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A method of modified coronal computed tomography (CT) was developed for examination of epiphyseal growth plate arrest and neoplasms near the growth plate. Twenty-four patients were examined with this technique (26 examinations). In all cases, the growth plate and adjacent regions were well demonstrated.
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.
The objectives of this study were to determine in horses 1) the ages at which viable cartilage canal vessels are present in the articular-epiphyseal cartilage complex of three predilection sites and one nonpredilection site of osteochondrosis (OC), 2) the prevalence of lesions of OC in these sites, and 3) whether there was an association of lesions of OC with necrotic cartilage canal blood vessels. The medial femoral condyle, lateral femoral trochlear ridge, and distal ends of the tibia and proximal phalanx were examined grossly, microradiographically, and histologically in 35 horses 18 months old or younger. Cartilage canals containing patent blood vessels were present in all sites examined in foals less than 3 weeks old and were absent from all sites by 7 months of age. The overall prevalence of lesions of OC at one or more of the sites examined was 12/35 (34%). Prevalence increased to 11/22 (50%) in horses 2 months old and older. These lesions occurred primarily in the medial condyle of the femur (n = 5) and the intermediate ridge of the distal tibia (n = 5). All lesions seen in horses between 3 weeks and 5 months of age were associated with necrotic cartilage canal blood vessels. In horses 7 months of age and older, lesions of OC were considered chronic because of extensive involvement of the subchondral bone and bone marrow. These results suggest that OC lesions develop prior to 7 months of age and that ischemic necrosis of cartilage secondary to a defect in vascular supply is an important factor in the pathogenesis of this disease in horses.
Epiphyseal fractures account for about one fifth of pediatric fractures. Approximately 10% cause major growth disturbances, depending on the location and type of the fracture and the skeletal maturity of the child. Intraarticular Salter-Harris type IV fractures are rare, carry a poor prognosis, and almost always need surgical reduction to prevent deformity. We present a case report of a pediatric patient who returned to normal function after the successful surgical reduction of a Salter-Harris type IV fracture in the proximal phalanx of the great toe.
The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.
Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.
We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcified hypertrophic region. Substantial levels of lactate were also present in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose-6-phosphate and the activity of LDH. Maximal levels of each of the three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.
The growth-promoting actions of GH are thought to be mediated by somatomedin(s) (Sms), but unilateral bone growth in hypophysectomized (HX) rats given local injections of human (h) GH has been reported. Using slightly different methods, we have confirmed these results with purified hGH and have extended them to show that four daily injections directly into the tibial epiphyseal plate of 1 or 5 micrograms/day purified rat GH or recombinant DNA-derived hGH, but not of hPRL (6 micrograms/day) caused significant cartilage growth compared to that of the vehicle-injected contralateral tibia in rats that had been HX 14 days before the first injection. Thus, it is unlikely that the effects of GH are due to contaminating growth factors in the GH preparations, because the bacteria-derived preparation of hGH, which is unlikely to have such contaminants, was also active. Furthermore, we have shown that similar injections of 100 or 500 ng/day purified hSm-C caused unilateral tibial growth in rats HX 8 days, but not 14 days, before the first injection. These results demonstrate that both GH and Sm-C have direct growth-promoting effects on cartilage in vivo and are compatible with the theory that GH may act by stimulating local SM synthesis.
Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.
Fibroblast growth factor-2 (FGF-2) is a potent autocrine mitogen for fetal epiphyseal growth plate chondrocytes and exhibits a transient nuclear translocation during G1 of the cell cycle. We have characterized an intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor. Chondrocytes were isolated from the proliferative zone of the ovine fetal proximal tibial growth plate at 50-130 days gestation by collagenase digestion and were maintained in monolayer at early passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cycle in the presence of 2% FBS. Cells were removed between 4-26 h of incubation, and fractions representing the plasma membrane, cytoplasm, nuclear membrane, and nuclear contents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using 125I-labeled FGF-2 showed that a FGFBP of 46-48 kDa (represented by a double band) was present on the nuclear membrane at mid to late G1, and Western blot showed this to be immunologically related to a part of the extracellular domain of the high affinity FGF receptor 1 (FGFR1). Immunocytochemistry with intact cell cultures showed that this protein underwent a juxtanuclear distribution through mid to late G1. Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing 35S-labeled methionine/cysteine, and the cellular compartments were separated before immunoprecipitation using an antibody raised against the extracellular domain of FGFR1. Newly synthesized FGFR1-related proteins appeared throughout G1 and migrated multidirectionally within the cell; intact receptor of 125-145 kDa accumulated at the plasma membrane, while both intact receptor and truncated FGFR1 of 46-48 kDa were detected on the nuclear membrane, but not within the nucleus. Cells were incubated with protamine sulfate to prevent the binding of endogenous, cell membrane-associated FGF-2 to high affinity FGFRs and their subsequent internalization. This did not alter the juxtanuclear accumulation of truncated FGFR1 in late G1, suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G1.
A growing body of evidence suggests that systemic hormones and peptide growth factors may exert their effects on cell growth and differentiation in part through regulation of the cell division cycle. We hypothesized that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through controlling cell cycle progression at the G1/S restriction point. Our results support this hypothesis by demonstrating that treatment of epiphyseal chondrocytes with thyroid hormone under chemically defined conditions results in the arrest of DNA synthesis and the onset of terminal differentiation, indicating that thyroid hormone is one factor capable of regulating the transition between cell growth and differentiation in these cells. This terminal differentiation process is associated with induction of the cyclin/cyclin-dependent kinase inhibitors p21(cip-1 waf-1) and p27kip1, suggesting that thyroid hormone may regulate terminal differentiation in part by arresting cell cycle progression through induction of cyclin-dependent kinase inhibitors.
Thioglycollate-elicited rat peritoneal macrophages and epiphyseal chondrocytes were cultured in vitro, treated with colchicine, and then studied by electron-microscopic and cytochemical techniques. Colchicine, but not lumicolchicine, caused disappearance of cytoplasmic microtubules and breakup of the Golgi complex with spreading of its dictyosomes from a well defined juxtanuclear area throughout the cytoplasm. There was also an altered distribution of lysosomes, which oriented themselves close to the dictyosomes both in control and colchicine-treated cells. Further, the structure of the individual dictyosomes was changed, especially in the chondrocytes. GERL equivalents were observed in control cells but were difficult to detect after exposure to colchicine. Reaction product for thiamine pyrophosphatase was found in narrow cisternae on the inner side of the dictyosomes in control cells but in vacuole-like structures in colchicine-treated cells. Reaction product for acid phosphatase was present in GERL equivalents and lysosomes in control cells but mainly in lysosomes in colchicine-treated cells. Nevertheless, the total specific activities of these enzymes as well as of 5'-nucleotidase, a plasma membrane marker, remained unaffected by the drug treatment. These observations show that cytoplasmic microtubules play an important and, in many respects similar, cytoskeletal role in two so functionally diverse cell types as macrophages and chondrocytes. They are particularly important for the structural integrity of the Golgi complex, which in both cells is normally organized in the area around the centrioles, from which numerous microtubules radiate into the cytoplasm. The observations further suggest that GERL is an integrated part of the Golgi complex in these cells.
Chrondroitin sulphate proteoglycans obtained from embryonic chick bone marrow and epiphyseal cartilage were compared using immunochemical and biochemical analyses. Proteoglycans from each tissue, separated on CsCl density gradients, under dissociative conditions, into high (1.6 g ml-1), medium (1.5 g ml-1) and low (1.4 g ml-1) buoyant density fractions, were immunochemically analysed, using a panel of monoclonal antibodies that specifically recognize chondroitin 4-/dermatan sulphates, chondroitin 6-sulphate, keratan sulphate, the hyaluronate binding region present on connective tissue proteoglycans, and link protein. The same antibodies were used in Western blot analyses to detect intact proteoglycan monomers and core proteins that had been fractionated by agarose-polyacrylamide and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Specific differences between marrow and cartilage proteoglycans were detected. In CsCl gradients, marrow proteoglycans displayed a higher degree of heterogeneity in terms of buoyant densities and hexuronate distribution. Keratan sulphate chains were constituents of the majority of 'large' proteoglycans in the marrow; however, a portion of the large proteoglycans in marrow middle buoyant density fraction either lacked keratan sulphate chains or were substituted with a form different from that found on cartilage proteoglycans. Marrow lacked 'small' chondroitin/dermatan sulphate proteoglycans that were present in cartilage and contained a more heterogeneous population of proteoglycans, particularly in the lower buoyant density fractions. Both marrow and cartilage were similar in that they contained, as their major components, large, aggregating proteoglycans and link proteins that were immunochemically and biochemically identical. The significance of these differences between marrow and cartilage proteoglycans remains to be determined, but they may, in part, be responsible for imparting unique characteristics to the haematopoietic extracellular matrices.
Three brothers with the X-linked variety of spondylo-epiphyseal dysplasia tarda are presented. Their clinical courses differed: one was relatively free of symptoms, the second had slowly progressive osteoarthritis of the hip, and the third had an acute, disabling arthritis necessitating total hip arthroplasty. The clinical features of this disorder are reviewed.
We have determined the natural history of hip development in 42 patients with multiple epiphyseal dysplasia (MED). Premature osteoarthritis was a frequent outcome and was almost inevitable before the age of 30 years in those with incongruent hips. There were two types of immature hips: type I, the more severe form, had a fragmented and flattened ossific nucleus and acetabular dysplasia, was misshapen at skeletal maturity and osteoarthritic by 30 years of age; the milder type II hip had a small, rounded, uniformly ossified nucleus and a more normal acetabulum. Type II hips were well formed at maturity and were less prone to premature osteoarthritis. Considerable variations were noted in the manifestations of MED between families but not within families. The prognosis of a child's hip could be predicted; in sporadic cases from the type of immature hip, and in familial cases by also taking into account the outcome of affected relatives.
As a continuation of our studies on mineralization in epiphyseal growth plate (GP) chondrocyte cultures, the effects of tri-iodothyronine (T3) in both beta-glycerophosphate-containing, serum-free (HL-1) and beta-glycerophosphate-free, serum-containing medium (DATP5) were studied. The GP cells responded to T3 in a serum-, stage-, and dosage-dependent manner. Added at graded levels (0.1-10.0 nM) to preconfluent cultures (from day 7) in both HL-1 and DATP5, T3 caused progressive decreases in protein, collagen, and DNA synthesis but increased mineral deposition. In postconfluent cultures, these effects of T3 were generally muted. In preconfluent cultures, proteoglycan (PG) levels were not significantly affected in DATP5, although in HL-1 they were decreased by approximately 50%. In postconfluent cultures, T3 increased PG levels in DATP5 but had no effect in HL-1. In HL-1, alkaline phosphatase (ALP) activity was progressively increased by 200-500% in both pre- and postconfluent cultures. In DATP5 in preconfluent cultures, T3 initially stimulated but later suppressed ALP; in postconfluent cultures, T3 also transiently increased ALP but did not suppress activity upon longer exposure. The inhibitory effects of T3 on protein, PG, and DNA levels of GP chondrocytes suggest that in vivo its effects on bone growth must occur primarily after cellular proliferation. Apparently by binding to the 50 kDa thyroxine-binding globulin, which cannot penetrate the PG barrier, accessibility of T3 to GP chondrocytes is limited until the time of vascular penetration when its stimulatory effects on ALP and mineral deposition become critical for continued bone development.
Localization and ultrastructure of proteoglycans during endochondral calcification or mineralization in the rat epiphyseal growth plate cartilage were investigated histochemically and by immunoelectron microscopy, using ruthenium hexammine trichloride and/or mouse monoclonal antibody (2-B-6) which recognizes chondroitin-4-sulphated glycosaminoglycans. The gold particles of the second antibodies were distributed solitarily, lineally or in small clusters in calcified cartilage as well as in the surrounding non-calcified matrix. The labelled matrix glycosaminoglycans in the longitudinal septa increased from the proliferative to the upper hypertrophic zones and remained constant in calcified cartilage. After fixation with ruthenium hexammine trichloride, matrix proteoglycans precipitated and were observed as a reticular network of strands. Immunogold-labelled antibodies for the demonstration of the glycosaminoglycans were localized on these strands. Proteoglycans in calcified cartilage were observed as a network of fine-filamentous structures after ruthenium hexammine trichloride, showing their non-precipitated native state. Immunostaining for chondroitin-4-sulphate indicated that there was no change in the concentration of glycosaminoglycans on the small calcified nodules in the areas where calcification was just beginning.
Longitudinal bone growth occurs in the epiphyseal growth plate and is regulated by a network of paracrine and autocrine interactions. Bone morphogenetic proteins (BMPs) are a family of growth factors whose potent osteogenic properties suggest that they may play an important role within this network, but direct evidence for this is lacking. To address this question, a cDNA encoding chick BMP-7 was cloned from a chick embryo cDNA library. Sequence homology and evolutionary arguments strongly suggested that we had cloned the chicken BMP-7 homologue. Using a reverse transcription-PCR assay, BMP-7 expression was readily detected in bone, growth plate cartilage, brain and heart, and was just detectable in liver, skeletal muscle and adipose tissue. In contrast to the pattern of BMP-7 expression in the rat and mouse, no BMP-7 expression was detected in the chick kidney. In situ hybridization was used to locate the site of BMP-7 expression more precisely within the growth plate. BMP-7 expression was confined to hypertrophic chondrocytes adjacent to and at the tips of the metaphyseal vessels. No expression was detected in the reserve zone or in proliferating chondrocytes. These results point to a specific role for BMP-7 in the growth plate, possibly in osteoblast activation or as a chemotactic agent for the metaphyseal vessels.