Electron-microscopic microprobe analysis on the initial stages of mineral formation in the epiphyseal growth plate

Summary Dry thin sections (300–500 nm thick) of shock-frozen, freeze-dried and embedded epiphyseal growth plates from the proximal tibia of guinea pigs were cut longitudinally from the plate. Dark round bodies (ø<0.5 m) were observed using the scanning transmission mode of the electron microscope initially directly in the vicinity of the chondrocytes. They gradually spread out in the direction of the metaphysis to the center of the longitudinal septum and represent most probably the matrix vesicles. By use of a microscan of 0.25×0.25 m the element-concentrations of these bodies were measured. The measurements started on those bodies that could be clearly recognized and were extended to a length of 30–40 m in the metaphyseal direction. To obtain approximate quantitative results the registered Ca_K and P_K x-ray counts were directly compared with counts of fully mineralized regions, the Ca and P contents of which are known. Ca as well as P could be detected in the first visible vesicle-like structures (Ca0.2%, P0.4%) and increased steeply in the metaphyseal direction, amounting to approximately 6% Ca and 3% P. These results may lead to the conclusion that P_i becomes split from phosphate esters and transformed into the matrix vesicles already in a very early stage of enrichment. Incorporation of Ca may be coupled with this process.The authors express their thanks to the Deutsche Forschungsgemeinschaft for financial support     

Energy dispersive X-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments

Summary Isolated cells of matrix fragments of freeze fractured and freeze dried growth plate from the four species was analyzed by EDX. Cells were removed from the tissue by stereoscopic microdissection using an SEM and mounted on thin film supports on TEM grids: this approach eliminated specimen X-ray background and reduced instrumental and support back-ground levels to insignificant proportions. Chondrocyte and matrix fragments were dissected and analyzed. Cell Ca reaches EDX detectable levels in hypertrophic cells close to the mineralization front. At all stages of maturation, the cells exhibit high P; however, matrix Ca levels are elevated before P. This data suggests that the early cartilage matrix is accumulating Ca and that the cells' role in this process may be to elevate the matrix Ca and P concentration. All cells showed clear S peaks although these are reduced in late hypertrophic cells. With mineralization, matrix S levels fall, indicating a loss of sulfated proteoglycans. Matrix before mineralization contains more K than would be expected from data of previous studies. It is suggested that while this K may be bound to fixed anionic sites in the matrix, reported values for cartilage lymph and extracellular fluid should be reviewed.     

Morphological studies of the epiphyseal growth zone in the brachymorphic (bm/bm) mouse

Light microscopy, including immunohistochemical techniques, and electron microscopy were performed on epiphyseal growth cartilage from brachymorphic (bm/bm) mice and age-matched phenotypically normal siblings aged 5, 16 and 25 days. In the bm/bm mice light microscopy showed a disturbed columnar arrangement and numerous chondrocytes with pronounced regressive changes. The normal development of proliferative cells into hypertrophic cells was halted and thus only a rather small and ill-defined hypertrophic zone was seen. The calcifying zone was irregular and the normal lacunae were replaced by a densely staining matrix. Using immunofluorescence techniques, the presence of considerable amounts of both type II and type V collagen was demonstrated in the bm/bm mice, while the cartilage from controls contained only type II. Ultrastructurally the lacunar matrix contained bundles of fine fibrils without the typical collagen periodicity which might indicate synthesis of a defective procollagen. Our observations together with the previously demonstrated deficiency of 3'-phosphoadenosine 5'-phosphosulphate, illustrate the complexity of the growth cartilage disturbance in the bm/bm mouse. Most of our findings are at variance with those described in the literature and possible pathogenetic mechanisms for the observed alterations in the growth cartilage are discussed.     

Distribution of lipids associated with mineralization in the bovine epiphyseal growth plate

Calcium-acidic phospholipid-phosphate complexes, known to induce in vitro hydroxyapatite formation from metastable calcium phosphate sotutions, have been isolated from the morphologically defined zones of the bovine epiphyseal growth plate. The changes in zonal distribution of these complexes in epiphyseal cartilage correlate directly with other biochemical changes which occur prior to cartilage calcification. The concentration of calcium-acidic phospholipid-phosphate complexes increases going from the morphologically defined reserve zone to the proliferative zone, peaking in the hypertrophic zone, where mineralization is initiated, and decreasing in primary spongiosa and diaphyseal bone. Expressed as milligrams of calcium-phospholipid-phosphate complex per milligram hydroxyproline the concentration ranged from 19 (articular cartilage) to 535 (hypertrophic cell zone) decreasing to 43 (diaphyseal bone) with parallel changes being seen when the concentration was expressed per gram of demineralized dry tissue, per total lipid, per DNA, or, per 5′-AMPase activity.     

Potassium concentration in membrane-associated particles in the epiphyseal growth plate

Summary Electron-dense particles with a diameter of 50–200 nm have been observed at the cell membrane of chondrocytes in the zone of the initiation and advance of mineralization, using the dark field STEM mode. Electronprobe x-ray microanalysis and laser microprobe mass analysis indicate that these particles contain predominantly K and Na. They appear only in dry thin sections of shock-frozen, freeze-dried embedded tissue and not in sections of water-treated samples; hence they contain water-extractable potassium and sodium. The function of the two elements at these special sites is not yet clear. On the one hand, they might reflect exocytotic processes connected with a Na-K-ATPase; on the other hand, they might exist as a transitory state before being replaced by Ca and phosphate in the mineralizing matrix and later transported elsewhere by the blood vessels.We thank the Deutsche Forschungsgemeinschaft for financial support     

The role of autocrine growth factors in radiation damage to the epiphyseal growth plate

Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated by the finding that the addition of 4.0 mM EGTA (a calcium chelator) to the cell cultures before irradiation prevented the decrease in PTHrP mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.     

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.     

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate

Summary 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 m. The half maximal transport rate occurred at 34 m 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 these 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.     

Stereological studies on matrix vesicle distribution in the epiphyseal growth plate during healing of low phosphate, vitamin D deficiency rickets

A model of the healing phase of low phosphate, vitamin D deficiency was used to investigate the initial stages of mineralization. The matrix vesicle distribution between the zones of the growth plate was found to be bimodal with high volume densities in the resting and hypertrophic zones and low volume densities in the proliferative and calcifying zones. Healing of the rachitic lesion was associated with a decrease in matrix vesicle volume density in the calcifying zone, compared with the lower hypertrophic zone in florid rickets. The volume density differences were due to differences in the number of vesicles, as the variation in mean caliper diameter was rather small. The findings are compatible with the dynamic cell debris theory for matrix vesicle origin and distribution presented earlier, which favours the view that a major part of matrix vesicles are formed from cell debris. A role of matrix vesicles in the mineralization process is indicated by the finding of an association between mineralization and matrix vesicle degradation.     

Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes

Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of ≥0.1 μM and Zn concentrations of ≥100 μM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype. J. Cell. Biochem. 68:110–120, 1998. © 1998 Wiley-Liss, Inc.     

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate

The goal of this review is to examine the fate of the hypertrophic chondrocyte in the epiphyseal growth plate and consider the impact of the cartilage microenvironment on cell survival and apoptosis. Early investigations pointed to a direct role of the hypertrophic chondrocyte in osteogenesis. The terminally differentiated cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens. We present a new concept: hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis.     

Structural study on the effect of 24,25-dihydroxyvitamin D3 on epiphyseal growth plate in suckling mice

The in vivo effects of 24,25(OH)2D3 on cellular structure and organization, matrix metachromasia and mineralization were studied in epiphyseal growth plate of normal neonatal mice. A relatively low dose of the metabolite, 40 ng/kg body weight, significantly increased the overall size of humeral growth plate and the zone of cellular proliferation. By and large, the tissue's response to the metabolite did not change with the increase in dose administered except for a decrease in the number of chondroblasts. 24,25(OH)2D3 led to significant increases in the metachromatic reaction of the cartilaginous matrix, but appeared to depress the mineralization process. Qualitative structural changes were noted in chondroblasts and hypertrophic chondrocytes. 24,25(OH)2D3 affected the osteoblastic and osteocytic populations of cells in the metaphysis and diaphysis of the humerus. High doses of 24,25(OH)2D3 brought about distinct atrophic changes in the above cells. These findings indicate that excessive doses of 24,25(OH)2D3 in an intact animal may lead to retardative effects upon bone growth.     

Use of microarray analysis to study gene expression in the avian epiphyseal growth plate

Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate.