A.L.J.J. Bronckers

MECHANICAL STIMULATION OF OSTEOPONTIN MRNA EXPRESSION AND SYNTHESIS IN BONE CELL CULTURES

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell‐matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast‐like cell line MC3T3‐E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3‐E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor‐like cells. Also, in both proliferating and differentiating MC3T3‐E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down‐regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997.

Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor α-1 and the protective factor matrix gla protein in human atherogenesis

OBJECTIVE Increasing evidence suggests that vascular calcification is a regulated process. We studied the vascular expression pattern of a key factor in mineralization and a counteracting, protective factor. Based on the phenotype of null mice, Core binding factor alpha-1 (Cbfa-1) plays a pivotal role in bone formation, whereas Matrix Gla Protein (MGP) is a potent inhibitor of vascular calcification. METHODS We investigated the expression of MGP and Cbfa-1 in cultured, human monocytic cells, endothelial cells and smooth muscle cells (SMC), as well as in normal and atherosclerotic vessel specimens. RESULTS In cultured cells MGP is expressed in endothelial cells and SMC, whereas Cbfa-1 mRNA is predominantly present in macrophages and to a lesser extent in SMC. In the normal vessel wall MGP expression is high at the luminal side and declines toward the center of the media, whereas Cbfa-1 is absent. Moderate, diffuse calcification of the aorta media was observed only in those regions where MGP is low or absent. In atherosclerotic lesions MGP is expressed in endothelial cells and SMC that form fibrous caps, but is never present in macrophages. Cbfa-1 is synthesized in regions without MGP, it is associated with calcified areas and Cbfa-1 may be considered a marker for osteoprogenitor-like cells in the vessel wall. CONCLUSIONS Our observations on MGP expression confirm and extend published data and are consistent with a protective function of MGP. Cbfa-1 expression is absent in normal medial SMC and co-localizes with neointimal macrophages and focal calcifications.

Phenotypic changes in dentition of Runx2 homozygote-null mutant mice

Genetic and molecular studies in humans and mice indicate that Runx2 (Cbfa1) is a critical transcriptional regulator of bone and tooth formation. Heterozygous mutations in Runx2 cause cleidocranial dysplasia (CCD), an inherited disorder in humans and mice characterized by skeletal defects, supernumerary teeth, and delayed eruption. Mice lacking the Runx2 gene die at birth and lack bone and tooth development. Our extended phenotypic studies of Runx2 mutants showed that developing teeth fail to advance beyond the bud stage and that mandibular molar organs were more severely affected than maxillary molar organs. Runx2 (−/−) tooth organs, when transplanted beneath the kidney capsules of nude mice, failed to progress in development. Tooth epithelial-mesenchymal recombinations using Runx2 (+/+) and (−/−) tissues indicate that the defect in mesenchyme cannot be rescued by normal dental epithelium. Finally, our molecular analyses showed differential effects of the absence of Runx2 on tooth extracellular matrix (ECM) gene expression. These data support the hypothesis that Runx2 is one of the key mesenchymal factors that influences tooth morphogenesis and the subsequent differentiation of ameloblasts and odontoblasts.

Sclerostin in Mineralized Matrices and van Buchem Disease

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.

Bone Morphogenetic Protein-7 (Osteogenic Protein-1, OP-1) and Tooth Development

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in maturation stage ameloblasts, odontoblasts and bone cells.

Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl(-) channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone. We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells.

In vitro gentamicin release from commercially available calcium-phosphate bone substitutes influence of carrier type on duration of the release profile

BackgroundPolymethyl-methacrylate (PMMA) beads releasing antibiotics are used extensively to treat osteomyelitis, but require surgical removal afterwards because they do not degrade.MethodsAs an alternative option, this report compares the in vitro gentamicin release profile from clinically used, biodegradable carrier-materials: six injectable cements and six granule-types. Cement cylinders and coated granules containing 3% gentamicin were submerged in dH2O and placed in a 48-sample parallel drug-release system. At regular intervals (30, 90, 180 min. and then every 24 h, for 21 days), the release fluid was exchanged and the gentamicin concentration was measured. The activity of released gentamicin was tested on Staphylococcus aureus.ResultsAll combinations showed initial burst-release of active gentamicin, two cements had continuous-release (17 days). The relative release of all cements (36–85%) and granules (30–62%) was higher than previously reported for injectable PMMA-cements (up to 17%) and comparable to other biodegradable carriers. From the cements residual gentamicin could be extracted, whereas the granules released all gentamicin that had adhered to the surface.ConclusionThe high release achieved shows great promise for clinical application of these biodegradable drug-carriers. Using the appropriate combination, the required release profile (burst or sustained) may be achieved.

OP-1 (BMP-7) Affects mRNA Expression of Type I, II, X Collagen, and Matrix Gla Protein in Ossifying Long Bones In Vitro

In long bone development, a regulating role of OP‐1 is suggested by the local correlated expression of both OP‐1 ligand and OP‐1 binding receptors in developing mouse hind limbs. OP‐1 is expressed in the interdigital mesenchyme, whereas OP‐1 binding receptors are found in the bordering perichondrium, and both OP‐1 ligand and receptors are present in the zone of (pre)hypertrophic chondrocytes. We investigated the role of OP‐1 in long bone development experimentally by treating organ cultures of embryonic mouse metatarsals with rhOP‐1. The mRNA expression patterns of type I, II, X collagen, and matrix Gla protein (MGP) were studied using in situ hybridization and cell proliferation using [3H]thymidine and BrdU labeling. In the epiphyseal perichondrium, treatment with 40 ng/ml OP‐1 enhanced cell proliferation after day 2, while 6‐day treatment caused a shift in expression from type I collagen to type II collagen mRNA. This supports previous histochemical findings that OP‐1 induced the transition of perichondrium into cartilage. In the center of the rudiment, OP‐1 inhibited the expression of type X collagen mRNA, indicating inhibition of chondrocyte hypertrophy. An arrest of differentiation at the prehypertrophic chondrocyte stage was also indicated by the large area of cells expressing MGP mRNA in the OP‐1–treated rudiments. We conclude that OP‐1 affected the expression of marker genes of chondrocyte differentiation by acting on two steps in endochondral ossification. First, cell proliferation was enhanced, particularly so in the perichondrium where cells started to express the chondrocyte phenotype. Second, the terminal differentiation of mature chondrocytes into hypertrophic chondrocytes was inhibited. These results, combined with the known pattern of OP‐1 ligand and BMP receptor expression in the embryo, suggest that OP‐1 plays a local role in the cascade of events during endochondral ossification.

Immunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis.

During dentinogenesis the expression of extracellular matrix (ECM) proteins by (pre)odontoblasts changes concomitantly with the stage of differentiation. Of these ECM proteins some are present throughout all stages of dentinogenesis, while others can only be demonstrated at particular stages of differentiation. Utilizing immunohistochemical techniques, positive detection of ECM proteins within the (pre)odontoblast or in their extracellular matrices has been demonstrated for (pro)collagen type I, III, IV, V and VI, fibronectin, tenascin, laminin, basement membrane heparan sulfate, nidogen, dentinophosphophoryns (DPP), osteocalcin (OC), osteonectin, osteopontin and 95 kDal glycoprotein. Early predentin before onset of dentin mineralization also reacts with antibodies to enamel matrix proteins. Of these ECM proteins, only DPP are exclusively synthesized by odontoblasts; DPP thus can be regarded as specific biochemical markers for odontoblast activity. A second marker for odontoblasts (but also synthesized by osteoblasts and osteocytes) is OC. In some species however OC levels in dentin seem very low. The initiation of dentin mineralization may be a matrix-mediated process in which preameloblasts also seem to be involved. Current data suggest that the DPP-collagen complex is associated with the mineralization process in dentin.