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J R Nefussi

Publications and source records attributed to J R Nefussi.

26 records · Page 2Linked to original sources
PubMed1989

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

Histochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization process in vivo but such studies have not yet been conducted in vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formed in vivo and bone nodules formed in 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 cells in vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix both in vivo and in 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 formed de novo in vitro and in vivo with the exception of the mineralization front, where the staining in vivo compared with in vitro was faint or absent.

Animals↗
PubMed1989

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.

Alkaline Phosphatase↗
PubMed1986

Collagen expression during teratocarcinoma cell line-induced endochondral bone tumor.

Collagen immunotyping by indirect immunofluorescence was performed in order to investigate the sequential development of bone formation. Osseous tumors were obtained after subcutaneous injection of 3/A/1D-1 teratocarcinoma cell line into 129/Sv mice (Nicolas et al., 1980). Frozen sections of developing tumors were incubated with specific antibodies directed against Types I, II, III, IV, and IX collagens. On Day 9, the expression of Type I and Type III collagens was correlated with the proliferation of mesenchymal cells. From Day 10, chondrogenesis was characterized by the occurrence of cartilaginous collagens, Types II and IX, in the cartilage matrix. Type IV collagen was also detected in focal areas and revealed vascular invasion of the tumor. On Day 13, osteogenesis was demonstrated by the presence of Type I collagen in the bone matrix coating the surfaces. Immunolocalization of Type III collagen on the hemopoietic elements corresponded with the bone remodeling. The sequential transitions of collagen types confirm the development of an endochondral bone tumor. These results suggest that 3/A/1D-1 teratocarcinoma cell line constitutes a valuable system for in vitro study of endochondral bone formation and cell differentiation.

Animals↗
PubMed1986

Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts.

Positive identification of osteoclast percursors has not yet been possible. The authors have, in the present report, used a model system in the rat in which it is possible to induce the formation of multinucleated osteoclasts at a predictable and reproducible site and time (Tran Van P, Vignery A, Baron R. Anat Rec 1982, 202:445-451; Cell Tissue Res 1982, 225:283-292). This system allowed the investigation of the cellular events occurring locally during the recruitment and differentiation of osteoclast precursors. Prior to the formation of multinucleated osteoclasts, mononuclear cells positive for fluoride-inhibitable nonspecific esterase and cells positive for tartrate-resistant acid phosphatase increase in number locally. Double staining procedures demonstrated the presence of both enzymes in a number of cells, thereby suggesting that they are steps in the differentiation of a single cell population. Ultrastructural studies show that lysosomal enzymes are present in every compartment of the biosynthetic pathway, in small primary lysosomes and various forms of storage granules. As these precursors arrive at the bone surface, the storage granule lysosomes are markedly depleted. It is concluded that mononuclear precursors of the osteoclast are members of the mononuclear-phagocyte lineage and differentiate early to synthesize, store, and later secrete large quantities of lysosomal enzymes. The mature osteoclast, which, as its precursor, is positive for the mononuclear-phagocyte marker enzyme nonspecific esterase, results from the fusion of these mononuclear precursors, which occurs only after their attachment to the bone surface to be resorbed.

Acid Phosphatase↗
PubMed1985

PGE2 stimulates both resorption and formation of bone in vitro: differential responses of the periosteum and the endosteum in fetal rat long bone cultures.

The ability of PGE2 to stimulate bone resorption in vitro and in vivo is well established but the effects of this compound on bone formation are still controversial. Recent clinical reports have suggested that long-term infusion of PGE in infants with cyanotic heart diseases led to a stimulation of periosteal bone formation and to hyperostosis. In the present report, we describe the effects of PGE2 (10(-5) M) in bone organ cultures on bone resorption, measured by the release of 45Calcium and the number of osteoclasts in sections of cultured bones, and bone volume, by measuring separately medullary and cortical areas. PGE2 induced a marked increase in 45Ca release and in cortical and medullary osteoclast numbers over 4 days in vitro; despite this increase in bone resorption, cortical bone volume remained constant, indicating a parallel increase in bone resorption and formation at this site. Morphological and quantitative data demonstrated a higher extent of osteoblastic surface along the periosteum of PGE2-treated bones when compared with control cultures. Medullary bone volume, on the other hand, decreased sharply during the culture period, demonstrating a lack of parallel increase in bone formation at this site. It is concluded that, under these experimental conditions, prostaglandin E2 stimulated both resorption and formation along the periosteum and only bone resorption along the endosteum of the cultured bones. The overall effect of PGE2 on bone as a whole, however, was net bone loss.

Animals↗
PubMed1985

Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion.

Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml ascorbic acid, with or without 10 mM beta-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of beta-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of beta-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of beta-glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo.

Alkaline Phosphatase↗
PubMed1982

Histomorphometry and autoradiography of cultured fetal rat long bones.

The behavior of fetal rat long bones cultured in vitro according to Raisz's technique (1969) was studied by histomorphometry and autoradiography for a period of four days. The changes were recorded daily both on the trabecular and cortical bone by measuring the bone volume, the number of osteoclasts, and the number of nuclei per osteoclast. Radioactive calcium release was measured and compared to the changes in bone volume and in the number of osteoclasts. An autoradiographic study, using 3H-proline and 3H-thymidine in flash labeling in the medium and 3H-thymidine in follow-up labeling after one injection in vivo was performed to evaluate the bone formation, the cellular proliferation rate and cell differentiation. After four days in culture, an increase in total calcified bone volume was observed which correlated with changes in the trabecular bone. No significant changes were recorded in the cortical bone. The results showed a good maintenance of the resorption and formation phenomena through an active process of cellular multiplication and differentiation. Undifferentiated cells were labeled in flash label and osteoblast, osteocyte and some osteoclast nuclei were labeled in follow-up studies.

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