysplasia in children.

OBJECTIVES: To develop a profile of impairment and activity limitation among children with epiphyseal dysplasia (ED) and to identify the relationship between these 2 domains. DESIGN: Cross-sectional study. SETTING: Acute, pediatric academic and health sciences center. PARTICIPANTS: Eleven subjects with multiple epiphyseal dysplasia (MED) and 17 subjects with spondyloepiphyseal dysplasia (SED), with a mean age of 12.9 years. INTERVENTIONS: Not applicable. Main outcome measures Anthropometric indexes of growth and nutrition, joint range of motion (ROM) and alignment, muscle strength, pain, and activity limitation. RESULTS: Subjects with SED had significantly shorter stature than the reference population (P<.01). Seventy-three percent of participants with MED and 77% of those with SED were above average or overweight for their height (P<.01). Both groups presented with moderate to severe joint ROM impairment, with greater lower-extremity involvement. Subjects with SED had worse outcomes with respect to overall ROM and alignment impairments (P<.01), particularly in their upper extremities (P<.01), than subjects with MED. Significant overall muscle weakness was noted in all subjects (z=-1.81; P<.01). Ninety-four percent of subjects with SED reported pain with activity, compared with 64% of those with MED (P=.04), although pain intensity did not differ between groups. Fifty percent of subjects had undergone orthopedic surgery. Mild activity limitation was reported by all subjects (mean score, 87.7+/-18.83). Significant correlations were identified between height for age and strength (r=.50) and pain and activity limitation (r=-.50). CONCLUSIONS: Despite moderate to severe impairments, all subjects reported surprisingly mild activity limitation. A positive correlation was identified between pain and activity limitation. This study also identified and described patterns of muscle weakness, pain, and nutritional concerns not previously reported in the ED literature.

Adolescent↗

Vascularisation and osteochondrosis of the epiphyseal growth cartilage of the distal femur in pigs--development with age, growth rate, weight and joint shape.

Until recently, the cartilage canals of the epiphyseal growth cartilage have not been associated with any specific disease. However, data support the hypothesis that osteochondrosis could be related to inadequate blood supply from vessels in these channels. We have done a study to investigate the relationship between the regression of cartilage canals and the formation of osteochondrosis latens in the epiphyseal growth cartilage of the distal femur in pigs, and the relationship between these events and age, growth rate, weight and femoral shape of the individual animals. This involved, in part, a comprehensive study of the distribution and pattern of regression of the cartilage canals. We found that the regression is a highly predictable process that follows an age-dependent pattern. However, we failed to prove any association between overall vascular regression and osteochondrosis, between vascular regression and weight, growth rate or femoral shape or between osteochondrosis and weight, growth rate or femoral shape. This may indicate that osteochondrosis latens is not caused by a general failure of vascular supply or general factors such as growth rate, but rather a consequence of local conditions affecting a limited number of vessels. A factor fitting this description is local compression.

Animals↗

ADAMTS5-mediated aggrecanolysis in murine epiphyseal chondrocyte cultures.

OBJECTIVE: Aggrecan degradation by aggrecanases [a disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) 1, 4, 5, 8, 9, 15] is considered to initiate much of the cartilage pathology seen in human arthritis, however, the proteinase responsible and its mode of control is unclear. The present work was done to examine mechanisms of aggrecanase control in a novel murine epiphyseal cell system and to determine whether ADAMTS5 alone is responsible for aggrecanolysis by these cells. METHODS: Epiphyseal cells from 4-day-old mice (wild type, TS-5 (-/-), CD44(-/-), syndecan-1(-/-), membrane type-4 matrix metalloproteinase [MT4MMP(-/-)]) were maintained in non-adherent aggregate cultures and aggrecanolysis studied by biochemical and histochemical methods. Confocal immunolocalization analyses were done with specific probes for ADAMTS5, hyaluronan (HA) and aggrecanase-generated fragments of aggrecan. RESULTS: Aggrecanolysis by these cells was specifically aggrecanase-mediated and it occurred spontaneously without the need for addition of catabolic stimulators. Chondrocytes from ADAMTS5-null mice were aggrecanase-inactive whereas all other mutant cells behaved as wild type in this regard suggesting that ADAMTS5 activity is not controlled by CD44, syndecan-1 or MT4MMP in this system. Immunohistochemical analysis supported the central role for ADAMTS5 in the degradative pathway and indicated that aggrecanolysis occurs primarily in the HA-poor pericellular region in these cultures. CONCLUSION: These findings are consistent with published in vivo studies showing that single-gene ADAMTS5 ablation confers significant protection on cartilage in murine arthritis. We propose that this culture system and the analytical approaches described provide a valuable framework to further delineate the expression, activity and control of ADAMTS-mediated aggrecanolysis in human arthritis.

ADAM Proteins↗

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: implications for endochondral ossification.

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.

Achondroplasia↗