Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system.

An in vivo system of membranous bone formation during distraction has been investigated in order to follow cells that express vascular markers with the objective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and invaded the central zone of the regenerating distraction tissue temporally preceded the growth of delicate woven bone trabeculae from both edges of the cut bone. Concentrically arranged 'onion-like' configurations were abundant in paracentral zones and in association with mesenchymal condensations, suggesting their de novo formation in situ. Vascular specific markers, the angiopoietin receptor Tie-2 and factor VIII-related antigen (FVIIIrAg), were localized immunohistochemically in order to follow cells of vascular origin. Endothelial cells of the new capillaries, centrally located cells of the concentric configurations, pericytes, and most of the adjacent polygonal mesenchymal cells stained positively with specific antibodies to both antigens. Moreover, preosteoblasts and osteoblasts that lie adjacent to or already embedded in the osteiod of the newly formed trabeculae were also FVIIIrAg and Tie-2 immunopositive. As the source of the bone-forming cells in regenerating tissue during distraction is not yet fully understood, this observation might support the possibility of their vascular origin.

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.

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.

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.

Midface membranous bone lengthening: A one-year histological and morphological follow-up of distraction osteogenesis.

Midface bone lengthening was performed on three young, adult sheep using distraction osteogenesis following osteotomy of the maxilla and mounting of an extraoral fixation device. The midface was gradually distracted, 2 mm/day, for 21 days, up to approximately 40 mm. A marked midface advancement was noted. Following a further 6 weeks of retention, the device was removed and the animals were followed for 1 year. Biopsies specimens were taken from the distracted area at the end of the distraction period, after the additional 6 weeks of retention, and finally 1 year later. A nondistracted area of the maxillary bone served as control. The specimens were analyzed histologically, histochemically, and by scanning electron microscopy for the ultrastructural pattern, mineralization, mineral content, and approximate Ca2+ concentration. Clinically and radiographically, all sheep fully bridged the experimental gap. Histologically, at the completion of distraction, collagen bundles and slender bone trabeculae oriented in the direction of the distraction could be seen. At the end of the retention period, the trabeculae thickened noticeably and were partially replaced by mature lamellar bone. At the end of 1 year and after completion of the process of remodeling, the pattern of the distracted area resembled the control area. The mineralization, as reflected by quantitative calcium analysis, compared with the nondistracted area, demonstrated a low rate of mineralization after 3 weeks of lengthening, increased 6 weeks later, and after 1 year became nearly the same as in the nondistracted area. In conclusion, distraction osteogenesis provides satisfactory quantitative and structural new bone.

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).

[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.

PTHrP(107-111) inhibits in vivo resorption that was stimulated by PTHrP(1-34) when applied intermittently to neonatal mice.

Stimulated resorption by PTH(1-34) and PTHrP(1-34) was studied in a neonatal mouse model by injecting a relatively high dose (0.2 microgram/g BW) of the peptides for 6 or 16 consecutive daily injections. 3H-Tetracycline was applied once, before the beginning of the injection period. Only PTHrP(1-34) increased resorption as revealed by decreased radioactivity remaining in the tibia when compared with vehicle-treated mice. This activity was related to increased numbers of mature osteoclasts in the metaphysis of the young bones. PTHrP(107-111) completely aborted the stimulated resorption when applied simultaneously with PTHrP(1-34).

[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.

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)

Morphometric analysis of atrial natriuretic peptide-containing granules in atriocytes of rats with experimental congestive heart failure.

The morphometric characteristics of atrial natriuretic peptide-containing granules were studied in atrial myoendocrine cells of rats with aorto-caval fistula, an experimental model of congestive heart failure. A total of 6680 granules of control and aorto-caval rats were analyzed by a computerized image analysis system that evaluated the number and sectioned surface area of granules and their subcellular location. Compared with control animals, rats with congestive heart failure displayed a slight increase in the number of peripheral granules, adjacent to the sarcolemma, but not centrally located in the Golgi areas. The mean sectioned surface area of granules in rats with congestive heart failure was about 50% of that in controls, both in the right and left atria. Rats with aorto-caval fistula had a higher percent of small granules and lower percent of large granules compared with controls. The data demonstrate different morphometric characteristics in atrial natriuretic peptide-containing granules in atriocytes in rats with experimental congestive heart failure; this may reflect the enhanced synthesis and release of atrial natriuretic peptide in heart failure.

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.

Growth hormone involvement in the regulation of tartrate-resistant acid phosphatase-positive cells that are active in cartilage and bone resorption.

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)

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.

Structural changes in condylar cartilage following prolonged exposure to the human parathyroid hormone fragment (hPTH) 1-34 in vitro.

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.

Structural characterization of the mandibular condyle in human fetuses: light and electron microscopy studies.

Mandibular condyles from 18- to 20-week-old human fetuses were examined in the light and electron microscope with particular attention to intratissue organization and extracellular matrix. In the human fetus the condyle has been divided into five layers: (1) the most superficial, articular layer, (2) chondroprogenitor cell layer, (3) condroblast cell layer, (4) nonmineralized hypertrophic cell layer, and (5) mineralized hypertrophic cell layer. The articular layer is rich in collagen fibers (mostly of the type I collagen), but the cells seldom divide. By contrast, in the chondroprogenitor cell layer and upper part of the chondroblastic cell layer mitosis gives rise to new cells. The matrix in the latter layer is composed of thick banded 'lucent' fibrils in a loose feltwork of granules representing cartilage proteoglycans. The daughter cells in the progenitor cell layer undergo differentiation which is apparently completed along the lower border of the mineralized hypertrophic cell layer--the ossification front. The matrix in the hypertrophic cell layer reveals distinct matrix vesicles that undergo mineralization and subsequently coalesce to form larger sheets of mineralized extracellular matrix. Mineralized cartilage serves as a backbone for new bone formation as marrow-derived osteoblasts and osteoclasts attach to remnants of mineralized cartilage, which enables the turning on of the remodeling cycles involved in new bone formation. It can be concluded that the process of endochondral ossification as has been reported in lower animals is recapitulated in the human fetus, thus the dynamics associated with condylar morphogenesis is maintained through phylogeny.

In vitro response of neonatal condylar cartilage to simultaneous exposure to the parathyroid hormone fragments 1-34, 28-48, and 53-84 hPTH.

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.

Microvascular invasion of rabbit growth plate cartilage and the influence of dexamethasone.

The normal morphological features of growth plate angiogenesis were examined in rabbits and compared with changes induced by dexamethasone. Penetration of growth plate cartilage was led by perivascular cells with some contribution by luminal capillary endothelial cells. There was a close relationship between the invasive perivascular cells and the luminal endothelial cells of the capillary tip. Growth plates from rabbits treated with dexamethasone underwent major changes in the pattern of capillary invasion. Most striking was the appearance of numerous narrow and tortuous channels which penetrated the cartilage, in some cases forming complete loops. These channels were filled with debris or red cells but did not contain capillaries. It is suggested that dexamethasone treatment leads to channel formation by disrupting the normal control of capillary invasion.