Biomedical subjects

D Magne

Publications and source records attributed to D Magne.

At least 19 recordsLinked to original sources

PubMed2007

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel.

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.

Aggrecans↗

PubMed2006

Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway.

Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

Animals↗

PubMed2006

Culture medium modulates the behaviour of human dental pulp-derived cells: technical note.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Alkaline Phosphatase↗

PubMed2006

The active metabolite of leflunomide, A77 1726, increases proliferation of human synovial fibroblasts in presence of IL-1beta and TNF-alpha.

OBJECTIVE AND DESIGN: Excessive synovial fibroblast (SF) proliferation is detrimental in rheumatoid arthritis. We therefore sought to determine the effects of A77 1726, the active metabolite of leflunomide, on SF proliferation. METHODS: Human SFs were used. Cell proliferation was investigated using MTS assay, by (3)H-thymidine incorporation and cell counts. RESULTS: Whereas A77 1726 alone had no effects, it significantly increased the mitogenic effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Cyclooxygenase inhibition might be at least partly involved, since indomethacin displayed similar effects, and since prostaglandin E2 inhibited SF proliferation. In contrast, the effect of A77 1726 did not appear to be mediated through depletion of the pyrimidine pool or inhibition of tyrosine kinases. CONCLUSION: A77 1726 displays proliferative effects in presence of IL-1beta and TNF-alpha. Further elucidation of involved mechanisms may prove useful for the utilization of leflunomide, the development of related compounds or elaboration of new therapeutic strategies.

Aniline Compounds↗

PubMed2005

Cartilage formation in growth plate and arteries: from physiology to pathology.

Vascular calcifications are the consequence of several pathological conditions such as atherosclerosis, diabetes, hypercholesterolemia and chronic renal insufficiency. They are associated with risks of amputation, ischemic heart disease, stroke and increased mortality. A growing body of evidence indicates that vascular smooth muscle cells (VSMCs) undergo chondrogenic commitment eventually leading to vascular calcification, by mechanisms similar to those governing ossification in the cartilage growth plate. Our knowledge of the formation of cartilage growth plate can therefore help us to understand why and how arteries calcify and, consequently, develop new therapeutic strategies. Reciprocally, thorough consideration of the events leading to ectopic chondrocyte differentiation appears crucial to further increase our understanding of growth plate formation. In this context, we will review the effects of known or suspected factors that promote chondrogenic differentiation in growth plate and arteries.

Animals↗

PubMed2005

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Animals↗

PubMed2004

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-beta1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-beta1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-beta1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.

Animals↗

PubMed2003

Noninvasive bone replacement with a new injectable calcium phosphate biomaterial.

The use of injectable calcium phosphate (CaP) biomaterials in noninvasive surgery should provide efficient bone colonization and implantation. Two different kinds of injectable biomaterials are presently under development: ionic hydraulic bone cements that harden in vivo after injection, and an association of biphasic calcium phosphate (BCP) ceramic granules and a water-soluble polymer vehicle (a technique particularly investigated by our group), providing an injectable CaP bone substitute (IBS). In our study, we compared these two approaches, using physicochemical characterizations and in vivo evaluations in light microscopy, scanning electron microscopy, and three-dimensional microtomography with synchrotron technology. Three weeks after implantation in rabbit bone, both biomaterials showed perfect biocompatibility and bioactivity, but new bone formation and degradation of the biomaterial were significantly greater for BCP granules than for ionic cement. Newly formed bone developed, binding the BCP granules together, whereas new bone grew only on the surface of the cement, which remained dense, with no obvious degradation 3 weeks after implantation. This study confirms that BCP granules carried by a cellulosic polymer conserve bioactivity and are conducive to earlier and more extensive bone substitution than a carbonated-hydroxyapatite bone cement. The presence of intergranular spaces in the BCP preparation, as shown on microtomography imaging, seems particularly favorable, allowing body fluids to reach each BCP granule immediately after implantation. Thus, the IBS functions as a completely interconnected ceramic with total open macroporosity. This new bone replacement approach should facilitate microinvasive bone surgery and local delivery of bone therapy agents.

Animals↗

PubMed2003

Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials.

This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation. Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 microm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.

Animals↗

PubMed2003

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

UNLABELLED: Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. INTRODUCTION: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. MATERIALS AND METHODS: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). RESULTS: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. CONCLUSIONS: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

Animals↗

PubMed2002

Fourier transform infrared microspectroscopic investigation of the organic and mineral constituents of peritubular dentin: a horse study.

Peritubular dentin (PTD) is a relatively dense mineralized tissue surrounding tooth dentin tubules, whose composition and mode of formation are still unclear. Fourier transform infrared microspectroscopic studies of the organic and mineral components of the highly developed horse PTD indicate that the peritubular matrix is less abundant than the intertubular matrix but is also mainly composed of collagen, which is more hydrated. These data suggest that most of the crystals are located outside the collagen fibrils and probably not associated with protein components. The crystals in PTD have nearly the same crystallinity as those in intertubular spaces, showing comparable amounts of carbonate ions, although some PO4 groups have different nonapatitic environments. Horse PTD composition is very similar to that of ITD, with collagen as the main protein component and carbonated apatite as the mineral fraction but the different proportions of these constituents and the greater water content in PTD suggest a different organization.

Animals↗

PubMed2002

In vitro characterization and in vivo properties of a carbonated apatite bone cement.

This study evaluated the in vivo behavior of an injectable calcium phosphate bone cement implanted in bone defects at the distal end of rabbit femora. After 3 weeks, samples were harvested and processed for undecalcified sectioning. Scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared microspectroscopy showed direct contact of bone and cement without soft tissue interposition, biocompatibility, and bioactivity with osteoconductive properties.

Animals↗

PubMed2001

Impairment of amino-acid absorption in suckling rats infected with Cryptosporidium parvum.

In the present study. we explored the nutritional consequences of cryptosporidiosis. In order to ascertain the direct responsibility of C. parvum for impairment of staturoponderal development observed during the infection in neonatal animals, we investigated the absorption of two major components of the total amino acids in dam's milk (leucine and glutamate) across the ileal mucosa. The infection resulted in significant (47% and 34%, respectively) reductions in leucine and glutamate fluxes (P<0.01). Moreover, the leucine aminopeptidase and alkaline phosphatase activities were reduced in the infected ileal mucosa. Interestingly, the reduction in weight gain, which began at day 6 post-infection (PI), persisted until day 20 PI, although no cryptosporidia were detected in the ileal mucosa after day 12 PI. We thus provide evidence that the malabsorption of amino acids during cryptosporidiosis contributes to impairing the development of neonatal animals, with consequences that persist beyond eradication of the parasite.

Alkaline Phosphatase↗

PubMed2001

Fourier transform infrared microspectroscopic investigation of the maturation of nonstoichiometric apatites in mineralized tissues: a horse dentin study.

Fourier transform infrared microspectroscopy (FTIRM) was used to study carbonated apatite/collagen interactions and maturation in horse secondary dentin. Unlike human dentin, this model contains no peritubular material around the odontoblastic processes and is thus quite similar to bone in composition, but not subject to tissue turnover. Crystals close to the mineralization front were very immature, showing high HPO(4) and very low CO(3) levels. Carbonate ions were located essentially in very labile, reactive environments, probably on the crystal surface. Removal of some of the HPO(4) ions from crystals during maturation was linked to an increase in total carbonate content. The CO(3) ions in labile environments decreased, probably after incorporation into more organized regions of the lattice. However, this increase of total carbonate content was associated with greater mineral crystallinity, confirming findings in other studies of synthetic apatite maturation in vitro. The good correlation between these results and those of in vitro experiments suggests that crystal maturation is essentially due to physicochemical processes and that the organic matrix controls only crystal size, multiplication, and/or organization.

Animals↗

PubMed2001

Study of the maturation of the organic (type I collagen) and mineral (nonstoichiometric apatite) constituents of a calcified tissue (dentin) as a function of location: a Fourier transform infrared microspectroscopic investigation.

Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The v1v3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO4(2-) groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.

Adolescent↗

PubMed2001

[Cryptosporidium parvum: functional study of the intestinal malabsorption syndrome].

Cryptosporidiosis is an important cause of diarrhea associated with growth retardation in children and severe malnutrition in immunocompromised patients. The pathophysiology is poorly understood. In the suckling rat model, we show that C. parvum infection impairs net electrogenic transport across the ileal mucosa without involvement of prostaglandins, as well as trans- and paracellular permeability and leucine and glutamate absorption. These results provide evidence for the development of an intestinal malabsorptive syndrome during cryptosporidiosis. Unspecific process such as villous atrophy and inflammatory cytokines secretion should be regarded as possible mediators of this syndrome. However, specific mechanisms have to be considered since C. parvum induces a rearrangement of the host enterocyte cytoskeleton which might impaired intracellular trafficking thus reducing the membrane expression of nutrient transporters. Infection and malnutrition are known to be tightly associated, making each other worse. As no specific efficient therapy exists, cryptosporidiosis-induced malnutrition must be taken into account when establishing therapeutic scheme.

Animals↗

PubMed1999

Cryptosporidium parvum infection in suckling rats: impairment of mucosal permeability and Na(+)-glucose cotransport.

Na(+)-glucose transport and transepithelial permeability were investigated during symptomatic acute cryptosporidiosis in newborn rats. The infection resulted in a significant (P < 0.01) decrease in the ileal short-circuit current and a nonsignificant fall in the transepithelial potential difference and conductance. In glucose-stimulated conditions, the rise in ileal short-circuit current and transepithelial permeability were significantly lower in Cryptosporidium parvum-infected rats than in controls (delta Isc = 3.24 +/- 1.21 microA.cm-2 vs delta Isc = 5.09 +/- 2.23 microA.cm-2 in infected and control animals, respectively; P < 0.001; delta PD = -0.35 +/- 0.13 mV vs delta PD = -0.44 +/- 0.14 mV for infected and control animals, respectively; P < 0.01). Electrical parameters were not affected by addition of the cyclooxygenase inhibitor indomethacin in either Cryptosporidium-infected newborn rats or controls. Horseradish peroxidase and mannitol flux studies demonstrated a significant decrease (P < 0.05) in transepithelial molecular permeability in infected enterocyte rats, HRP flux = 380, range 68-5570 ng.cm-2, and mannitol flux = 1.06, range, 0.34-1.44%.cm-2.min-1, compared with controls rats, HRP flux = 4446 range, 1121-124,363 ng.cm-2, and mannitol flux = 1.99, range, 0.57-5.09%.cm-2.min-1; P < 0.05. These effects could originate from C. parvum-induced alteration of intracellular trafficking of pinocytosis vesicles and therefore account for the decrease in permeability to solute and macromolecules, together with impaired transcellular nutrient transport, in suckling rats.

Animals↗

PubMed1999

Fecal tumor necrosis factor alpha, eosinophil cationic protein and IgE levels in infants with cow's milk allergy and gastrointestinal manifestations.

Infants with atopic eczema exhibit a specific fecal protein pattern after oral challenge with cow's milk, characterized by an increase in both eosinophil cationic protein (ECP) and tumor necrosis factor (TNF)alpha. The aim of our study was to determine the pattern of these proteins in allergic infants with intestinal manifestations. TNFalpha, ECP and immunoglobulin E (IgE) were measured in stools from 13 infants with intestinal symptoms and 10 healthy infants. The allergic infants underwent two stool collections, one before a cow's milk challenge and the other after the challenge, either at the onset of clinical manifestations (n=6) or 15 days after the challenge if no clinical manifestations occurred (n=7). Baseline TNFalpha, ECP and IgE levels were low in all infants. The concentration of TNFalpha increased after the challenge in infants positive to challenge (p<0.05) but not in those negative to challenge. ECP and IgE levels remained low after the challenge in all the allergic infants. These data confirm that fecal TNFalpha and ECP levels indicate various reaction types of food allergy and that different immunologic disturbances lead to atopic eczema or intestinal symptoms during food allergy. Fecal protein pattern can thus be a useful tool in diagnosing food allergy in infants with intestinal manifestations.

Blood Proteins↗