Differential effects of hypothyroidism on the cartilage and the osteogenic process in the mandibular condyle: recovery by growth hormone and thyroxine.

Hypothyroidism was induced in young female Sprague-Dawley rats by the addition of methimazole (50 mg/kg BW.day) to drinking water for a period of 7 weeks (7-14 weeks of age). Replacement therapies of 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 were introduced during the last 2 weeks of the experiment. The responses of the cartilage and subchondral spongiosa of mandibular condyles were assessed by morphological and morphometric parameters. In addition, immunohistochemical localization of the GH receptor and insulin-like growth factor-I was determined by the streptavidin-biotin-peroxidase technique. In the hypothyroid rats, the trabecular bone volume of the subchondral spongiosa increased by 49%, indicating osteopetrosis. Along the ossification front, bone trabeculae occupied 18% and vascular elements 82% in mandibular condyles of control rats, whereas in hypothyroid rats, the percentage occupied by bone trabeculae increased to 89%. The cellularity of the cartilage in hypothyroid condyles was markedly reduced and was fully restored by T4, but not by GH replacement. Immunohistochemistry revealed the presence of GH receptors throughout the condyle regardless of the thyroid state of the animal. On the other hand, insulin-like growth factor-I immunohistochemical localization revealed the peptide to be present at all maturational stages of the cells in condyles from control and T4-treated rats, but to be lacking in young chondrocytes of hypothyroid and hGH-treated rats. This result demonstrates that the hypothyroid cartilage is compromised in its response to hGH.

In vivo anabolic effects of parathyroid hormone (PTH) 28-48 and N-terminal fragments of PTH and PTH-related protein on neonatal mouse bones.

We developed a neonatal mouse model to investigate in vivo anabolic effects of intact PTH (1-84) and its two fragments PTH (1-34) and PTH (28-48) and of the N-terminal fragment of PTH-related peptide [PTHrP (1-34)]. Two-day-old mice were injected with low-dose (0.05 microg/g body weight) and high-dose (0.2 microg/g body weight) of each of these peptides daily for 6 or 16 consecutive days. Long bones (tibias and femurs) and mandibular condylar cartilages were harvested. Total DNA and protein were analyzed as parameters for anabolic effects. DNA was increased significantly in tibias only by low doses of PTH (1-84) and PTH (1-34), but by both doses of PTH (28-48). In the cartilages of the mandibular condyles, both doses of all three peptides increased DNA. Total protein was increased in the tibia by the low dose of the three peptides, whereas in the condylar cartilage high doses of PTH (1-34) and PTH (28-48) also caused a 2- to 4-fold increase. When the effects of PTH (1-34) and PTHrP (1-34) on the tibias were compared, it became apparent that PTH (1-34) was more effective than PTHrP (1-34) when injected in low doses, but the latter caused a severalfold increase in DNA and protein at both doses. The outstanding anabolic effect of PTH (28-48) was further investigated using [3H]thymidine autoradiography, analysis of insulin-like growth factor I (IGF-I) protein, and localization of IGF-I messenger RNA (mRNA) by in situ hybridization. PTH (28-48) increased by 3-fold the number of [3H]thymidine-labeled cells in the epiphyseal cartilage of tibias removed from 8-day-old injected mice, and in the proliferative zone of the epiphyseal growth plate of tibias removed from 18-day-old injected mice. Femurs from the latter showed a 20% increase in their IGF-I content. In parallel, only tibias from 18-day-old injected mice showed IGF-I mRNA localization in proliferating chondrocytes, whereas those from vehicle-injected control mice did not exhibit IGF-I mRNA. In summary, our study showed that the neonatal mouse is a sensitive model to examine anabolic effects of different PTH and PTHrP fragments. It also revealed that PTH (28-48) has strong anabolic effects on this model, and suggests that IGF-I might mediate the anabolic effects of PTH (28-48).

Effect of thyroid hormone and growth hormone on recovery from hypothyroidism of epiphyseal growth plate cartilage and its adjacent bone.

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.

Ontogenesis of chondro/osteoclasts and their precursors in the mandibular condyle of the mouse.

Tartrate-resistant acid phosphatase (TRAP) histochemistry has been used to follow chondro/osteoclasts and their precursors during ontogenesis of the mandibular condyle of the mouse (from day 16 of gestation until day 15 of neonatal life). TRAP+ mono-, bi-, and multinuclear cells were counted separately in the perichondrium, along the resorption front and in the subchondral spongiosa. Index of cellular density was calculated by dividing the absolute numbers of cells by the length of resorption front or the area of spongiosa, respectively. The study revealed that TRAP+ cells are present in the perichondrium of the mandibular condylar cartilage from the first day of its existence as an organ, namely day 17 of gestation. These cells are more numerous in the posterior part of the condyle, and reach their maximal number on the third day of neonatal life. Along the resorption front, mono-, bi-, and multinucleated TRAP+ cells were counted. Their total number and their respective indexes of cellular density changed variably during the observation period: (a) the number of mononuclear cells increased gradually and, by the end of the observation period, was 13 times greater than it was at the beginning; (b) the number of binuclear cells increased threefold; and (c) the number of multinuclear cells stayed constant, except for a small peak around the time of birth. The changes in the spongiosa showed an opposite trend. In the spongiosa, the index of cellular density of the mononuclear TRAP+ cells increased slightly during ontogenesis. The number of binuclear cells increased twofold, while the number of multinuclear cells increased 17-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 modulates trabecular and endochondral bone turnover in the nude mouse by stimulating osteoclast differentiation.

A great deal of evidence has been accumulating that implicates the immune system in normal and pathological bone turnover. The objective of the present study was to examine the possible involvement of cytokines produced by T lymphocytes in bone metabolism. We have chosen the immunologically compromised athymic mouse, which demonstrate sclerotic features in its trabecular bone, as the animal model for assessment of possible modulation effects of interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6) on bone and cartilage metabolism. The cytokines were applied by daily subcutaneous injections for 3 consecutive days. Histomorphometry, measuring epiphyseal trabecular bone volume (ETBV), metaphyseal trabecular bone volume (MTBV), and the width of the growth plate, and tartrate-resistant acid phosphatase (TRAP) histochemistry were used to assess parameters of bone turnover in the proximal tibia. IL-6, but not IL-1alpha, reduced ETBV and MTBV. Both IL-6 and IL-1alpha reduced the width of the growth plate. IL-6, but not IL-1alpha, increased the number of chondroclasts and osteoclasts in the primary spongiosa of the proximal tibia, as well as the number of nuclei. The resultant bone resembled that of the wild-type mouse. The results point to IL-6 as a possible regulator of bone turnover in vivo. It is suggested that the athymic mouse has a deficiency somewhere in the cascade of events leading to the production of IL-6 or, alternatively, that IL-6 replaces other factors that are supplied by T lymphocytes directly or indirectly. As T lymphocytes interact with B lymphocytes it is suggested that the athymic mouse might be appropriate for studying the in vivo effects of the immune system on normal bone metabolism.

Evaluation of atrial natriuretic peptide and brain natriuretic peptide in atrial granules of rats with experimental congestive heart failure.

The natriuretic peptides are believed to play an important role in the pathophysiology of congestive heart failure (CHF). We utilized a quantitative cytomorphometric method, using double immunocytochemical labeling, to assess the characteristics of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in atrial granules in an experimental model of rats with CHF induced by aortocaval fistula. Rats with CHF were further divided into decompensated (sodium-retaining) and compensated (sodium-excreting) subgroups and compared with a sham-operated control group. A total of 947 granules in myocytes in the right atrium were analyzed, using electron microscopy and a computerized analysis system. Decompensated CHF was associated with alterations in the modal nature of granule content packing, as depicted by moving bin analysis, and in the granule density of both peptides. In control rats, the mean density of gold particles attached to both peptides was 347.0 +/- 103.6 and 306.3 +/- 89.9 gold particles/microm2 for ANP and BNP, respectively. Similar mean density was revealed in the compensated rats (390.6 +/- 81.0 and 351.3 +/- 62.1 gold particles/microm2 for ANP and BNP, respectively). However, in rats with decompensated CHF, a significant decrease in the mean density of gold particles was observed (141.6 +/- 67.3 and 158.0 +/- 71.2 gold particles/microm2 for ANP and BNP, respectively; p<0.05 compared with compensated rats, for both ANP and BNP). The ANP:BNP ratio did not differ between groups. These findings indicate that the development of decompensated CHF in rats with aortocaval fistula is associated with a marked decrease in the density of both peptides in atrial granules, as well as in alterations in the quantal nature of granule formation. The data further suggest that both peptides, ANP and BNP, may be regulated in the atrium by a common secretory mechanism in CHF.

[Characterization of the biological process of aseptic loosening of joint implants in orthopedics].

The purpose of orthopedic joint implants is to improve joint movement. Within the past 30 years, biomechanical improvements affecting the life-span of the implants has brought a new type of problem: the biological response to debris from the implant material contributes greatly to aseptic loosening of the prosthesis. The process is mediated by osteotropic factors, cytokines released from mononuclear cells, and osteoblasts and osteoclasts in the bone-cement interface or bone-implant interface.

[Distraction osteogenesis for hypoplastic facial bones].

Distraction osteogenesis is a well-known method for bone lengthening which stretches callus to generate new bone in the distracted area. The method was developed by Ilizarov for the lengthening of long enchondral bones. In recent years the method has also been applied to the facial bones and to the jaw.

Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: further evidence for the enhancement of intracellular glycogen and visualization of matrix components.

The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde-osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde-osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.

Glucocorticoid-induced alterations in collagen of neonatal mouse condylar cartilage.

Neonatal mice were treated with a single dose of triamcinolone hexacetonide, a long-acting synthetic analogue of cortisol, and their mandibular condyles were studied ultrastructurally ten days thereafter. A pronounced decrease in the number and size of matrix granules (proteoglycans) was found in the cartilaginous matrix of triamcinolone-treated condyles. In contrast, a marked increase concomitant with significant structural changes was noted in collagen fibrils. An obvious enhancement of collagen fibrillogenesis was noticed in the pre-mineralizing extracellular matrix. Atypical, wider than normal, banded collagen fibrils were found to form dense meshworks which appeared to lack any specific orientation or organization. It is proposed that glucocorticoid hormones, given systemically to neonatal mice, interfere with regulatory mechanisms involved with the biosynthesis of cartilaginous matrical macromolecules, i.e., proteoglycans and collagen and thereby promote certain aging processes within active growth centers.

An electron microscopic study of the premineralizing zone of the condylar cartilage of the mouse mandible.

The mandibular condylar cartilage of young ICR mice was used for ultrastructural studies with special emphasis on the cells and matrix in the premineralizing zone. It became evident that cytodifferentiation of chondroblasts was accompanied by deposition of intracellular lakes of glycogen, followed within the premineralizing hypertrophic chondrocytes by a gradual reduction in the glycogen content of the lakes and its replacement by lipid-like droplets. The premineralizing condylar cartilage, like that of other cartilage involved in mineralization, showed numerous matrix vesicles (Types I and II) unassociated with appreciable amounts of collagen.

Landmarks in chondrocyte differentiation and maturation as envisaged by changes in the distribution of calcium complexes: an ultrastructural histochemical study.

Organ cultures of fetal condylar cartilages have been examined using the K-pyroantimonate-osmium fixation method to follow alterations in cellular calcium distribution in maturing cartilage. Special attention has been given to analyzing the temporal and spatial relationships among intracellular calcium accumulation, cell maturation, and matrix mineralization. The findings of this study suggest that concomitant with the change in cellular metabolism from aerobic respiration to glycolysis there is a distinctive change in the distribution of cellular and matrical calcium complexes. This ultrastructural histochemical method can thus be used to evaluate the maturational state of chondrocytes actively involved in endochondral ossification.

Parathyroid hormone stimulates proliferation of chondroprogenitor cells in vitro.

Mandibular condylar explants of newborn ICR mice were maintained as serum-free organ culture systems and were used to study the effects of 0.1-10.0U/ml parathyroid hormone (PTH) on the morphology of the organ and the ultrastructure of the chondroprogenitor cells. Parameters of proliferation such as 3H-thymidine autoradiography and incorporation into the explants were also studied. The chondroprogenitoric zone gradually increased with increasing dosages of the hormone up to a maximum of 5-fold of the control with 5.0 U/ml PTH. Autoradiographic studies showed a 3-fold increase in the number of 3H-thymidine-labeled cells in the chondroprogenitoric zone of PTH-treated explants. This was matched by a dose-dependent stimulation of 3H-thymidine incorporation, reaching maximal values at 5.0 U/ml PHT. At this concentration, the stimulated incorporation of 3H-thymidine was found to be dependent on the Ca2+ concentration of the medium. Chondroprogenitor cells located adjacent to the chondroblastic zone tended to pile up and aggregate in "syncytium"-like clusters, establishing intercellular gap junctions. All PTH-treated chondroprogenitor cells demonstrated large deposits of glycogen and highly elaborated stacks of their Golgi systems; the latter were associated with large numbers of vesicular elements. On the other hand, the chondroblastic zone was significantly reduced in size. Hence, it seems that PTH possesses a rather intense mitogenic effect upon chondroprogenitor cells and might possibly interfere with their normal pattern of differentiation into mature cartilage cells.

Variation in the branching of the axillary artery. A description of a rare case.

A case is described in which a rare variation of a branch arising from the axillary artery is defined as a thoracoepigastric artery. This variation was observed in the cadaver of a 72-year-old man. This artery branched from the axillary artery, passing as a common trunk between the roots of the median nerve, and divided into two branches. The lateral one gave rise to muscular branches supplying the shoulder and fasciae, while the medial one descended on the anterior aspect of the axillary fossa, reaching the hypogastric region, and anastomosed with the superficial epigastric artery, which is a branch of the femoral artery. To our best knowledge, no variation similar to this one has been described. We suggest naming this artery the thoracoepigastric artery.

Chondroclasts and endothelial cells collaborate in the process of cartilage resorption.

The condylar cartilage of the young rat is a major growth center of the craniofacial complex. Differences between the mechanism that results in bone formation from growth centers in the epiphyseal plates of long bones are dictated primarily by the different character of the mineralization of the cartilage. In this ultrastructural study we demonstrate that the terminal hypertrophic chondrocytes undergo apoptosis and disintegration while simultaneously chondroclasts dissolve gaps in the calcified cartilage that engulfs them. The latter are also phagocytizing debris of the chondrocytes. The chondroclasts are intimately followed by tube-forming endothelial cells that most probably coalesce to create extensions of the invading capillaries into the evacuated lacunae. The chondroclasts have ultrastructural features similar to osteoclasts. They are multinucleate, are rich in mitochondria and vacuoles, form clear zones that adhere to the spicules of the calcified cartilage, and also form a sort of ruffled border. The latter is not as elaborate and orderly arranged as is known from osteoclasts. The capillaries that follow orient the stroma cells to the evacuated lacunae and, together with the calcified cartilaginous scaffold, supply the adequate environmental conditions for the stroma cells to differentiate into osteoblasts and to build up trabecular bone.

In vitro precocious accumulation of calcium and matrix vesicles formation in young cartilage cells: specific effects of corticosteroids.

The present study examined the effects of various corticosteroid and noncorticosteroid hormones upon the ultrastructure of chondroprogenitor cells and chondroblasts in an organ-culture system of late fetal condylar cartilage. Corticosteroids, (triamcinolone acetonide, dexamethasone, corticosterone) at concentrations of 10(-6) - 10(-8)M stimulated markedly a precocious formation of matrix vesicles by chondroblasts. This stimulation was accompanied by a significant accretion of calcium complexes intra- and extracellularly in both the chondroprogenitor cell population and chondroblasts in vitro, as well as in the newly induced matrix vesicles. Nonglucocorticoid steroids (progesterone, estradiol, testosterone, cortexolone) did not evoke similar effects. Progesterone and testosterone, however, seemed to adversely affect the ultrastructure of the cartilage cells, whereas estradiol appeared to have a favorable effect on the morphology of cultured condylar cartilage.

Glucocorticoid-induced changes in the activity of cartilage alkaline phosphatase.

Glucocorticoid hormones are known to exert distinct inhibitory effects upon cartilage metabolism and endochondral bone growth. This study examined the influence of triamcinolone hexacetonide, a long-acting synthetic analogue off cortisol, on the activity of non-specific alkaline phosphatase i condylar cartilage of neonatal mice. Four-day-old mice received a single dose (10 mg/kg) of the hormone and the activity of beta-glycerophosphatase was assayed 3 and 6 days thereafter. Whereas no significant changes were noted in the enzyme's specific activity, distinct alterations were observed in the latter's distributional pattern. By 48 hours cells along both the proliferative and chondroblastic zones exhibited a significant enhancement of alkaline phosphatase activity. This increase in enzyme activity was most prominent along the cells' plasmalemma and within their adjacent matrix. Thus, glucocorticoid hormones possess a significant stimulatory effect upon alkaline phosphatase activity in very young cartilage cells which in turn might affect the mineralization process.