Jean-Raphaël Nefussi

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.

In vitro bone formation on coral granules.

SummaryWe investigated the ability of fetal rat bone cells isolated after collagenase digestion to differentiate in vitro and to produce a mineralized matrix on coral granules. Scanning electron microscopy examination of the surface of the seeded coral granules revealed that cells attached, spread, and proliferated on the material surface. Bone nodule formation was studied in this in vitro system by direct examination under an inverted phase contrast microscope. The initial event observed was the appearance of cells with phosphatase alkaline activity arranged in several layers and forming a three-dimensional organization around the coral particles. By Day 7, nodule formation began and a refringent material appeared and extended to the background cells during the following days. By Day 15, some coral granules were embedded in a mineralized matrix. Histologic results demonstrated the formation of a mineralized tissue with the appearance of woven bone.

Surface-reactive biomaterials in osteoblast cultures : an ultrastructural study

The tissue/biomaterial interface reactions of three biomaterials selected as candidates for hard tissue replacement were studied at the electron microscopical level after incubation with enzymatically isolated rat bone cells. An electron-dense layer was routinely observed between hydroxyapatite, coral, cytodex polymer and the neighbouring cells. This layer was visible before bone formation occurred, and was collagen free. The ultrastructural features revealed a needle-shaped filamentous layer continuous with coral material, whereas hydroxyapatite or cytodex/tissue interface was granular in appearance. These different structures may indicate reactive surfaces, depending on the composition of the substrate.

Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: Matrix formation and mineralization

Matrix formation and mineralization have been reported in vitro with cells isolated from rat calvaria bones by collagenase digestion (Nefussi et al., 1985). In the current study, kinetics of bone nodule formation and osteoblastic cell differentiation were studied in this in vitro system using an improved microcinematographic device and flash and follow-up labeling autoradiographic techniques. Microcinematographic analysis showed the formation of bone nodules within 24 h. The initial event observed was the change in the top cells layer which became alkaline phosphatase positive. Matrix synthesis occurred a few hours after this. The autoradiographic results demonstrated the formation of an integrated system where osteoblasts and osteocytes were active and synthesized a collagen matrix and mineralized it in a similar time sequence than in vivo.

Mineralization and bone formation on microcarrier beads with isolated rat calvaria cell population

SummaryUsing enzymatically isolated rat bone cells in the presence of cytodex microcarrier beads, osteoblastic cell differentiation and bone nodule formation were studied at the optical and electron microscopic level. Cytochemical method showed an intense alkaline phosphatase activity mainly around the microcarriers where the cells have formed multilayers on day 4 of cultures. On day 7 of experiment cultures. Von Kossa method stained positively only the cytodex microcarriers. During the following days, bone nodule formation was closely associated with cytodex microcarriers. In contrast, in control cultures with negatively charged glass beads, cells failed to pile up around the glass beads, and bone nodule formation occurred randomly in the culture dishes with 24 hour delay. Light microscopy observations of experiment cultures revealed the formation of nodular structures, with active osteoblastic cells forming a mineralized matrix in which osteocytes were present. Transmission electron microscopy revealed first, a mineralization process of the surface of the cytodex microcarriers which appeared like a granular electron-dense, collagen-free layer followed by the deposit of a collagenous matrix. These results indicated that cytodex microcarriers provided an excellent matrix for bone cell differentiation and mineralization.

A comparative ultrahistochemical study of glycosaminoglycans with cuprolinic blue in bone formed in vivo and in vitro.

SummaryHistochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization processin vivo but such studies have not yet been conductedin vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formedin vivo and bone nodules formedin vitro from osteoblastic cells in culture. For this investigation, we used a cationic phthalocyanin dye, cuprolinic blue, in a critical electrolyte concentration which simultaneously stained the glycosaminoglycans and demineralized the bone. This histochemical technique demonstrated (1) osteoblast cellsin vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix bothin vivo andin vitro. In the uncalcified matrix, PG were either free with a granular or rodlike structure or tightly connected to the periphery of the collagen fiber. Contrarily, in the calcified matrix, PG formed dense filamentous reticular patches between the collagen fibers. (3) Similarities in localization, organization, and morphology were noted in PG of bone formedde novo in vitro andin vivo with the exception of the mineralization front, where the stainingin vivo compared within vitro was faint or absent.

Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro

SummaryAn electron histochemical study was carried out on bone nodules formed in vitro in collagenase-released calvarial cells in order to visualize the lipid components of the extracellular matrix (EM). The malachite green aldehyde fixative technique, which allows both preservation and staining of some phospholipids of the extracellular matrix, was used. Controls were performed on sections demineralized, and then submitted to lipid extraction with a chloroformethanol mixture (2/1 v/v) and to glycosaminoglycans digestion with 0.5% bovine testicular hyaluronidase to verify specificity for lipid staining. This allowed us to visualize the lipids (1) in the osteoid as granules associated to ribbon-like structures connected to the collagen fibers, (2) as electrondense deposits seen as dots on the outer surface membrane of the matrix vesicles, and (3) in the mineralized matrix as roundish patches formed of needle-shaped materials and at the mineralization front as individual ones. This study demonstrated that at the EM level, the lipids are present in the osteoid at locations very similar to what have been observed for the glycosaminoglycans, and in the mineralized matrix as components of the crystal ghosts.

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β‐GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto‐ALP at physiological pH range. β‐GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto‐ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β‐GP. In both types of cultures, β‐GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β‐GP on 45Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β‐GP hydrolyses, which were likely catalyzed by the same ecto‐enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β‐GP, was inhibited by 50% by levamisole. In cells grown in the absence of β‐GP, the 45Ca incorporation rate increased progressively after β‐GP addition, reaching after 12 h the value of cultures grown in the presence of β‐GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β‐GP‐derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. β‐GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto‐ALP to hydrolyze β‐GP in cultured fetal calvaria cells, yet it influenced the β‐GP‐stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.

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.