Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Process heterochronies in endochondral ossification

Heterochrony, evolutionary changes in developmental rates and timing, is a key concept in the construction of a synthesis of development and evolution. Heterochronic changes in vertebrate evolution have traditionally been identified through plesiomorphic-apomorphic comparisons of bone growth. This methodological framework assumes that observed heterochronies are the outcome of dissociations of developmental processes in time. Recent findings of non-heterochronic developmental changes underlying morphological heterochrony invalidate this assumption. In this paper, a function for bone growth (at the organ level) has been mathematically deduced from the underlying developmental mechanisms. The temporal domain of the model spans from the time at maximum growth rate, after the formation of growth plates, to the time at atrophy of the proliferating stratum of cells. Three organizational levels were considered: (a) cell kinetics of endochondral ossification, (b) variation of bone growth rates and (c) variation of accumulated bone growth with increasing age. This quantitative model provides an excellent tool to deal with the problem of the developmental basis of morphological change. I have modelled potential evolutionary changes on the system at different levels of biological organization. This new framework involves an epistemological shift in heterochronic analysis from a pattern-oriented inductive way to a process-oriented deductive way. The analysis of the relationships between the evolutionary alterations of endochondral ossification and the morphological expression of these changes reveals that observed pattern heterochronies can be the outcome of different process heterochronies. Moreover, I discuss at length the heteroposic hypothesis, that evolutionary changes in the tight regulation of the amount of protein synthesized by a cell population during development would underlie acceleration or deceleration in cases of evolutionary changes in the initial number of proliferating cells at growth plates. Future research on the genetic basis of process heterochronies and heteroposies will complete our understanding of these evolutionary phenomena.     

Histological, histometrical and fluoride electrode studies of the effects of fluoride on the mandibular condyles in growing newborn rats

The effects of fluoride on the mandibular condyles in growing newborn rats were studied by histological, histometrical and fluoride electrode methods. The layer of cartilage of the mandibular condyle in the animals administered 5, 15, 25 and 35 mg/kg of fluoride for 3 weeks displayed a significant increase in thickness when compared with that of the mandibular condyle in the control animals. The thickening of the cartilage layer was proportioned to the amounts of fluoride administered. The volumetric density of cancellous bone of the condyle in the animals administered 25 and 35 mg/kg of fluoride also increased significantly when compared with that of the condyle in the control animals. The trabeculae of cancellous bone of the condyle in these animals contained large amounts of osteoid. The cancellous bone of the condyle in the animals of the four fluoride groups showed a significantly higher fluoride concentration when compared with that of the condyle in the control animals. The fluoride concentration proportionally increased with the amounts of fluoride administered. The results of the present study indicate that the morphologic changes and the fluoride concentrations in the mandibular condyles of rats receiving fluoride were closely correlated with each other.     

Inhibitory effect of bFGF on endochondral heterotopic ossification

Basic fibroblast growth factor (bFGF) is reported to stimulate repair of fracture and bony defects in in vivo animal studies. However, most studies performed in vitro demonstrate inhibitory effect of bFGF on cartilage and bone differentiation. To understand the discrepancy observed in in vivo and in vitro studies, we evaluated the effect of bFGF on chondro-osteogenesis initiated by bone matrix powder (MP). MP was implanted in the murine hamstring muscles with or without administration of bFGF. Injection of 1 microg of bFGF markedly reduced the size of heterotopic bone induced by MP, as detected by X-ray. Injection of 10 microg of bFGF completely inhibited ossification and only fibrous tissues were observed at the site of MP implantation. The expressions of alkaline phosphatase and osteocalcin mRNAs, markers for bone differentiation, were completely suppressed by 10 microg of bFGF. These results demonstrate the inhibitory effect of bFGF on endochondral ossification in vivo, implicating a precaution for its use in musculo-skeletal disorders.     

High serum levels of IGF-I contribute to promotion of endochondral ossification in mandibular condyle and cause its specific elongation in acromegaly-like rats.

Mandibular protrusion accompanies acromegaly or acrogigantism. To clarify the detailed mechanisms, we used an acromegaly-like rat model recently developed by exogenous administration of insulin-like growth factor I (IGF-I). Human recombinant IGF-I (640 microg/day) continuously was infused subcutaneously to 10-week-old male rats (n=12) for four weeks. Control, sham-operated animals (n=12) were injected with saline alone. Twelve rats (six from each group) were killed immediately after ending administration at age 14 weeks. Another 12 rats (six from each group) were housed for an additional four weeks after treatment ended. Mandibular condylar length increased significantly in the IGF-I rats compared with the control rats, but no significant intergroup difference was found in the lengths of the coronoid and angular processes. Cartilaginous layer width, bone matrix volume, and the number of osteoblasts in the mandibular condyle increased significantly in the IGF-I group. These histopathological changes in the condyle disappeared after IGF-I administration was discontinued; however, the morphological change in condylar length remained. These findings suggest that mandibular protrusion in patients with acromegaly or acrogigantism may be evoked by superfluous elongation of the mandibular condyle and that such elongation can be induced by endochondral ossification caused by high IGF-I serum levels.     

Potential role of leptin in endochondral ossification.

Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.     

The role of hypertrophic cartilage in endochondral ossification.

Normal stages of histogenesis of long bones show that the hypertrophy of cartilage cells is the pre-requisite for the perichondrium to take up osteoblastic activity, (Fell 1925, Lutfi 1971). Cooper (1965) found the cartilage cells from epihysis of the long bones of chick failed to induce chondrogenesis in somites in mice and chick whereas flat cells and early Peripheral cells could do same. Fell and Landauer (1935) noticed that in avian phocomelia the hypertrophied cartilage cells fail to hypertrophy leading subsequently to deformities of long bones. Presently an attempt is made to analyse this process further by culture experiments. It is found that complete tibial rudiment or part of it grows normally in vitro with good differentiation of various zones and the development of osteoid tissue. However it is noticed that when cartilage and the associated perichondrial tissues are grown separately, there is no patterned growth of cartilage and the absence of development of osteoid tissue in either types of cultures. The role of perichondrium and cartilage is discussed in the light of experimental findings.     

Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification

Summary This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine, which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.     

Effects of alendronate on tooth eruption and molar root formation in young growing rats

Tooth eruption consists of the movement of teeth from the bony crypt in which they initiate their development to the occlusal plane in the oral cavity. Interactions between the tooth germ and its surrounding alveolar bone occur in order to offer spatial conditions for its development and eruption. This involves bone remodeling during which resoption is a key event. Bisphosphonates are a group of drugs that interfere with the resorption of mineralized tissues. With the purpose of investigating the effects of sodium alendronate (a potent bisphosphonate inhibitor of osteoclast activity) on alveolar bone during tooth development and eruption, we gave newborn rats daily doses of this drug for 4, 14, and 30 days. Samples of the maxillary alveolar process containing the tooth germs were processed for light, transmission, and scanning electron microscopy and were also submitted to tartrate-resistant acid phosphatase histochemistry and high-resolution colloidal-gold immunolabeling for osteopontin. Inhibition of osteoclast activity by sodium alendronate caused the absence of tooth eruption. The lack of alveolar bone remodeling resulted in primary bone with the presence of latent osteoclasts and abundant osteopontin at the interfibrillar regions. The developing bone trabeculae invaded the dental follicle and reached the molar tooth germs, provoking deformities in enamel surfaces. No root formation was observed. These findings suggested that alendronate effectively inhibited tooth eruption by interfering with the activation of osteoclasts, which remained in a latent stage. This work was supported by grants from Fapesp (04/05831-9 and 06/60094-5) and CNPq (Brazil).     

FGF9 can induce endochondral ossification in cranial mesenchyme

Background  

The flat bones of the skull (i.e., the frontal and parietal bones) normally form through intramembranous ossification. At these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. This type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone.     

Immunoelectron microscopic studies of type X collagen in endochondral ossification

Immunofluorescence and immunoelectron microscopy were used in conjunction with a monoclonal antibody to investigate the localization of type X collagen in the proximal tibial growth plate of 7-d-old chicks. This molecule was detected throughout the hypertrophic zone first appearing when chondrocytes exhibited hypertrophy: it was absent from the proliferative zone. Type X collagen was primarily associated with type II collagen fibrils as demonstrated by immunogold staining. Type X collagen was not concentrated in the focal calcification sites nor was it associated with matrix vesicles. These observations suggest that type X collagen may play a role other than that directly related to the nucleation of calcification.     

Stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification

High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1(-/-) mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1(-/-) mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.     

Heterochronic patterns in primate evolution: evidence from endochondral ossification

Heterochrony (evolutionary modifications in developmental timing and/or rates) is widely recognized as an important agent of morphological change. The adaptive significance of heterochronic changes might lie either in the advantages of the derived morphologies (organ size and shape) or the derived growth parameters themselves (rate and duration of growth). We have tested these hypotheses by comparing the growth rate, the duration of growth and the relative length of the adult tibia in Primates in a phylogenetic context. We report an evolutionary decrease in growth rates (paedochronocline) and an increase in the duration of growth (perachronocline), lying in the cline from the last common ancestor of Primates, passing through the last common ancestor of Haplorhini, that of Catarrhini, to the last common ancestor of the Hominidae. However, the variation in the relative length of the adult tibia does not show any phylogenetic pattern. The derived growth parameters in themselves (slower rate, longer duration) would be of adaptive significance and they would have been selected because a prolonged learning period prior to maturity conferred advantage. The proximate (developmental) causation of differences in bone growth rate were also investigated and it was found that cell production rate in the growth plates rather than the chondrocyte size, underlies the variation in bone growth rate.     

Type X collagen synthesis during endochondral ossification in fracture repair

Collagen synthesis in normal connective tissue development and repair is integral to tissue stability. The appearance of a short chain collagen, designated Type X, was studied in experimental fractures created in the chicken humerus. Biosynthetic studies using [14C]proline incorporation coupled with histologic examination of the cartilaginous callus demonstrated that Type X collagen synthesis occurs during endochondral ossification in the fracture callus. Type X synthesis occurred in the areas of cartilaginous callus composed of hypertrophic and degenerative chondrocytes that were associated with increased vascularity and matrix mineralization. Synthesis of short chain collagen was not detected in either skeletal muscle or bone. Two-dimensional peptide mapping of cyanogen bromide and proteolytic fragments derived from fracture callus short chain collagen confirmed the identity of this collagen as Type X. The synthesis of Type X collagen by fracture callus is further evidence supporting its close association with the process of endochondral ossification.