Biomedical subjects

J Guicheux

Publications and source records attributed to J Guicheux.

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

Injectable bone substitute to preserve alveolar ridge resorption after tooth extraction: a study in dog.

The aim of the present study was to assess the efficacy of a ready-to-use injectable bone substitute on the prevention of alveolar ridge resorption after tooth extraction. Maxillary and mandibular premolars were extracted from 3 Beagle dogs with preservation of alveolar bone. Thereafter, distal sockets were filled with an injectable bone substitute (IBS), obtained by combining a polymer solution and granules of a biphasic calcium phosphate (BCP) ceramic. As a control, the mesial sockets were left unfilled. After a 3 months healing period, specimens were removed and prepared for histomorphometric evaluation with image analysis. Histomorphometric study allowed to measure the mean and the maximal heights of alveolar crest modifications. Results always showed an alveolar bone resorption in unfilled sockets. Resorption in filled maxillary sites was significantly lower than in control sites. Interestingly, an alveolar ridge augmentation was measured in mandibular filled sockets including 30% of newly-formed bone. It was concluded that an injectable bone substitute composed of a polymeric carrier and calcium phosphate can significantly increase alveolar ridge preservation after tooth extraction.

Alveolar Process↗

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

Cartilage and bone tissue engineering using hydrogels.

Tissue engineering is an emerging field of regenerative medicine which holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity and trauma. During the past decade, tissue engineering has evolved from the use of naked biomaterials, which may just replace small area of damaged tissue, to the use of controlled three-dimensional scaffolds in which cells can be seeded before implantation. These cellularized constructs aims at being functionally equal to the unaffected tissue and could make possible the regeneration of large tissue defects. Among the recently developed scaffolds for tissue engineering, polymeric hydrogels have proven satisfactory in cartilage and bone repair. Major technological progress and advances in basic knowledge (physiology and developmental biology) are today necessary to bring this proof of concept to clinical reality. The present review focuses on the recent advances in hydrogel-based tissue engineered constructs potentially utilizable in bone and cartilage regenerative medicine.

Animals↗

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↗

PubMed2005

The effect of bisphosphonates and titanium particles on osteoblasts: an in vitro study.

In an attempt to increase the life of cementless prostheses, an hydroxyapatite-coated implant which releases a bisphosphonate has been suggested as a drug-delivery system. Our in vitro study was designed to determine the maximum dose to which osteoblasts could be safely exposed. Our findings demonstrated that zoledronate did not impair the proliferation of human osteoblasts when used at concentrations below 1 microm. Murine cells can be exposed to concentrations as high as 10 microm.A concentration of 0.01% of titanium particles did not impair the proliferation of either cell line. Zoledronate affected the alkaline phosphatase activity of murine osteoblasts through a chelation phenomenon. The presence of titanium particles strongly decreased the alkaline phosphatase activity of murine osteoblasts. We did not detect any synergic effect of zoledronate and titanium particles on the behaviour of both human and murine osteoblasts.

Alkaline Phosphatase↗

PubMed2004

Calcium phosphate drug delivery system: influence of local zoledronate release on bone implant osteointegration.

Despite total hip replacement (THR) gives generally satisfactory results, the quality of outcome in young patients is markedly decreased compared to the average THR outcome. For this population, pharmacological treatment with bisphosphonate would be beneficial to decrease the peri-implant osteolysis. However, as this population does not necessarily suffer from osteoporosis, a nonsystemic treatment would be preferable. Zoledronate was then grafted to hydroxyapatite (HA) coating of titanium implants. The implants were inserted in rat condyles with various zoledronate concentrations. A positive concentration-dependent effect was observed on the peri-implant bone density and on different histomorphometric parameters. Importantly for the outcome of the implants, the mechanical fixation was increased by the local presence of zoledronate. The obtained results open the way of an easy transformation of currently existing HA-coated implants by grafting bisphosphonate onto the coating in order to increase their service life in the patients.

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

Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation.

UNLABELLED: Signaling involved in osteoblastic cell differentiation remains largely unknown. This study further investigates mechanisms involved in BMP-2-induced osteoblastic cell differentiation. We report that BMP-2 can activate JNK and p38 in osteoblastic cells and provide evidences that these MAP kinases have distinct roles in regulating alkaline phosphatase and osteocalcin expression. INTRODUCTION: Bone morphogenetic protein (BMP)-2 exerts many of its biological effects through activation of the Smad pathway. Cooperative interactions between the Smads and the stress-activated protein kinase (SAPK) p38 and c-Jun-NH2-terminal kinase (JNK) pathways have recently been observed in TGF-beta signaling. MATERIALS AND METHODS: Activation of mitogen-activated protein (MAP) kinases by BMP-2 and the role of these signaling pathways for cell differentiation induced by BMP-2 was investigated in mouse MC3T3-E1 and primary cultured calvaria-derived osteoblastic cells using immunoprecipitation, in vitro kinase assay and Western blot analysis, as well as specific MAP kinase inhibitors. RESULTS: Associated with the rapid activation of Smads, BMP-2 barely affected extracellular-signal regulated kinase (ERK) activity, whereas it induced a transient activation of p38 and JNK. The role of p38 and JNK in mediating BMP-2-induced stimulation of osteoblastic cell differentiation was evaluated using the respective specific inhibitors SB203580 and SP600125. Inhibition of p38 by SB203580 was mainly associated with decreased alkaline phosphatase (ALP) activity, whereas inhibition of JNK by SP600125 was associated with a marked reduction in osteocalcin (OC) production induced by BMP-2. Corresponding alterations in ALP and OC mRNA levels were found in cells treated with BMP-2 and inhibitors, suggesting an implication of p38 and JNK pathways in BMP-2-induced osteoblastic cell differentiation at a transcriptional level. CONCLUSION: Data presented in this study describe p38 and JNK as new signaling pathways involved in BMP-2-induced osteoblastic cell differentiation with evidences for a distinct role of each MAP kinase in the control of alkaline phosphatase and osteocalcin expression.

3T3 Cells↗

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

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation.

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.

3T3 Cells↗

PubMed2001

Il-4 and IL-13, but not IL-10, protect human synoviocytes from apoptosis.

Interleukin-4, which has been contemplated for the treatment of rheumatoid arthritis and/or osteoarthritis because of its anticatabolic properties, has also been shown to modulate apoptosis. Because inadequate apoptosis is thought to contribute to synovial hyperplasia, we have investigated the ability of IL-4 and other Th2 cytokines to protect human synovial cells from apoptosis. Human synoviocytes or synovial explants were pretreated with IL-4, IL-10, and IL-13 before exposure to NO donor sodium-nitro-prusside (SNP). Apoptosis was evaluated by microscopy, annexin V-FITC, 3-(4,5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2H: tetrazolium inner salt (MTS) test, pulse field gel electrophoresis, and a method proposed in this study based on (32)P Klenow end labeling of high m.w. DNA. Pretreatment by IL-4 or IL-13, but not IL-10, protected human synoviocytes from apoptosis induced by SNP. Even at doses as high as 2 mM SNP, up to 86% and 56% protection was achieved, after IL-4 and IL-13 treatment, respectively. Cell survival was dependent on IL concentration. IL-4 and IL-13 also had antiapoptotic effects on SNP-treated human synovial explants. Effects of IL-4 and IL-13 varied in the presence of phosphatidylinositol-3 kinase and protein kinase C inhibitors, implying the involvement of these pathways in antiapoptotic signaling. Antiapoptotic effects were dramatically inhibited by LY294002, and partially by the protein kinase C inhibitor Gö 6976, while insulin-like growth factor increased synoviocyte survival. The possibility that IL-4 and IL-13 may enhance synovial expansion in vivo by their antiapoptotic effects is discussed.

Adjuvants, Immunologic↗

PubMed2001

Macroporous biphasic calcium phosphate ceramics versus injectable bone substitute: a comparative study 3 and 8 weeks after implantation in rabbit bone.

Macroporous biphasic calcium phosphate ceramics (MBCP) and a calcium phosphate injectable bone substitute (IBS), obtained by the association of biphasic calcium phosphate (BCP) ceramic granules and an aqueous solution of a cellulosic polymer, were compared in the same animal model. The two tested biomaterials were implanted in distal femoral osseous defects in rabbits. Qualitative and quantitative histological evaluation was performed three and eight weeks after implantation to investigate bone colonization and ceramic biodegradation associated with the two bone substitutes. Both biomaterials expressed osteoconduction properties and supported the apposition of a well-mineralized lamellar newly-formed bone. Bone colonization occurred much earlier and faster for IBS than for MBCP implants, although the respective rates of newly-formed bone after eight weeks of implantation did not differ significantly. For both biomaterials, ceramic resorption occurred regularly throughout the implantation period, though to a greater extent with IBS than with MBCP implants. The associated polymer in IBS produced intergranular spaces allowing body fluids to reach each BCP ceramic granule immediately after implantation, which may have favored osteoblastic activity, new bone formation and ceramic resorption. This completely interconnected open macroporosity could account for the earlier and more satisfactory bone substitution achieved with IBS.

Journal Article↗

PubMed2001

Ultrastructural evidence in vitro of osteoclast-induced degradation of calcium phosphate ceramic by simultaneous resorption and phagocytosis mechanisms.

Osteoclasts are physiological polykaryons specialized in the resorption of calcified tissue. In the context of the clinical use of calcium-phosphate (CaP) ceramics as bone substitutes, this study used transmission electron microscopy to investigate the in vitro mechanisms of CaP ceramic degradation by osteoclastic cell types. Osteoclasts cultured on CaP ceramic developed typical ultrastructural features of bone osteoclasts, such as a polarized dome shape, a clear zone and a ruffled border. Modification of the shape and density of CaP crystals under the ruffled border indicated an acidic microenvironment. Moreover, osteoclasts were able to degrade ceramic by simultaneous resorption and phagocytosis mechanisms. Phagocytosis did not alter the ability of osteoclasts to resorb CaP ceramic. The phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. The common mechanism of phagocytosis described here is similar to that observed with the monocyte/macrophage lineage, confirming that osteoclasts are part of the mononuclear phagocyte system. Osteoclasts are thus clearly involved in CaP degradation by means of resorption and phagocytosis.

Animals↗

PubMed2000

A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification.

In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.

Biological Transport↗