Biomedical subjects

P Hardouin

Publications and source records attributed to P Hardouin.

At least 19 recordsLinked to original sources

PubMed2004

Migration of polyethylene debris along well-fixed cemented implants.

Implants, consisting of smooth Inox cylinders, were cemented into the lower femur and upper tibia of nine sheep to study the distal migration of polyethylene particles. Some implants had a titanium-bead porous coat at the proximal end. These were of three types: In the first type, the porous coat was covered with hydroxyapatite to obtain a bony seal; the second type was prepared for a polymethylmethacrylate seal; in the third type, the porous zone was surrounded by a 2-mm-thick space to allow the formation of a fibrous seal. Small polyethylene particles were injected into the knees once a week during the third and fourth months after implantation. The animals were euthanized 2 months later. Major longitudinal sections of the implants and the surrounding bone were examined under a polarized light microscope. Birefringent particles were counted at the cement-bone and cement-implant interfaces. Osteolysis was not observed. None of the seals significantly decreased the migration of particles around the cemented part of the implants. Particles were observed in cement fissures and vacuoles. They migrated at both interfaces and in the bone itself. They were visible in marrow spaces between bone trabeculae.

Animals↗

PubMed2004

Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography.

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.

Adaptation, Physiological↗

PubMed2004

Effect of dexamethasone on moesin gene expression in rabbit bone marrow stromal cells.

The influence of dexamethasone on rabbit bone marrow stromal cells differentiation was studied by screening the action of dexamethasone on gene expression. Using differential display, we observed some differential amplifications. The use of five of thirteen different primers combination allowed to identify one or more differential bands. One of them was identified as moesin gene. Real-time PCR confirmed a significant reduction of moesin gene expression following dexamethasone treatment. The decrease of expression for this protein, involved in cytoskeletal organization, could explain the effects of dexamethasone treatment on bone marrow stromal cells differentiation.

Animals↗

PubMed2004

Kinetic study of the expression of beta-catenin, actin and vinculin during osteoblastic adhesion on grooved titanium substrates.

Intercellular adhesions are known to play an important role in differentiation of osteoblasts and in the development of bone tissue architecture. However, to our knowledge, they have never been studied during the formation of bone tissue in contact with a biomaterial surface. In an in vitro kinetic study, we followed the expression of proteins involved in cell-cell interactions (beta-catenin), in cell-material interactions (vinculin) and in cytoskeleton (actin) of human osteoblastic cells cultured on grooved titanium-based substrates during 1, 2, 4, 6, 24, 48, and 72 hours. The human osteoblasts aligned themselves in the 150 microm wide grooves only after 24 hours. The distribution of vinculin-positive focal contacts, actin cytoskeleton and beta-catenin positive-adherens junctions was not significantly influenced by the cell alignment. beta-catenin-positive adherens junctions were expressed by human osteoblasts as soon as 1 hour after inoculation. At this time, they showed a patch-like aspect along cytoplasmic processes in contact with an underlying or an adjacent cell. After 2 hours, the patches were more and more numerous underlining the connections between cells. After 4 hours and more, the patches were organised in a parallel arrangement perpendicular to the two connected cells forming a "zip-like" aspect. Additionally, using double immuno-staining, we demonstrated that sometimes beta-catenin and vinculin appeared co-localised and sometimes not. The linkage of catenin/cadherin complex and vinculin-positive focal contacts with actin filaments may explain this apparent co-localisation.

Biocompatible Materials↗

PubMed2003

Influence of polymeric additives on the biological properties of brushite cements: an experimental study in rabbit.

The resorbability and ability of calcium phosphate hydraulic cements to promote new bone formation was investigated in vivo. The effects of two hydrosoluble polymeric additives (hyaluronic acid, and xanthan gum,) on the biological response of two brushite cement formulations (BHC-A vs BHC-B) was investigated. The brushite cements differed in P/Ca (0.71 vs 0.98) and S/Ca (0.10 vs 0.005) atomic ratios and by the presence of calcium sulfate hemihydrate in BHC-A. Polymer-free cements were used as controls. Cement specimens were injected in cylindrical bone defects manually drilled in the distal condyle of rabbit femora. The implants were harvested at 12 and 24 weeks after implantation and subjected to quantitative histomorphometry. The study showed a significantly lower resorption rate for cement BHC-A, which induces the formation of well-mineralized bone in close apposition to the residual material. In contrast, cement BHC-B showed a significant increase of bone formation period and the formation of a thick layer of unmineralized osteoid tissue at the bone/residual cement interface. The presence of xanthan gum made the biological response even worse, particularly in the case of cement BHC-B. The presence of hyaluronic acid has little effect, except for a slight decrease in initial resorption rate, in the case of cement BHC-A.

Animals↗

PubMed2002

Effect of grooved titanium substratum on human osteoblastic cell growth.

Various surface treatments have been developed to increase the clinical performance of titanium-based implants. Many in vitro tests have been carried out on substrates with varied surface topography for a complete understanding of osteoblasts. In previous research, we made the observation that surface roughness must be taken into account, not only in terms of amplitude but also in terms of organization. In this study, we tested the adhesion and proliferation of human primary osteoblasts on grooved titanium surfaces with various amplitudes and organizations of topography. The roughness was described at a scale above (macro-roughness) or below (micro-roughness) the cell size. We observed better orientation and proliferation of human osteoblasts on surfaces with a micro-roughness characterized by a lower Order (parameter describing the organization of topography) and by a higher Ra and Rz (parameters describing the amplitude of topography). It appears that cultured human osteoblasts prefer surfaces with relatively high micro-roughness amplitude and with a low level of repeatability.

Biocompatible Materials↗

PubMed2002

Bone colonization of beta-TCP granules incorporated in brushite cements.

Injectable calcium phosphate hydraulic cements are known to have a high clinical potential in bone reconstruction for mini-invasive orthopaedic surgery, interventional radiology, and rheumatology. Previous in vivo experiments in rabbit have shown that the presence of beta-TCP granules in injectable bone cement help maintain the transient biomechanical function of the implanted bone and promote the formation of good-quality new bone. Histomorphometric analysis of two brushite hydraulic cement (BHC) mixtures selected from previous results (referred to in this work as BHC-A and BHC-B) was performed at three postoperative delays (0, 12, and 24 weeks): histomorphometric analysis of bone colonization within beta-TCP shows that, just before implantation, the beta-TCP granule area is significantly higher in BHC-B; the residual granule area decreases steadily over time in BHC-A, whereas it goes through a maximum of 30% at 12 weeks in BHC-B; the residual granule porosity increases steadily up to 35% in BHC-A, whereas it goes through a maximum of 35% at 12 weeks and decreases somewhat until 24 weeks in BHC-B. New bone formation within granules appears higher in BHC-A (58% Area) compared to BHC-B (38% area) at 12 weeks. At 24 weeks bone colonization levels off in both cements at about 50% area. Irrespective of the cement matrix composition, beta-TCP granules contribute actively to the conduction of new bone formation.

Animals↗

PubMed2002

Improvement in the morphology of Ti-based surfaces: a new process to increase in vitro human osteoblast response.

Surface roughness has been shown to be an influencing parameter for cell response. In this experience we attempted to compare the effect of roughness organization of Ti6A14V or pure titanium substrates on human osteoblast (hOB) response (proliferation, adhesion). Surface roughness was extensively analyzed at scales above the cell size (macro-roughness) or below the cell size (micro-roughness) by calculation of relevant classic amplitude parameters (Ra, Rt) and original frequency parameters (Order, Delta). We developed a new process to prepare isotropic surfaces (electro-erosion), which were compared to isotropic surfaces obtained by polishing and anisotropic surfaces obtained by machine-tooling. The hOB response on electro-eroded (EE) Ti6A14V surfaces or pure titanium (Ti) surfaces was largely increased when compared to polished or machine-tooled surfaces after 21 days of culture. Moreover, the polygonal morphology of hOB on these EE surfaces was very close to the aspects of hOB in vivo on human bone trabeculae. By a complete description of the surface topography of EE surfaces, we concluded that when the topography was considered below the cell scale, hOB appreciated their isotropic smooth aspect, although when the topography was considered above the cell scale they appreciated their rough isotropic 'landscape' formed by many 'bowl-like nests' favouring cell adhesion and growth. Electro-erosion is a promising method for preparation of bone implant surfaces, as it could easily be applied to preparation of most biomaterials with complex geometries.

Algorithms↗

PubMed2002

An unscaled parameter to measure the order of surfaces: a new surface elaboration to increase cells adhesion.

We present a new parameter to quantify the order of a surface. This parameter is scale-independent and can be used to compare the organization of a surface at different scales of range and amplitude. To test the accuracy of this roughness parameter versus a hundred existing ones, we created an original statistical bootstrap method. In order to assess the physical relevance of this new parameter, we elaborated a great number of surfaces with various roughness amplitudes on titanium and titanium-based alloys using different physical processes. Then we studied the influence of the roughness amplitude on in vitro adhesion and proliferation of human osteoblasts. It was then shown that our new parameter best discriminates among the cell adhesion phenomena than others' parameters (Average roughness (Ra em leader )): cells adhere better on isotropic surfaces with a low order, provided this order is quantified on a scale that is more important than that of the cells. Additionally, on these low ordered metallic surfaces, the shape of the cells presents the same morphological aspect as that we can see on the human bone trabeculae. The method used to prepare these isotropic surfaces (electroerosion) could be undoubtedly and easily applied to prepare most biomaterials with complex geometries and to improve bone implant integration. Moreover, the new order parameter we developed may be particularly useful for the fundamental understanding of the mechanism of bone cell installation on a relief and of the formation of bone cell-material interface.

Alloys↗

PubMed2001

Should percutaneous vertebroplasty be used to treat osteoporotic fractures? An update.

Acrylic cement vertebroplasty is being increasingly used to treat osteoporotic vertebral compression fractures (VCFs), although no controlled studies supporting this trend have been published. Vertebroplasty remains controversial as a treatment for osteoporotic fractures because it is a local response to a systemic disease and because the pain caused by osteoporotic fractures usually subsides within a few days or weeks. Current data suggest that pain severity may decrease by half, on average, in 90-100% of patients. Although vertebroplasty is usually well tolerated, serious neurological complications have been reported in a few patients. The most common adverse event is nerve root pain, usually caused by leakage of the cement into the intervertebral foramen. Whether vertebroplasty is followed by an increased risk of osteoporotic fractures in the adjacent vertebras remains unclear. Resorbable cements are being developed and may provide better results than the acrylic cements used today. At present, acrylic cement vertebroplasty to treat osteoporotic VCFs is appropriate in only a minority of patients selected carefully by a multidisciplinary team including a rheumatologist.

Arthroplasty↗

PubMed2001

Porous HA ceramic for bone replacement: role of the pores and interconnections - experimental study in the rabbit.

Hydroxyapatite (HA) porous ceramics are increasingly used in biomedical applications. Their physical characteristics, such as porous volume, require perfect control of the pore shape, as well as the number and the size of their interconnections. The aim of our study was to evaluate a new HA ceramic using polymethylmethacrylate microbeads (PMMA) as the porous agent. Four interconnection sizes (30, 60, 100 and 130 microm) with a 175-260 microm pore size and three pore sizes (175-260, 260-350 and 350-435 microm) for a 130 microm interconnection size were tested. Various HA implants were appraised by microscopic evaluation in a 4.6 x 10 mm rabbit femur cancellous bone defect 12 weeks after implantation. The best osteoconduction result was obtained in the center of the ceramic by means of a 130 microm interconnection size and a 175-260 microm mean pore size. Bone formation obtained within the pores was double that obtained in our previous study where naphtalen microbeads were used as the porous agents.

Journal Article↗

PubMed2000

Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses.

We quantitatively evaluated the adhesion of human osteoblasts on orthopedic metallic substrates (Ti6Al4V alloy) with various surface roughnesses at several times after inoculation and studied its correlation with qualitative changes in the expression of adhesion proteins and with parameters extensively describing the surface topographies. Cells were orientated in a parallel order on polished surfaces. This orientation was not affected by residual grooves after polishing. On sandblasted surfaces the cells never attained confluence and had a stellate shape, and the cell layer had no particular organization. Extracellular matrix (fibronectin, type I collagen, osteopontin) and cytoskeletal protein (actin, vinculin) orientation reflected the cell layer organization. In our experiment human osteoblasts expressed alpha3beta1 integrin but not alpha2beta1 integrin. In addition to currently analyzed roughness magnitude parameters, we calculated roughness organization parameters (fractal dimension parameters) of the substrates. We observed lower adhesion and proliferation on less organized surfaces (i.e., sandblasted ones). The significant statistical correlation observed between fractal dimension parameters (describing surface roughness organization) and cell parameters adds a new concept to the studies of substratum roughness influence on cell behavior. An attempt at modelization of the cell-surface interaction was made that includes the influence of fractal dimensions parameters.

Alloys↗

PubMed2000

The relative influence of the topography and chemistry of TiAl6V4 surfaces on osteoblastic cell behaviour.

Proliferation and adhesion of mouse (MC3T3-E1) osteoblastic cells and primary human osteoblastic cells were carried out on Ti6Al4V titanium alloy samples with varied surface roughnesses. Mechanically or manually polished surfaces were prepared to produce respectively non-oriented or oriented residual polishing grooves. Sand-blasted surfaces were prepared using 500 microm or 3 mm alumina particles. Surface roughness parameters showed a negative correlation in comparison to proliferation and adhesion parameters. X-ray microprobe chemical surface microanalysis showed complete disturbance of the surface element composition of the Ti6Al4V alloy following sand-blasting treatment. An AlOx-enriched layer was observed on sample surfaces. This may lead to the suspicion that the concomittant effect of surface roughness amplitude and AlOx surface concentration has an effect on osteoblastic cell proliferation and adhesion. These findings show the significance of chemical surface analysis after any surface treatment of titanium-based implants before any biological use.

3T3 Cells↗

PubMed2000

Comparative study of the in vitro characteristics of osteoblasts from paralytic and non-paralytic children.

STUDY DESIGN: Cerebral palsy is known to provoke a high loss of bone in children and adults. The potential interest of human osteoblastic cell culture for assessing the osteoblastic function in metabolic bone disorders has been demonstrated by many previous studies. Few studies have attempted to evaluate the capacities of osteoblasts isolated from immobilized or normal bones by in vitro culture methods. Moreover, a few teams did make the distinction between young spastic and flaccid patients. OBJECTIVES: We attempted to characterize mature osteoblasts (OB) and bone marrow-stromal cells (BM) originating from 56 immobile and normal children. Spastic and flaccid patients formed the paralytic group. SETTING: France. METHODS: Osteoblasts and bone marrow cells were isolated from iliac crests obtained during pelvic osteotomies of young control and paralytic patients. The in vitro viability, proliferation and differentiation parameters of the cells from paralytic patients were compared with those of cells coming from normal controls. RESULTS: No significant differences in the cell proliferation parameters were observed between the two groups. Only initial cell viability before inoculation was lower for the paralytic group, compared to the control group. On the other hand, contrary to expectations, we found that fresh and thawed OB cells from flaccid patients synthesized more osteocalcin and more collagen respectively than those of the spastic and control groups. Opposite results were obtained from BM cultures. CONCLUSION: A negative feedback mechanism by systemic or local factors, which is not conserved in vitro but controls the in vivo osteocalcin and collagen synthesis of flaccid paralytic OB cells, is hypothesized. Because these flaccid patients are known to have a high fat/lean mass, we suggest that leptin may be the potential regulating factor implicated in the hypothesized negative feedback mechanism.

Bone Marrow Cells↗

PubMed2000

[Histologic and biomechanic evaluation of posterolateral arthrodesis using a biphasic ceramic of calcium phosphate as bone substitute. Experimental study with sheep].

The effectiveness of a macroporous biphasic calcium phosphate ceramic was studied after laterovertebral arthrodesis in sheep. A ceramic with a TCP/HAP ratio of 35/65 was compared with autologous bone graft in an histomorphometric and biomechanical point of view. Quantitative analysis of the results indicated that the biphasic ceramic allows an arthrodesis after 12 months, although control graft is effective after six months. A large decrease of flexibility in all directions was obtained with the ceramic, similarly to autologous graft. No nonfusion case was observed. In conditions very close to the human surgery, a posterolateral fusion can be obtained using biphasic phosphate ceramic as bone substitute.

Animals↗

PubMed2000

Tissue engineering and skeletal diseases.

Tissue engineering, a cross between the science of the living organism and that of engineering, aims to replace, maintain or improve human tissue functions, by means of tissue substitutes containing living elements. Thus, it is about production of artificial tissue, using (alone or in combination) cells, matrix or bioactive factors. Their association gives rise to a hybrid biomaterial combining biological components (cells, growth factors or adhesion proteins) and materials (polymers, ceramics). The applications are wide-ranging, from the skin, to the liver, or to the cornea as well as to the locomotor system. Bone tissue engineering has advanced the most in this field, partly because of the progress made by research into bone substitutes, although cartilage and tendons are also concerned. This technology requires cell culture (committed cells or more often bone marrow stem cells), biomaterials (porous materials with controlled architecture and cements), growth factors (such as 'Bone Morphogenetic Proteins'), the proteins implicated in cell adhesion (such as fibronectin or the aminoacid sequences specifically recognised by integrin subunits) or gene therapy (notably using transfected stem cells). Tissue engineering and regenerative stimulation of tissue are now booming on experimental and industrial levels and clinical applications are increasingly numerous. Considering the potential of these technologies, they should continue to develop widely.

Biomedical Engineering↗

PubMed1999

Association of porous hydroxyapatite and bone marrow cells for bone regeneration.

The preparation of hybrid material with osteoinductive capacity may be achieved by association of cultured autologous bone cells with a porous ceramic vehicle. We optimized culture conditions for rabbit marrow stromal stem cells (MSCs), notably by selection from batches of fetal calf serum. Rabbit MSCs formed colony-forming unit-ribroblastic (CFU-Fs) in vitro. Their alkaline phosphatase (ALP) activity was doubled in the presence of dexamethasone. Autologous rabbit serum allowed the formation of ALP-positive CFU-Fs, but results were highly variable depending on the rabbit. We tested the osteogenic potential of autologous cultured (with or without dexamethasone addition in the culture medium) and noncultured rabbit MSCs associated with a porous hydroxyapatite ceramic after a dorsal intramuscular implantation. Nucleated cells (10(7) or 10(8)/mL) were used for the preparation of autologous hybrid material. A significantly higher number of implants containing bone was obtained with a suspension of 10(7) cells/mL cultured in the presence of 10(-8) M dexamethasone. Some positive implants were also obtained with a suspension of 10(8) noncultured cells/mL. We demonstrated the feasibility of preparing rabbit autologous hybrid materials following a process for controlling culture conditions, cell characterization and cell/material association.

Alkaline Phosphatase↗

PubMed1999

Raman microspectrometry studies of brushite cement: in vivo evolution in a sheep model.

Calcium phosphate hydraulic cements are promising synthetic bone grafting materials. Brushite-based cements were implanted for 6 and 12 months in the distal condyle of sheep femur, and their in vivo evolution was investigated by Raman microspectrometry. This new technique can probe small volumes in the cubic micrometer range. Its resolution allows a very fine analysis of crystalline changes in calcium phosphate mixtures at the microscopic level. First, Raman spectra of pure brushite, monetite, and beta-tricalcium phosphate (beta-TCP) were recorded, in order to set a data base for the basic components of brushite cements. These spectra show significant differences in the vibration mode v1 for the phosphate ion (988 and 878 cm(-1) for brushite, 988 and 900 cm(-1) for monetite, 968 and 948 cm(-1) for beta-TCP). These differences are strong enough as to allow the qualitative and quantitative analysis of these crystalline phases in the cement. Implanted sheep femur samples were harvested after 24 and 52 weeks post-op, and prepared for Raman analysis in the form of 1-mm-thick sections. Implants at 24 weeks show a core of residual cement isolated from the surrounding bone by fibroconnective tissue. No trace of brushite was detected by micro-Raman analysis in this area, but instead, a mixture of beta-TCP and Type-B carbonated apatite, the latter being very close in composition and structure to the mineral fraction of normal bone in the vicinity of the implant. Implants recovered after 52 weeks show a decrease of the bone/residual cement perimeter, whereas new trabeculations are formed in the implanted zone; the small amounts of residual cement still present are substantially transformed into Type-B carbonated apatite containing small amounts of proteins. In the same area, some beta-TCP particles are also detected showing that, contrary to brushite, the excess beta-TCP originally present in the cement is not completely metabolized. In the implanted zone already converted into trabecular bone, Raman microspectrometry shows the characteristic spectrum of normal bone.

Animals↗