M. Oboeuf

Bioactive Glass Stimulates In Vitro Osteoblast Differentiation and Creates a Favorable Template for Bone Tissue Formation

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step‐by‐step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron‐dense layer composed of needle‐shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X‐ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X‐ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X‐ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model.

The present study is devoted to Msx1 distribution and function from birth to 15 months, events and periods still unexplored in vivo using Msx1 knock in transgenic mice. The study is focused on the mandible, as an exemplary model system for Msx1-dependent neural crest-derived skeletal unit. The transgenic line enabled study of morphological abnormalities in Msx1 null mutation mice and Msx1 protein expression in Msx1+/ m heterozygous mice. In Msx1 null mutation, the most striking feature was an inhibition of the mandibular basal convexity, the absence of teeth and alveolar bone processes, and absence of endochondral ossification in the mandibular condyle. At birth, in Msx1+/ m heterozygous animals, we identified for the first time a double Msx1 aboral-oral and disto-proximal gradient field developmental pattern located in the low border of the mandibular bone in relation with this bone segment modeling. Msx1 expression involved both osteoblast and osteoclast cells. A distinct pattern characterized bone surfaces: Periosteum osteoblast differentiation was related to Msx1 downregulation, while in the endosteum both differentiated osteoblasts and osteoclasts expressed the homeoprotein. In postnatal stages, Msx1 expression was maintained in the alveolar bone processes and dento-alveolar cells in relation with tooth function. Our data suggest that Msx1 play a role in a site-specific manner not only in early patterning but also in skeletal growth and modeling by acting on heterogenous bone cell populations.

Bioactive glass-ceramic containing crystalline apatite and wollastonite initiates biomineralization in bone cell cultures

Rat bone cells were cultured in the presence of bioactive glass-ceramic containing crystalline apatite and wollaston te. Scanning electron microscopy observations of the surface of the seeded ceramic disks revealed that cells attached, spread, and proliferated on the material surface. Soaking in cell-free culture medium showed that no change occurred in the surface structure. However, when cultured with bone cells and observed under a transmission electron microscope, an electron-dense layer was noted initially at the surface of the material, before bone formation occurred. In addition, energy-dispersive X-ray microanalysis demonstrated the presence of calcium and phosphorus in this layer. Progressively, during the following days of culture, active osteoblasts synthetized and laid down an osteoid matrix composed of numerous collagen fibrils arranged either parallel or perpendicularly to the first-formed electron-dense layer. Mineralization initiated on the ceramic surface dispersed then along the collagenous fibrils, leading to a mineralized matrix which surrounded the ceramic particles. These results demonstrate the capacity of apatite-wollastonite glass ceramic to initiate biomineralization in osteoblast cultures and to achieve a direct bond between the surface apatite layer of the bioactive glass-ceramic and the mineralized bone matrix.

Involvement of Desmoplakin Phosphorylation in the Regulation of Desmosomes by Protein Kinase C, in HeLa Cells

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

Desmosomes are Regulated by Protein Kinase C in Primary Rat Epithelial Cells

In the present study, we addressed the possible relevance of protein kinase C (PKC) in the regulation of intracytoplasmic desmosome assembly. Treatment of cultured rat lingual and epidermal keratinocytes with a potent and highly selective PKC inhibitor (GF109203X) induced an increase in granular labelling for major desmosomal proteins, desmoplakins, desmoglein and plakoglobin, both intracellularly and at the cell surface. This was associated with the formation of ultrastructurally recognizable desmosomes deep in the cytoplasm and increase in intercellular desmosome number. In contrast, PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in altered cell morphology, loss of intercellular contact and accumulation of desmosomal proteins in the juxtanuclear zone. On the other hand, PKC depletion by long term TPA treatment re-established cell-cell contact, where desmosomal markers were exclusively redistributed. Taken together, these results suggest that inhibition of PKC is required for intracytoplasmic as well as intercellular desmosome assembly, whereas its activation may regulate disassembly process.

Rapid nodule evaluation computer-aided image analysis procedure for bone nodule quantification

Using bone cell cultures, the effects of drugs on cell activities such as proliferation, differentiation, matrix formation, and mineralization can be explored. To quantify these parameters accurately and quickly, a kinetic reproducible computed image analysis procedure of culture dishes is proposed which could be conjointly used with biochemical analysis of the medium. In the present article, different mathematical procedures coupled either with or without histochemical staining are investigated and analyzed. Using serial cross sections and microradiographies of bone nodules, we demonstrated that the gray-level parameter is well correlated with bone mass and/or the mineralization status of the nodules. The procedure selected is a multistep procedure called rapid nodule evaluation (RNE), which uses a binary reconstruction program with different thresholds. To challenge this RNE procedure with the classical Von Kossa staining and quantification procedure, we cultured the cells in the presence of 10 nmol/L dexamethasone and compared the results using the two procedures. The RNE procedure appeared to be accurate and reproducible, and also has the advantage of speed and dynamic analysis over the classical Von Kossa quantification procedure.

Modulation of 1α,25‐dihydroxyvitamin D3‐membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1α,25‐(OH)2D3

The rapid, nongenomic effects of 1α,25‐dihydroxyvitamin D3 (1α,25‐(OH)2D3) have been related to a 1,25D3‐membrane associated, rapid response steroid binding protein or 1,25D3‐[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1α,25‐(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3‐[MARRS]bp in dental cells, in the presence or absence of 1α,25‐(OH)2D3, we have used rabbit polyclonal antibodies directed against the N‐terminus of the 1,25D3‐[MARRS]bp (Ab099) that recognizes the 1α,25‐(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast‐like cell line (MO6‐G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3‐[MARRS]bp in MO6‐G3 cells. Moreover, 1,25D3‐[MARRS]bp was up‐regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6‐G3 cells with different doses of 1α,25‐(OH)2D3 for 36 h resulted in an inhibition of 1,25D3‐[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6‐G3 cells contains immunoreactive 1,25D3‐[MARRS]bp. Immunogold positive membrane vesicle‐like structures are present in the extracellular matrix of MO6‐G3 cells. Altogether, these results indicate that the 1,25D3‐[MARRS]bp expression in MO6‐G3 cells is modulated by 1α,25‐(OH)2D3. In conclusion, this 1α,25‐(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1α,25‐(OH)2D3 in dental cells.

Chondrogenic differentiation during midfacial development in the mouse: in vivo and in vitro studies

Abstract Because the mouse is now the main model for developmental research of all types, it is important to understand the basic developmental pattern of various organs. The first aim of the present study was to establish normal prenatal developmental standards of the cartilaginous nasal capsule during embryonic development of the mouse. For this purpose we have performed sagittal and coronal sections ranging from E12.5 to E18.5 in gestation age. The primordia of the nasal septal cartilage is recognizable around the 14th embryonic day as demonstrated by the metachromatic toluidine blue staining and by immunostaining of type II collagen. Northern blot analysis of the transcription factors Cart‐1 and Sox‐9 indicated maximum mRNA levels at E12.5 then a decreased expression during the following days of gestation. Type II collagen and aggrecan mRNA levels are constant during the embryonic period. In the second part of this study, we have established a primary culture system where chondrocytes were isolated from E.18 mouse embryo nasal septum. The purpose of this second part was to assess if chondrocytes could further differentiate in vitro until the hypertrophic phase and matrix mineralization. After the condensation phase, the cells synthesize an extracellular matrix including type II collagen and aggrecan. Progressively, typical cartilaginous nodules composed of clusters of round cells are visible, then increase in size and finally mineralize at day 12 of culture. Cart‐1 and Sox‐9 mRNA levels remain constant throughout the cultures, whereas type II collagen and aggrecan gradually decrease. Ultrastructural observations of the nodules show typical chondrocytes embedded in a dense network of fibers with matrix vesicles and mineralized foci. Other ultrathin sections revealed the presence of chondrons, typical of hyaline cartilage. Results from this study provide useful tools to further investigate morphogenesis and differentiation of the cartilaginous nasal capsule, and could in the future serve as a basic developmental standard.

The Biomimetics of Bone: Engineered Glass-Ceramics a Paradigm for In Vitro Biomineralization Studies

In this study, we investigated the behavior of fetal rat osteoblasts cultured up to 23 days on a bioactive apatite-wollastonite glass-ceramic (AW) and on the same material on which a carbonated apatite layer was formed by a biomimetic process (AWa). The specific activity of alkaline phosphatase activity was about 30% increased on AWa compared to AW disks at the last day of culture. Scanning electron microscopic (SEM) observations of the material surfaces after scrapping off the cell layers revealed that mineralized bone nodules remained attached to both surfaces but in larger numbers on AWa. The AWa/bone interfaces were also analyzed after fracturing the disks and by transmission electron microscopy (TEM). All these results indicated the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix. Furthermore, prefabrication of a biological apatite layer by a biomimetic method could improve our knowledge of biomineralization processes and could find application as bone-repairing material.

Cytoplasmic desmosome formation by H-7 and EGF treatment in cultured fetal rat keratinocytes

Cytoplasmic desmosomes (CD) are classically found in dyskeratotic cells of many epithelial tumors. Their significance and mechanism of formation remain largely speculative. Recently, we have reported the induction of these structures in rat keratinocytes following a brief treatment with acrylamide, and proposed that protein kinase inhibition may be implicated in their formation. In the present study, we show that protein kinase inhibitor H-7 in the presence of EGF is able to induce CD in rat keratinocytes within half an hour. In serum free medium containing 20 ng/ml of EGF, desmosomal structures at different stages of assembly were obtained using H-7 at concentrations ranging between 20 and 80 microM. No such structures were found at lower concentrations. The plaque diameters were significantly small in comparison with plasma membrane plaques. EGF induced plakoglobin positive membrane invaginations and in the presence of H-7, desmosomal plaques assembled on these membranes as either half desmosomes or as symmetric ones. The present results implicate protein kinase inhibition in CD formation and suggest that EGF provides tubular membrane structures in the cytoplasm on which desmosomes may assemble.