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PubMed2002

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP(i)). PP(i) suppresses the formation and growth of hydroxyapatite crystals. PP(i) is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP(i) content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP(i) concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP(i) insufficiency.

Alkaline Phosphatase↗

PubMed1991

Musculoskeletal ontogeny, phylogeny, and functional adaptation.

Physical forces applied to connective tissues may cause significant changes in cell metabolism and gene expression. Theoretical investigations indicate that mechanical loading histories beginning very early in skeletal development may guide endochondral ossification patterns and the initial architectural construction of bones. Developmental patterns and structures of bones can be emulated using mathematical algorithms or "rules of construction" which relate developmental processes to tissue stress (or strain) histories. Skeletal forms and tissues are well-designed for their mechanical function primarily because their histomorphological construction has been guided by mechanical loading during growth and development. Construction rules of developmental mechanics can also be used to describe many of the histological and morphological adaptations of mature skeletal tissues to changes in customary physical activity. Over many generations, changes in the heritable genetic information occurs by mutation and genetic variability. The range of skeletal forms that are possible in evolution due to such variations, however, is constrained by the developmental rules of construction that reflect biophysical processes associated with the tissue mechanical loading.

Adaptation, Physiological↗

PubMed2001

Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of Rana catesbeiana.

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.

Alkaline Phosphatase↗

PubMed1991

Role of proteoglycan in the provisional calcification of cartilage. A review and reinterpretation.

Study of the calcification of cartilage in endochondral ossification has yielded two apparently contradictory views of the role of proteoglycan in this process. The ability of proteoglycan to act as a calcium-concentrating agent (Kalksalzfänger) in cartilage is consistent with the view that proteoglycans are promoters of calcification. However, study of their effect on hydroxyapatite formation in vitro suggests that proteoglycans are inhibitors of cartilage calcification. A resolution of this paradox is now proposed. Proteoglycans inhibit hydroxyapatite formation under in vitro conditions of limited calcium availability (in part) by binding calcium ions. However, under in vivo conditions of essentially infinite calcium availability, proteoglycans may promote hydroxyapatite formation, since binding of calcium to proteoglycan will not decrease the free calcium concentration, and the bound calcium may easily be displaced. Therefore, it is proposed that the role of proteoglycans in the calcification of cartilage is to function as a cation-exchanging calcium reservoir.

Animals↗

PubMed1993

Two patterns of calcification in primary (physeal) and secondary (epiphyseal) growth cartilage.

It is generally stated that the process of cartilage calcification and ossification is the same in the physeal (primary growth center) and epiphyseal (secondary growth center) growth plates. Reexamination of osteophyte growth led to the proposal that these growth plates differ. This proposition was tested by examining rabbit and human material from both sites before and after maceration; both tissue preparations were processed for light microscopy, and the macerated tissues were also studied by scanning electron microscopy (SEM). The physis showed the well-recognized, longitudinal calcified tubal structures in which capillaries make direct contact with and resorb the nonmineralized, transverse septal cartilage. In the epiphysis, the transverse septal cartilage was also mineralized, producing a continuous calcified sheet, perforated only by a limited number of channels and susceptible only to osteoclastic removal. This variation in mineral distribution modifies the way in which the primary spongiosa is formed during growth.

Animals↗

PubMed2004

Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival.

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.

Angiography↗

PubMed2002

Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis.

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.

Animals↗

PubMed2007

Delivery mode and efficacy of BMP-2 in association with implants.

Bone healing may be improved in implant patients by the administration of osteogenic agents, such as bone morphogenetic protein 2 (BMP-2). But the efficacy of BMP-2 depends upon its mode of application. We hypothesized that BMP-2 is capable of a higher osteogenic efficacy when delivered physiologically, viz., when incorporated into a calcium-phosphate carrier that mimics mineralized bone matrix, than when administered via simple pharmacological modes, such as by adsorption onto a carrier surface. Using an ectopic rat model, we compared the osteoinductive efficacies of calcium-phosphate implant-coatings bearing either incorporated, adsorbed, or incorporated and adsorbed BMP-2. When adsorbed directly onto the naked implant surface, BMP-2 was not osteogenic. When adsorbed onto a calcium-phosphate coating, it was osteoinductive, but not highly efficacious. When BMP-2 was incorporated into calcium-phosphate coatings, it was a potent bone-inducer, whose efficacy was compromised, not potentiated, by the additional deposition of an adsorbed pool.

Adsorption↗

PubMed2000

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage.

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage mitrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.

Animals↗

PubMed2000

Developmental control of chondrogenesis and osteogenesis.

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.

Animals↗

PubMed1997

Osteogenic response to hydroxyapatite-fibrin implants in maxillofacial bone defects.

Bone formation in hydroxyapatite-fibrin implants has been reported several times. However, available studies refer to experimental animals, or are limited to short periods after implantation. We report the results of histological, histochemical and ultrastructural studies carried out 2.5-8 yr after implantation of non-resorbable, porous hydroxyapatite (HA) and fibrin glue in human maxillofacial bones. Prominent ossification was found in all cases, with the presence of normally structured spongy bone. HA granules were embedded in the calcified bone matrix. They had not elicited inflammatory reactions and did not induce bone resorption. Ossification was preceded by the appearance of alkaline phosphatase activity on fibroblast-like cells, and by the formation of dense collagenous layers, similar to osteoid borders, on the surface of HA granules. The early phases of the calcification process occurred in these borders, with the appearance of calcification nodules adjacent to alkaline phosphatase-positive osteoblast-like cells. A remodeling process similar to that occurring in normal bones was found in the newly formed bone. These results justify the conclusion that HA-fibrin implants lead to the formation of long-lasting bone that does not differ from that of the normal maxillofacial skeleton. Mixing the HA granules with fibrin has the advantage of creating an easily mouldable material which can be adapted to any skeletal surface and stays in place after surgery.

Alkaline Phosphatase↗

PubMed1956

Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the newborn infant.

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.

Bone Matrix↗

PubMed2003

Endochondral bone formation in the heart: a possible mechanism of coronary calcification.

During the atherosclerotic process, calcification occurs and is associated with a high likelihood of adverse events. Coronary calcification has been perceived as a passive precipitation of mineral. Recently, calcification associated with atherosclerosis has been found to be the result of an organized, regulated process that is similar to the process of calcification in bone. Mineralization in skeletal tissue can form by endochondral ossification in which mesenchymal cells differentiate into chondroblasts and produce a cartilage matrix which then degenerates and is remodeled to form bone. In this study, hearts from oophorectomized, aged female Sprague Dawley rats were found to contain areas of cartilage. Micro-computerized tomography radiogrammetry provided quantitative images of the architecture and confirmed the calcified tissue. Histological analysis revealed staining for several markers consistent with cartilage and bone tissue: acid phosphatase and bone matrix proteins, osteocalcin, osteopontin, osteonectin, and bone sialoprotein. In addition, cartilage types II, X, and procollagen type I were present. The presence of chondrocytes in the aged rat heart provides insights into the process of calcification in coronary arteries. Many proteins associated with calcification in bone are present in the cartilage that is present in vascular tissue, suggesting that endochondral calcification is another possible mechanism by which calcification of vascular tissue may occur.

Aging↗

PubMed2005

Gracile bones, periostal appositions, hypomineralization of the cranial vault, and mental retardation in brothers: milder variant of osteocraniostenosis or new syndrome?

We report on two brothers with ossification anomalies of membranous and cranial bones, remodeling defect of long bones leading to dense, overtubulated, narrow diaphyses, metaphyseal flare, periostal hyperosotosis that increased during the first months of life, thoracic dystrophy and severe hypotonia. One boy had hypospadias and cleft palate. Follow-up of the surviving boy documented progressive osteopenia, slow healing of the periostal anomalies, liver angiomatosis, mental and motor delay, thoracic deformity, delay in tooth eruption, and progressive microcephaly with enlargement of the cerebral ventricles. This disorder shares some traits with osteocraniostenosis, but lacks the cranial deformity and acromelic micromelia of the latter, in which periostal anomalies are not described. The syndrome reported here may represent a milder form of osteocraniostenosis, or a new entity belonging to the same "family." Genealogical data are consistent with AR or XLR inheritance. No mutations were found in the coding sequence of filamin A.

Abnormalities, Multiple↗

PubMed2004

PHOSPHO1-A novel phosphatase specifically expressed at sites of mineralisation in bone and cartilage.

Mineralisation of bone and cartilage is essential for skeletal development and function. We have previously reported a novel gene (PHOSPHO1); a member of the large haloacid dehalogenase superfamily of hydrolases which has an active site indicative of a phosphatase. Its high expression in skeletal tissues has led us to speculate that PHOSPHO1 may be involved in the mineralisation process. Therefore, in this study, we have determined that PHOSPHO1 is localized to sites of mineralisation in both cartilage and bone. Recombinant derived PHOSPHO1 protein was produced and affinity purified PHOSPHO1 antiserum was generated and used to immunostain a range of skeletal and soft avian tissues. In addition, PHOSPHO1 gene expression was determined in SaOS-2 and MG-63 osteoblast-like cells by RT-PCR. In diaphyseal cortical bone, immunohistochemistry localized PHOSPHO1 protein to the osteoid layer of the periosteum, forming surfaces of growing osteons, and newly formed osteocytes, whereas the endosteum and closed osteons were negative. In growth plate cartilage, immunoreactivity was limited to the early hypertrophic chondrocytes and the ossification groove of Ranvier. Cartilage remnants and trabecular bone within the primary spongiosa exhibited strong immunoreactivity on their mineralising surfaces. In 17-day-old embryonic calvaria, the osteoid present on the intramembranous and periosteal bone surfaces stained positively for PHOSPHO1. All soft tissues examined were negative. PHOSPHO1 gene expression was detected in mineralising SaOS-2 but not in the non-mineralising MG-63 osteoblast-like cells and gene expression levels were unchanged by dexamethasone, estradiol, 1,25-dihydroxyvitamin D3 or PTHrP treatment. Western analysis of chick growth plate cell lysate yielded bands (30.4 and 28.6 kD) corresponding to transcripts initiated at each of two possible initiation codons indicating the presence of alternative transcripts for PHOSPHO1 in growth cartilage. These results confirm that the PHOSPHO1 protein and gene expression profile is consistent with a role for PHOSPHO1 in bone and cartilage matrix mineralisation.

Animals↗

PubMed2006

The Fifth European Growth Plate Working Group Symposium. 21st September 2005, Lyon, France.

The regulation of longitudinal bone growth at the growth plate occurs through the tightly controlled interaction of circulating systemic hormones and locally produced peptide growth factors, the net result of which is to trigger changes in gene expression by growth plate chondrocytes. These molecular events lead to carefully orchestrated alterations in chondrocyte size, extracellular matrix components, secreted enzymes and growth factors, and receptor expression. The result of these events is calcification of the matrix, chondrocyte apoptosis, and the completion of endochondral bone formation. Although over the past decade advances in molecular and cellular biology have increased our understanding of skeletal biology and the process of endochondral ossification, many of the signaling pathways and their interactions remain poorly understood in the growth plate. The European Growth Plate Working Group (EUROGROP) was established in 2000 with the aim to bring together groups of basic and clinical researchers from Europe with an interest in the normal biology and pathophysiology of the growth plate. The 5th EUROGROP Symposium, held in Lyon, France, as a satellite symposium to the 7th Joint Meeting of ESPE/ LWPES 2005, enabled researchers, coming from all parts of the world, including South America, Australia, Asia and Japan, to discuss their ongoing studies and exchange technical information. Topics ranged from the search for the molecular mechanisms of growth plate senescence to IGF-I and sugar metabolism in skeletal growth centers, while techniques discussed included microarray analysis of rat growth plates and the use of human mesenchymal stem cells as a model of chondrocyte differentiation.

Animals↗

PubMed1998

Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system.

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10(5) cell/cm2 and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and beta-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro.

Aggrecans↗

PubMed1988

The effects of cadmium, copper or zinc on formation of embryonic chick bone in tissue culture.

Femurs from 9-day-old chick embryo were cultivated for 6 days by the roller-tube method in the presence of Cd, Cu or Zn. Cd (5.0 microM and above) and Cu (2.5 microM and above) caused a decrease in collagen content of both diaphysis and epiphysis, mainly due to inhibition of collagen synthesis. In addition, Cd and Cu each showed a tendency to inhibit an increase in Ca content of diaphysis, where intraperiosteal ossification could be observed. Alkaline phosphatase (ALP) activity was decreased by Cd (5.0 microM and above) or Cu (10 microM and above) in diaphysis. On the other hand, Zn at 50 microM and above inhibited an increase in Ca content of the diaphysis with a remarkable elevation of ALP activity in the medium. At this time, Zn did not decrease the collagen content of the diaphysis so strongly. Histological observations revealed that Cd and Cu each decreased both calcified and uncalcified osteoid tissue at 2.5 microM, while Zn at 100 microM decreased calcified tissue but increased uncalcified osteoid tissue. As Zn accumulated particularly in diaphysis and deposited at the edge of calcified tissue, it was suggested that Zn inhibited calcification physicochemically. It was concluded that Cd or Cu would induce bone damage represented by osteoporosis, whereas Zn would induce osteomalacia.

Alkaline Phosphatase↗