Hypothyroidism was induced in young female Sprague-Dawley rats by the addition of methimazole (0.67 mg/ml) to drinking water for a period of 7 weeks (7-14 weeks of age). The responses of the articular cartilage, epiphyseal growth plate cartilage, epiphyseal trabecular bone, and metaphyseal trabecular bone in the proximal tibia were assessed by structural parameters. In addition, replacement therapies were introduced for the last 2 weeks of the experimental period. These included 0.7 U/kg BW human GH (hGH), 15 micrograms/kg BW L-T4 (T4), and a combination of hGH and T4 at the same doses. In the hypothyroid rats, the width of epiphyseal growth plate cartilage decreased by 27%, that of articular cartilage by 35%, epiphyseal trabecular bone volume by 30%, and metaphyseal trabecular bone volume by 66% relative to those in age-matched control tissues. T4 treatment led to a full restoration of the epiphyseal trabecular bone and surpassed by 40% the control value. The magnitude of the articular cartilage and the epiphyseal trabecular bone volume returned to control values, while that of metaphyseal trabecular bone was 68% of control values. Treatment with hGH did not improve the epiphyseal growth plate cartilage or articular cartilage. It did restore epiphyseal trabecular bone to almost normal values, but metaphyseal trabecular bone improved to only a small though significant level (45% of control value). The combination of T4 and hGH resulted in an additional enlargement in the width of the epiphyseal growth plate cartilage and an increase in metaphyseal trabecular bone volume compared to those in the T4 group. Qualitative examinations indicated that it was only in the T4 and T4 plus hGH groups that the lowest chondrocytes in the epiphyseal growth plate cartilage resumed their normal hypertrophied size. These results suggest that the change in the hypothyroid state do not rely solely on the lack of pituitary GH synthesis and secretion, as replacement by exogenous GH did not restore normal epiphyseal growth plate cartilage morphology or its remodeling into metaphyseal trabecular bone. Treatment with T4 (which restored endogenous pituitary GH to 30% of control levels) results in full recovery of the epiphyseal growth plate cartilage morphology along with its associated metaphyseal trabecular bone. In addition, it can also be concluded that the decrease in epiphyseal trabecular bone volume observed in the hypothyroid animals was due solely to the GH-deficient state that accompanied hypothyroidism.
Young male Sprague-Dawley rats (5-7 weeks old, 80-120 g) were hypophysectomized (HX) and maintained on thyroxin and dexamethasone replacement therapies. Ten days after surgery, some HX rats received a single injection of human growth hormone (hGH), and others five daily injections of hGH. Tartrate-resistant acid phosphatase (TRAP) histochemistry was employed in order to evaluate the number of cells of resorptive potential in the metaphyseal bone of the proximal tibiae of HX rats and was compared with normal rats and HX rats that further received hGH replacement therapy. In normal rats, two populations of TRAP-positive cells were identified: multinuclear cells, which showed histological characteristics of osteoclasts, and small mononuclear cells, the number of which was overwhelming when compared with the number of TRAP-positive multinuclear cells. Both populations were reduced in the HX rat, but more so the mononuclear cells, which were assumed to represent the precursor pool of mature osteoclasts and chondroclasts (P < 0.005). Five daily injections of hGH to HX rats brought about a significant increase in the number of TRAP-positive multinuclear cells, the number of nuclei of these cells, and the number of mononuclear TRAP-positive cells, throughout the metaphyseal bone (P < 0.05). A single injection of hGH increased only the number of TRAP-positive multinuclear cells in the trabecula/bone marrow interface (P < 0.05), indicating a very rapid fusion of precursor cells into mature osteoclasts in that particular location.(ABSTRACT TRUNCATED AT 250 WORDS)
Mandibular condylar explants of neonatal ICR mice were maintained as serum-free organ culture systems and were used to study the effects of three synthetic fragments of human parathyroid hormone (hPTH) on the morphology of the organ and its ability to incorporate [3H]thymidine. Forty-eight-hour incubation with hPTH (1-34), at a concentration of 0.5 microgram/ml caused an increase of 88% in DNA synthesis and a marked increase in the size of the chondroprogenitor zone. The mitogenic effect of hPTH (1-34) was decreased to 34% over control levels when the fragment hPTH (28-48) was added to the system. However, the addition of the latter fragment brought about a marked enhancement in the mineralization of the cartilaginous extracellular matrix along with the formation of an appreciable amount of new bone. The de novo osseous tissue was attached to the mineralized cartilage. When the carboxyl-terminal fragment hPTH (53-84) was added together with the other two fragments, the mitogenic effect of hPTH (1-34) was completely abolished and the respective cultures incorporated [3H]thymidine even less than untreated control cultures. Moreover, the addition of hPTH (53-84) to the culture system led to distinct structural features throughout the mineralized hypertrophic cartilage. The latter contained a mixture of cells within an unorganized extracellular matrix. Untreated control cultures lacked such structures, but contained the various cell zones as normally seen in neonatal condylar cartilage. Therefore, it seems reasonable to suggest that each of the three fragments tested induces a biological effect on neonatal cartilage and might be involved in the normal process of endochondral ossification.
The present study showed that the fragment hPTH (1-34) is mitogenic in organ cultures of neonatal mandibular condylar cartilage, and even more so in late fetal condyles. Three fragments of hPTH were used to clarify which part of the molecule possesses a mitogenic effect alike that of the native hormone: hPTH (1-34), and (53-84). [3H]thymidine incorporation into trichloracetic acid insoluble material and quantitative autoradiography were employed in a serum-free medium to assess the effects of these fragments while light and electron microscopy studies served for morphological evaluations. It became evident that the fragment hPTH (1-34) enhanced the incorporation of [3H]thymidine, a fact that could be noted only in serum-free medium. The putative target cells for the effects of hPTH (1-34) were the chondroporogenitor cells which also appeared to have experienced a blockage in their differentiation into chondroblasts. Ultrastructurally, the latter cells responded in the formation of adherent profiles of plasma membranes, whereas the differentiated zone of the cartilage reduced its size. Using serum-free medium, hPTH (1-34) also brought about an inhibition in alkaline phosphatase activity, a fact that was not encountered in medium containing serum. By contrast, hPTH (28-48) had no mitogenic effect, although treated specimens revealed morphological changes in the chondroprogenitor cell zone along with an enhancement of cartilage cells hypertrophy. No significant effects on either mitogenecity or morphology could be noted in hPTH (53-84)-treated cultures.Various in vivo studies with parathyroid hormone have shown that the epiphyseal growth plate usu¬ ally responds in a marked widening and in an in¬ crease in the number of chondroclasts along its ossification zone (1,2). These findings, along with others in earlier studies, appeared to suggest that PTH possesses a growth-stimulating effect on car¬ tilage. However, in order to gain further under¬ standing as to the mechanisms involved in the ac¬ tivity of PTH, one had to turn to in vitro studies. Kawashima et al. (3) carried out a series of organculture studies whereby the growth of cartilage of 9-day-old chick embryo femur was stimulated by PTH (500 U/l). In a subsequent study, Kawashima et al. (4) indicated that the stimulatory effects of PTH were exerted selectively upon the more mature, non-dividing hypertrophie chondrocytes, whereas younger cells did not respond to this hor¬ mone. Suzuki et al. (5) were the first to investigate the effects of PTH in tissue cultures of chon¬ drocytes isolated from growth cartilage of young male rats, finding that the hormone (100 (tg/1) in¬ duced a marked stimulatory effect upon the syn¬ thesis of proteoglycans. Takigawa et al. (6), who extended the original studies of Suzuki et al.,showed that PTH also increased the level of cAMP in chondrocytes, but had no effect upon DNA syn¬ thesis in cultured chondrocytes. Burch 8c Lebovitz (7) using chick pelvic cartilage in vitro, reported that PTH (10 nmol/1) increased the DNA content by 5...
This investigation presents the structural changes in condylar cartilage incubated in the presence of human parathyroid hormone (1-34) in an organ culture system for 6 to 12 days. Control cultures maintained their cartilaginous characteristics whereas human parathyroid hormone (1-34)-treated cultures revealed the following modifications: (1) The chondroprogenitor cell zone at the apical region of the explant underwent a substantial enlargement. The cells changed from a mesenchyme-like morphology into polygonal, glycogen-rich cells that were tightly attached to each other by a fibrillar intercellular matrix, but even by 12 days the apical region was comprised of healthy cells. (2) The mineralizing zone in the hypertrophic cartilage revealed a change in its cellular population. Hypertrophic chondrocytes were replaced by cells with amoeboid extensions and large numbers of secretory granules or vesicles. Based upon the above findings it appears that the chondroprogenitor cells that are initially stimulated to proliferate, are being suppressed from subsequent differentiation into chondroblasts; and that hypertrophic chondrocytes apparently undergo a dedifferentiation process followed by development into an as yet unknown cell population.
The purpose of the present study was to examine the effects of various concentrations of serum on the behavior of neonatal condylar cartilage when cultured in an organ culture system. Mandibular condylar cartilages were obtained from newborn ICR mice, of which the zone of undifferentiated chondroprogenitor cells along with a few layers of young cartilage cells were cultivated at the medium-air interface. The incubation medium included fetal bovine serum at concentrations ranging from 0 to 10%, and the explants were kept in vitro up to 10 d. The serum-free medium maintained the chondrogenic expression, and the overall size of the cartilagenous portion of the explants increased with the decrease of the concentrations of serum in the medium. When explants were labeled with [3H]thymidine and were then processed for autoradiography, the peak of labeling was noticed at 48 h, a feature that recapitulated itself in all cultures (73, 140, 175, 201, and 129 labeled cells per chondroprogenitor zone in explants grown in 0, 1, 2.5, 5, and 10%, respectively). It can be concluded that serum-free medium maintains the chondrogenic phenotype of condylar cartilage in vitro.
9033 Background: The aim of this pilot study was to determine VEGF serum levels (S-VEGF) at diagnosis and at restaging in children diagnosed with Hodgkin’s disease, and to investigate whether this parameter provides prognostic information for remission after 2 courses of chemotherapy Methods: S-VEGF levels of 9 consecutive pediatric patients (pts) with Hodgkin’s disease were assayed at diagnosis and at restaging. Levels of VEGF were determined using a commercially available ELISA anti-human VEGF immunoassay kit. PET-CT fusion was performed for each child at diagnosis and after 2 courses of chemotherapy in order to assess response to treatment. Results: 8 children went into complete remission or very good partial response after 2 courses of chemotherapy according to the protocol, one child developed tumor progression and respond to second line chemotherapy. At diagnosis average S-VEGF level was 655.7pg/ml (range, 1078.7–29.22 pg/ml) and at restaging decreased to 237.6 pg/ml (range, 0–453 pg/ml). (p=0.0039). One child with Hodgkin’s disease who had a higher level at first restaging and developed progressive disease responded to reinduction therapy and had a significantly lower level at the second restaging. The comparison between the levels of S-VEGF at diagnosis and at restaging showed a significant difference for the pts who responded to treatment with decreased S-VEGF and the pt who developed tumor progression with increased S-VEGF. Conclusions: Changes in S-VEGF levels correlated with response to treatment for most of the children diagnosed with Hodgkin’s disease. This provides a rationale for exploring clinical interest in S-VEGF measurements of a larger group of children with Hodgkin, and using the test for clinical trials of antiangiogenic therapies. No significant financial relationships to disclose.
MA 01655, USA arid Dcpt of Pacd~atrics, Free Unlv Brusseis, 81020 Brussels, Belgium.In adult rats, selenium (Se) dcficiency markedly decreases liver type I outcr-ring 5' dclod~nasc (5'D-I, n sclcnacnzymc convcrtlng T4 into T3), roult~ng In a 40% increase ~n scrum T4 conc~~ntr.~tions Scrum T3 and revcisu T3 (rT3) ionccnlr.,tlt,trh arc c>ncI~angcd or marginally decreased or ~ncreascd, respectively. In rat fetuses, scrum T4 and rT3 concentrations are not affected by selenium dcfic~uncy. We studled the effect of Sc deficiency on thyroid function in the rat neonate. 28 weanling female rats werc fed a Sc replete (Sct) or Sc dcficicnt (Se-) dlct for 4 wk pilor to mating and throughout gcstatlon. 2-3 pups from each litter wcrc sacr~ficcd 7, 14 and 21 days after delivery Serum T4, T3, rT3 and TSH conccnlrallons and liver 5'D-I scllvlty, to assess Se dcf~c~cncy, rverc measured.
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