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PubMed1975

[The cell-independent influence on calcification in living bone and in vitro (author's transl)].

While there is no doubt that collagen is an important extra-cellular factor in the calcification of bone tissues, the exact nature of the process remains unclear. It has been explained in turn by the nucleation effect of corresponding lattice distances of apatite and collagen, that is, an oriented crystal overgrowth of the two substances which decreases the energy of nucleus formation, or by conformity between fibril-bundles and calciumphosphate crystal, or by the binding of phosphate to anionic positions of the collagen, as well as by the bone-forming effect of a non-collagen protein which is, however, separable from collagen. Because of their high viscosity proteo-polysaccharides inhibit crystal formation. Alkaline soluble proteo-pholysaccharides, however, appear to promote calcification in vitro and also in tissue of living bone. Lathyrogene and penicillamine impair the cross-linking in the bone collagen; this probably explains their disturbing effect on ossification. Diphosphonates would, like anorganic pyrophosphate, inhibit both the formation and dissolution of apatite crystal. Our research shows, however, that methanie-bis-phosphonate clearly promotes calcification, in vitro and in vivo. In vitro, and probably also in vivo, magnesium inhibits the formation of apatite crystal nucleus. Our research indicates moreover that magnesium also slows down the reduction of calcified bone tissue on account of its inhibiting effect on parathormone emission.

Apatites↗

PubMed1990

A suitable culture medium for ossification of embryonic chick femur in organ culture.

To establish a culture medium which allows ossification in organ culture, 9-day-old embryonic chick femurs were cultured in variously supplemented BGJb-HW2 media. Changes of Ca and Pi concentrations in the BGJb-HW2 medium or the 10% addition of chick embryo extract (CEE) did not induce ossification. Furthermore, combinations of the 10% CEE with a high Ca x Pi product or with 5 mM beta-glycerophosphate (beta-GP) or with 10% horse serum plus a high Ca x Pi product often caused pathological abnormalities in the periosteum. On the other hand, BGJb-HW2 medium supplemented with 5 mM beta-GP induced development of ossification. The Ca content of femurs and the diaphysial hydroxyproline content were markedly increased. Histological observation showed a formation of a thick and active periosteum, numerous osteoblastic cells, a sufficient amount of osteoid tissue and well developed calcified trabeculae without any pathological changes. Thus, the organ culture system using this medium was considered to be an appropriate one for studies on osteogenesis in vitro.

Animals↗

PubMed2003

[Calcifications in temporal arteries -- their morphogenesis in comparison to physiological osteogenesis].

OBJECTIVE: Vascular calcification, traditionally regarded as a dystrophic process, has recently been interpreted as a bone-like biologically regulated phenomenon. Because temporal arteries which also contain calcifications are easily available from biopsies of older individuals with suspected giant cell arteritis, we studied the morphogenesis of this calcification in comparison with the development of fetal bone. MATERIAL AND METHODS: Formaldehyde fixed arteries were processed in paraffin sections and investigated by light-, transmission-, and scanning electron microscopy. The atomic composition of the calcifications was estimated by X-ray microanalysis. Fetal bone, also fixed with formaldehyde, was investigated using identical methods. RESULTS: Early calcifications are often present as focal or diffuse granular mediacalcinosis. With increasing age, calcified granules appear in association with the internal elastic membranes and progress to sheet-like calcifications. Ultrastructurally calcospherites, not rarely exhibiting the Liesegang phenomenon, are the hallmark of early calcifications. In advanced sheet-like calcification calcospherites could be detected as components of the calcified lumps. Calcospherites also appear in endochondral calcification, but are absent in calcified osteoid. The ultrastructure of calcospherites in provisional endochondral ossification differs from that in vascular calcification. Instead of corpuscles as with the Liesegang phenomenon, radial clusters of needle-like crystals resembling apatite are present. While calcospherites of the tunica media often contain a high amount of magnesium, calcified sheets as well as bone exhibited in contrast a low magnesium content. CONCLUSIONS: It is concluded that, comparable to granular mediacalcinosis of the aorta, development of calcified arteriosclerotic plaques, and Mönckeberg's disease, calcification of small muscular temporal arteries is also initially a calcospherite-dependent process. It is generally accepted that these calcified corpuscles represent remnants of calcified necrotic or apoptotic cells. In the phase of confluence with the appearance of sheet-like calcification, a low bone-like magnesium content that differs from the high amount in the isolated medial calcospherites was measured. This finding, together with the observation that calcified sheets contain calcospherites, supports the hypothesis that vascular calcification in general is different from bone formation and has to be regarded as a biphasic process initiated by the appearance of calcospherites and followed by a secondary calcifying phase with the formation of bone-like apatite that leads to rock hard lumps of calcified vessels.

Adult↗

PubMed1979

[Physiology and pathology of the epiphyseal cartilage (author's transl)].

Knowledge of the physiology of the epiphyseal cartilage, respectively epiphyseal plate, is essential for an understanding of defective growth and abnormal modeling of the long bones. The epiphyseal cartilage develops from the embryonal, cartilaginous long bone structure. The histology of the epiphyseal cartilage is characterised by definable zones representing the individual differentiation steps from the reformation of cartilage to chondrolysis. Modeling of the ends of the long bones is also influenced by a transversal and longitudinal direction of growth in the epiphyseal cartilage. The intercellular substance mainly contains collagin, proteoglycanes and non-collagenic proteins. These macromolecules are compounded by means of physicochemical bonds and are responsible for the special mechanical qualities of the hyaline cartilage. The process of mineralisation at the base of the epiphyseal cartilage is an essential differentiating step for the ossification processes which take place in the metaphysis. Two pathogenetic principles at the epiphyseal cartilage appear to be important for the defective growth of the long bones. On the one hand, the flowing equilibrium between the differentiation steps of cartilage reformation, transformation of the hyaline cartilage into a mineralised cartilaginous tissue and chondrolysis is changed, whereas on the other hand the turnover of these differentiation steps is retarded or accelerated.

Animals↗

PubMed2005

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development.

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Animals↗

PubMed1995

Assessment of bone formation during osteoneogenesis: a canine model.

Distraction osteoneogenesis, callotasis, has been demonstrated to be an effective means of lengthening long bones. A variation of Ilizarov's technique produces a transport disk from one cut surface of bone within a defect and advances the disk to the opposite surface to close the defect. This process, previously described by Costantino et al. (Arch Otolaryngol Head Neck Surg 1990; 116:535-45), demonstrated bone formation within the distraction site. The precise mechanism of bone formation has not yet been described for the mandible. Four conditioned beagles were studied, with one control dog maintained in neutral fixation and three dogs distracted at 0.25 mm every 8 hours. A two-cm defect was closed, and dogs were kept in fixation for 1 week after closure, after which they were killed. Three sites were evaluated: (1) the distraction seam, (2) the interface of the cortical and distracted bone, and (3) the cortexes at the closed defect. Each site was bisected, and one half was decalcified for immunohistochemical and hematoxylin and eosin pathologic evaluation. The vascular basement membrane was labeled for laminin and type IV collagen. Both of these substances demonstrate the differentiation of the vascular matrix component predisposing primary bone formation. Labels were intense at the distraction seam where intense angiogenesis occurred. No hyalin cartilage was observed at the distraction site, which indicates that the fixation was stable and that ossification occurred primarily without intermediate callous formation. This model demonstrated that osteoclasts within the canine model produce bone through primary bone formation within an angiogenic matrix rich in basement membrane laminin and type IV collagen. Likewise, bone is species specific in mineral composition for dog mandible. Understanding the formation and composition of distracted bone is essential for understanding application of this technique within the clinical setting.

Animals↗

PubMed1990

The nature of bones and joints: a new perspective.

In human ontogeny recapitulating phylogeny, bones arrive late on the scene--long after neurogenesis, musculogenesis, organogenesis and so on are over--as islands of ossification in an ocean of collagen. This study confirms this developmental sequence by demonstrating, in cadavers, the rather independent nature of bone, to which nothing--muscle, tendon, ligament or articular cartilage--is attached. Bone is like the air in a tubeless tyre; it gives rigidity and shape to the tyre, and in return takes the shape of the tyre. The tibia, for example, is the bony tissue that is contained in tyre-like casing made of peritibial soft tissues whose inner limit is the periosteum, which continues proximally and distally as capsules of knee/ankle joint, and to which only are the articular cartilages of the knee and ankle attached, being clearly free from the bones. This study also exposes the truer nature of a joint wherein the articular cartilage assumes anatomic and physiologic significance hitherto unthought of.

Bone and Bones↗

PubMed1996

Ossification of the distal phalanx of the first digit as a maturity indicator for initiation of orthodontic treatment of Class III malocclusion in Japanese women.

The influence of mandibular growth on the stability of orthodontic treatment has been well established. A particular problem is late mandibular growth in patients with Class III malocclusions, because of skeletal jaw discrepancies that may influence the timing and course of treatment, as well as the stability of posttreatment. We have used the ossification of the distal phalanx of the first digit as an indicator of the skeletal maturity of the patient and of their potential for further growth. Our previous studies have shown that fusion of the epiphysis and the diaphysis of the distal phalanx of the first digit occurs from 1 to 3 years after the pubertal growth maximum in Japanese women. In these case reports, the orthodontic treatment of two female patients with mild Class III skeletal malocclusions is presented to show the possible clinical application of the ossification of the first digit as an indicator of the completion or near cessation of mandibular growth in the timing of treatment of Class III malocclusions. The case reports show that, although both patients experienced some mild degree of mandibular growth after treatment, this method can be helpful in determining residual mandibular growth potential in Japanese female patients with Class III malocclusions and mild skeletal discrepancies.

Age Determination by Skeleton↗

PubMed1999

Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification.

The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction.

Animals↗

PubMed1986

[Diseases of the Achilles tendon].

In this report diseases of the Achilles tendon are discussed. First an anatomical survey of this region is presented including anatomical variations together with the results of the author's own investigations on corpses. Certain positions and insertions of the plantaris tendon with respect to the Achilles tendon may have pathological influence. Pathological aspects are discussed after a review of the physiology of the Achilles tendon, including functional and tensile tests. The clinical picture changes according to the location of the pathological disorder--tendon, paratenon, insertion and bursae--and whether the disease is acute or chronic. There are various reasons for degenerative changes in the tendon, which can even lead to rupture. Surgical and non-surgical treatment and their indications are presented. In conclusion treatment methods and their results for 36 cases are given.

Achilles Tendon↗

PubMed1978

Studies on the mechanism of callus cartilage differentiation and calcification during fracture healing.

The morphologic and biochemical events during fracture callus cartilage differentiation and calcification are presented. 1. Histologic studies have demonstrated that unimmobilized fractures heal through endochondral ossification. 2. Biochemical studies have demonstrated an increase in the activities of alkaline phosphatase, enzymes involved with aerobic glucose metabolism, and lysosomal enzymes and a decrease in activities of enzymes involved with glycogen synthesis and anaerobic glycolysis. Hexosamines and hydroxyproline show a net decrease with cartilage differentiation. 3. Electron microscopic studies have demonstrated the intracellular origin and aggregation of collagen molecules, the cellular origin of matrix vesicles, and the early sites of calcification in the fracture callus.

Alkaline Phosphatase↗

PubMed2005

Age-dependent changes in plasma biochemistry of yellow-legged gulls (Larus cachinnans).

The study of avian plasma chemistry is providing useful reference values for the management of endangered and game species, supporting veterinarians in their diagnostics, and also bringing to light relevant physiological adaptations during periods of food-shortage. Age is an important source of variability for plasma chemistry. Here I report plasma chemistry of yellow-legged gulls Larus cachinnans from different ages, between post-independence and adulthood, a 5-year interval. Increase in plasma cholesterol concentration and decreases in uric acid, inorganic phosphorus and alkaline phosphatase values were seen. Body mass corrected by body size (i.e. body condition) increased with age, plasma cholesterol being positively correlated in females, but not in males. Moreover, cholesterol was also positively correlated to gonad size in both sexes. Long-term developmental changes in this species, such as gonad development and the acquisition of an optimal body mass for reproduction, could explain these findings. Finally, inorganic phosphorus and alkaline phosphatase, both traditionally related to osteogenesis, were not associated to deferred skull ossification, as originally was suggested in other species.

Aging↗

PubMed2001

A molecular analysis of matrix remodeling and angiogenesis during long bone development.

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.

Animals↗

PubMed1994

Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells.

Animals↗

PubMed1998

Osteogenesis of electrically stimulated bone cells mediated in part by calcium ions.

Culture selected and expanded osteoblastic cells may be able to be reintroduced into massive skeletal defects to accelerate cell mediated regeneration of skeletal tissues, especially in bone ingrowth in total joint replacement, fracture healing, and osteoporosis. In vitro osteogenic cell culture is a useful model in studying the mechanism of bone metabolism under direct current stimulation. In this study, an osteoblastlike cell line was isolated from newborn rat calvaria. The osteogenic processes of the in vitro cultured cell line were studied by cytochemical, electron microscopic, and energy dispersive x-ray analysis techniques that resembled those observed in membrane bone ossification centers in vivo. Direct current stimulation of 100 microA/cm2 accelerated greatly the proliferation and calcification of the in vitro cultured cells. Intracellular free calcium ion metabolism was measured with an Adherent Cell Analysis and Sorting Machine. Under direct current stimulation, intracellular free calcium ion concentration increased an average of 2.3 times of the original level, which may play a key role in regulating osteogenesis and bone metabolism.

Alkaline Phosphatase↗

PubMed2006

Evaluation of fetal bone structure and mineralization in IGF-I deficient mice using synchrotron radiation microtomography and Fourier transform infrared spectroscopy.

The role of insulin like growth factor-I (IGF-I) during pre-natal development has not been evaluated in detail. However, the high degree of growth retardation and peri-natal mortality in IGF-I deficient mouse models indicates that it plays a critical role during this time. Techniques to assess the structure and quality of bone in small animal fetuses could be beneficial in better understanding its role in bone metabolism and skeletal development. Synchrotron microtomography (SR-microCT) and Fourier transform infrared spectroscopy (FTIR) may provide methods to visualize and quantify differences in the structure and mineral density of bone in small animal fetuses. Tibia and spine from IGF-I deficient and wildtype fetal mice (18th day gestation) were imaged using SR-microCT. Three-dimensional structural indices and the degree of mineralization were determined for each sample. Mineralization was also assessed using FTIR and von Kossa staining. Bone volume was systematically lower in IGF-I -/- animals (tibia: -15%, p<0.05) while both sites were found to have a more rod-like architecture (24%, p<0.05; 113%, p<0.01) and lower trabecular separation (-16%, p<0.05; -21%, p<0.05). These structural results were mostly consistent with those seen in adult models of IGF-I deficiency. The degree of mineralization as measured by SR-microCT was higher in the IGF-I tibial metaphysis (11.7%, p<0.0001), while FTIR of the whole bone showed mineralization to be lower in the knockout group (-11%, p<0.05). Interestingly, von Kossa staining revealed no mineral content in the IGF-I -/- spinal ossification center while SR-microCT clearly indicated the presence of highly attenuating components, if somewhat lower in IGF-I -/- animals (-2.2%, p<0.05). This indicates that IGF-I deficiency is linked to subtle differences in the mineral environment and mineralization progression. The advantages unique to SR-microCT allow for 3D visualization and quantification of pre-natal bone microstructure and mineral density in mice which was not previously possible.

Animals↗

PubMed2004

Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells.

Supraphysiological concentrations of exogenous growth factors are typically required to obtain bone regeneration, and it is unclear why lower levels are not effective. We hypothesized that delivery of bone progenitor cells along with appropriate combinations of growth factors and scaffold characteristics would allow physiological doses of proteins to be used for therapeutic bone regeneration. We tested this hypothesis by measuring bone formation by rat bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels. The alginate was gamma-irradiated to vary the degradation rate and then covalently modified with RGD-containing peptides to control cell behavior. In the same delivery vehicle, we incorporated bone morphogenetic protein-2 (BMP2) and transforming growth factor-beta3 (TGF-beta3), either individually or in combination. Individual delivery of BMP2 or TGF-beta3 resulted in negligible bone tissue formation up to 22 weeks, regardless of the implant degradation rate. In contrast, when growth factors were delivered together from readily degradable hydrogels, there was significant bone formation by the transplanted BMSCs as early as 6 weeks after implantation. Furthermore, bone formation, which appeared to occur by endochondral ossification, was achieved with the dual growth factor condition at protein concentrations that were more than an order of magnitude less than those reported previously to be necessary for bone formation. These data demonstrate that appropriate combinations of soluble and biomaterial-mediated regulatory signals in cell-based tissue engineering systems can result in both more efficient and more effective tissue regeneration.

Animals↗

PubMed1994

Pedicle and antler regeneration following antlerogenic tissue removal in red deer (Cervus elaphus).

The physiological control mechanisms underlying the histogenesis of the pedicle, which precedes antler growth, from the frontal bones of male deer are poorly understood. The aim of this study was to investigate the extent of pedicle/antler regeneration after removal (for histological evaluation) of the antlerogenic region or pedicle tissue during pedicle development to determine whether the subsequent pattern of regeneration could contribute to the understanding of pedicle histogenesis and the mechanism of the transition between permanent pedicles and deciduous antlers. The contralateral pedicles of six stags were not removed and these data serve as controls. All deer were observed frequently and measurements of regeneration were made in March after all antler growth had ceased for that season. The development stage of the pedicle was determined by a combination of pedicle height measurements and histological studies. The results showed that pedicle formation histologically covers three stages: a period of intramembranous ossification (when the pedicle is less than 5 mm in height), a period when the ossification pattern changes (when the pedicle is between 5 and 28 mm in height) and a period of modified endochondral ossification (when the pedicle is over 30 mm in height). In all ossification stages some deer regenerated neither antlers nor pedicles. The pedicles which regenerated following the loss of antlerogenic tissue at the intramembranous ossification stage were shorter than the controls (P < 0.01), but longer than the pedicles regenerated after pedicle removal at the transitional stage in the pattern of ossification (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗