Biomedical subjects

Paul Ducheyne

Publications and source records attributed to Paul Ducheyne.

18 recordsLinked to original sources

PubMed2007

Adhesion of MC3T3-E1 cells to RGD peptides of different flanking residues: detachment strength and correlation with long-term cellular function.

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (tau(50)) of RGD peptides of different flanking residues was found: RGDSPK > RGDSVVYGLR approximately RGDS > RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily alpha(v)-integrin-mediated. In contrast, the tau(50) value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both alpha(5)beta(1) and alpha(v)beta(3) integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides.

Amino Acid Sequence↗

PubMed2006

Controlled release of vancomycin from thin sol-gel films on titanium alloy fracture plate material.

Risk of infection is considerable in open fractures and its management is challenging, especially when fracture fixation material is used. Thus, it may be desirable to use a device from which antibiotics can be released in a controlled way. Room temperature processed silica sol-gels are novel, resorbable and biocompatible, controlled release materials. Vancomycin, a potent antibiotic used in treating osteomyelitis, can be released from silica sol-gels. Herein, we describe the synthesis of thin, resorbable, controlled release bactericidal sol-gel films on a Ti-alloy substrate and determine the effect of processing parameters on its degradation and vancomycin release. A close correlation between release and degradation rates suggests that film degradation is the main mechanism underlying the control of release. Using a multi-layer process and various concentrations of vancomycin, released concentrations exceed the minimal inhibitory concentration (MIC) of vancomycin against Staphylococcus aureus. The findings enable the tailoring of release and degradation properties of the films to therapeutic needs by controlling sol-gel processing parameters. Given the bactericidal properties of released vancomycin, and the biocompatibility of the sol-gel films, the present data suggest great promise to prevent and treat bone infections in a clinical setting.

Alloys↗

PubMed2006

The effect of starch and starch-bioactive glass composite microparticles on the adhesion and expression of the osteoblastic phenotype of a bone cell line.

There is a clear need for the development of microparticles that can be used simultaneously as carriers of stem/progenitor cells and as release systems for bioactive agents, such as growth factors or differentiation agents. In addition, when thinking on bone-tissue-engineering applications, it would be very useful if these microparticles are biodegradable and could be made to be bioactive. Microparticles with all those characteristics could be cultured together with adherent cells in appropriate bioreactors to form in vitro constructs that can then be used in tissue-engineering therapies. In this work, we have characterized the response of MC3T3-E1 pre-osteoblast cells to starch-based microparticles. We evaluated the adhesion, proliferation, expression of osteoblastic markers and mineralization of cells cultured at their surface. The results clearly show that MC3T3-E1 pre-osteoblast cells adhere to the surface of both polymeric and composite starch-based microparticles and express the typical osteoblastic marker genes. Furthermore, the cells were found to mineralize the extracellular matrix (ECM) during the culture period. The obtained results indicate that starch-based microparticles, known already to be biodegradable, bioactive and able to be used as carriers for controlled release applications, can simultaneously be used as carriers for cells. Consequently, they can be used as templates for forming hybrid constructs aiming to be applied in bone-tissue-engineering applications.

Biocompatible Materials↗

PubMed2005

Solution-mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells.

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSCs) on PLGA by promoting the formation of a calcium-phosphate-rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells was tested. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSCs, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell-derived factors that promoted distant MSC differentiation. Thus PLGA-BG composites demonstrate significant potential as a bone-replacement material.

Adult↗

PubMed2005

Effect of biomaterial surface properties on fibronectin-alpha5beta1 integrin interaction and cellular attachment.

The ability of fibronectin (Fn) to mediate cell adhesion through binding to alpha(5)beta(1) integrins is dependent on the conditions of its adsorption to the surface. Using a model system of alkylsilane SAMs with different functional groups (X=OH, COOH, NH(2) and CH(3)) and an erythroleukemia cell line expressing a single integrin (alpha(5)beta(1)), the effect of surface properties on the cellular adhesion with adsorbed Fn layers was investigated. (125)I-labeled Fn, a modified biochemical cross-linking/extraction technique and a spinning disc apparatus were combined to quantify the Fn adsorption, integrin binding and adhesion strength, respectively. This methodology allows for a binding equilibrium analysis that more closely reflects cellular adhesion found in stable tissue constructs in vivo. Differences in detachment strength and integrin binding were explained in terms of changes in the adhesion constant (psi, related to affinity) and binding efficiency of the adsorbed Fn for the alpha(5)beta(1) integrins (CH(3) approximately NH(2)<COOH approximately OH) and the resulting average bond strength. Fn interacted more strongly with alpha(5)beta(1) integrins when adsorbed on COOH vs. OH surfaces suggesting that negative charge may be a critical component of inducing efficient cellular adhesion. As evident by the low psi values, Fn adsorbed on NH(2) and CH(3) surfaces interacted inefficiently with alpha(5)beta(1) integrins and also possessed significant non-specific components to adhesion. Lastly, comparison of cellular adhesion to Fn adsorbed onto smooth and rough surfaces showed that nano-scale roughness altered cellular adhesion by increasing the surface density of adsorbed Fn.

Biocompatible Materials↗

PubMed2005

Excretion of resorption products from bioactive glass implanted in rabbit muscle.

Bioactive glass granules were implanted in the paraspinal muscle of rabbits to determine the pathway of the silicon released from bioactive glass in vivo. We traced and quantified the silicon released by obtaining 24-h urine and blood samples for up to 6 months after implantation. Furthermore, local muscle tissue as well as the following organs were resected for chemical and histopathological analyses: brain, heart, kidney, liver, lung, lymph nodes, spleen, and thymus. The urinary silicon of the group with implanted granules was significantly higher than in the sham-operated, control group. The average excretion rate was 2.4 mg/day, and as such, 100% of the implanted silicon was excreted in 19 weeks. No elevated concentrations of silicon were found at the implant sites or in the other organs at sacrifice, that is, 24 weeks. The histological appearance of all organs was normal throughout. The concentrations of silicon measured in the urine were well below saturation and because no significant increase in silicon was found in any organ, including kidney, the increased silicon excretion rate was within the physiological capacity of rabbits. Therefore, it can be concluded that the resorbed silica gel is harmlessly excreted in soluble form through the urine.

Animals↗

PubMed2005

A three-dimensional nonlinear finite element analysis of the mechanical behavior of tissue engineered intervertebral discs under complex loads.

The use of tissue-engineering method holds great promise for treating degenerative disc disease [Gan JC, Ducheyne P, Vresilovic E, Shapiro IM. J Biomed Mater Res 2000; 51(4): 596-604]. This concept typically implies that nucleus pulposus (NP) cells are seeded on a scaffold, while the NP tissue is regenerated. Such hybrid implant is inserted into the host intervertebral disc. Because the success of a tissue engineering approach depends on maintenance or restoration of the mechanical function of the intervertebral disc, it is useful to study the initial mechanical performance of the disc after implantation of the hybrid. A three-dimensional finite element model (FEM) of the L2-L3 disc-vertebrae unit has been analyzed. The model took into account the material nonlinearities and it imposed different and complex loading conditions. In this study, we validated the model by comparison of its predictions with several sets of experimental data; we determined the optimal Young's modulus as well as the failure strength for the tissue-engineered scaffold under different loading conditions; and we analyzed the effects of implanted scaffold on the mechanical behavior of the intervertebral disc. The results of this study suggest that a well-designed tissue-engineered scaffold preferably has a modulus in the range of 5-10 MPa and a compressive strength exceeding 1.67 MPa. Implanted scaffolds with such properties can then achieve the goal of restoring the disc height and distributing stress under different loading conditions.

Biomechanical Phenomena↗

PubMed2005

In vivo tissue response to resorbable silica xerogels as controlled-release materials.

Biodegradable, controlled-release carrier materials with non-toxic degradation products are valuable for local delivery of biologically active molecules. Previously, it was shown that room-temperature processed silica sol-gels (or xerogels) are porous, resorbable materials that can release molecules of various sizes in a controlled, time dependent manner. Previous in vitro studies also demonstrated benefits of silica xerogels as controlled-release materials for the treatment of bone infections. Herein the tissue and cell response to xerogels is documented using a subacute implantation procedure. The tissue response was correlated to composition, surface properties, resorption rate and incorporation of the antibiotic vancomycin. Ca- and P-free and Ca- and P-containing xerogels, with and without apatite (AP) surface, were used. Xerogels were implanted either as discs in a subcutaneous site, or as granules in the iliac crest of New Zealand white rabbits. The samples with surrounding tissue were retrieved after 2 and 4 weeks of implantation. Silica xerogels implanted either as discs subcutaneously or as granules in the iliac crest showed a favorable tissue response. The granules, either with or without Ca and P content, gradually resorbed over time. The resorption was accompanied by extensive trabecular bone growth and a minimal inflammatory response. Ca- and P-containing granules with an AP-surface layer showed a slower resorption rate and more extensive new bone growth than those without AP layer. Among AP-coated granules, those with incorporated vancomycin showed the most favorable tissue response. The present in vivo data together with prior in vitro data suggest that these xerogels have potential as controlled-release materials for the treatment of bone infections and as carrier materials for a variety of other applications.

Absorbable Implants↗

PubMed2005

The effect of non-specific interactions on cellular adhesion using model surfaces.

The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH(2)), and methyl (CH(3)) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18 h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH(3) < COOH approximately epoxide << NH(2). The NH(2) SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.

Adsorption↗

PubMed2005

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic-co-glycolic acid)/bioactive glass substrate for tissue engineering.

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.

Animals↗

PubMed2004

Fibronectin adsorption on surface-activated poly(dimethylsiloxane) and its effect on cellular function.

This article reports that surface modification of poly(dimethylsiloxane) (PDMS) influences fibronectin (Fn) adsorption and enhances cell attachment. Controlled adsorption of Fn on chemically activated polymer substrates is known to influence cellular function. Thin films of PDMS were spun cast on silicon wafers to obtain homogeneous and molecularly smooth surfaces. The films were made hydrophilic by exposure to ultraviolet ozone activation (PDMS*). The films then were characterized by contact angle goniometry, ellipsometry, atomic force microscopy (AFM), Rutherford backscattering spectrometry and X-ray photoelectron spectroscopy. Contact angle measurements indicated higher hydrophobicity of the nonactivated PDMS substrates than PDMS*. AFM scans of the substrates indicated higher surface roughness of PDMS* (Ra = 0.55 nm) than PDMS (Ra = 0.25 nm). Although Fn surface density (Gamma) was slightly higher on PDMS than on PDMS*, due to hydrophobic interactions between substrate and Fn, cell function was greatly enhanced on the Fn-coated PDMS* (PDMS*-Fn) than on PDMS (PDMS-Fn). Higher attachment of MC3T3-E1 osteoblast-like cells was observed on PDMS*-Fn than on PDMS-Fn. Moreover, cell spreading and cytoskeleton organization after 72 h was clearly favored on the Fn-coated PDMS* surfaces.

Actins↗

PubMed2004

Apoptosis and survival of osteoblast-like cells are regulated by surface attachment.

We tested the hypothesis that RGDS peptides regulate osteoblast survival in culture. Osteoblast-like MC3T3-E1 cells were allowed to attach to RGDS peptides that had been tethered to a silicone surface utilizing a previously described grafting technique. The RGDS-modified surface caused up-regulation of alpha(v)beta(3) integrin. We noted that there was an increase in expression of activated focal adhesion kinase and activated Akt. There was no change in the expression level of the anti-apoptotic protein Bcl-2, the pro-apoptotic protein Bad, or the inactivated form of Bad, pBad. Attachment to the RGDS-treated membrane completely abolished apoptosis induced by staurosporine, the Ca(2+).P(i) ion pair, and sodium nitroprusside. However, the surface modification did not interfere with apoptosis mediated by the free RGDS peptide or serum-free medium. When the activity of the phosphatidylinositol 3-kinase pathway was inhibited, RGDS-dependent resistance to apoptosis was eliminated. These results indicated that the binding of cells to RGDS abrogated apoptosis via the mitochondrial pathway and that the suppression of apoptosis was dependent on the activity of phosphatidylinositol 3-kinase.

3T3 Cells↗

PubMed2003

Intervertebral disc tissue engineering I: characterization of the nucleus pulposus.

The characteristics of the nucleus pulposus cells from adult rabbits maintained in in vitro cultures were described in another study. Herein, the authors provide a parallel profile of adult rabbit nucleus pulposus in situ, therefore allowing direct comparisons between in vitro and in situ investigations. Nucleus pulposus specimens from adult rabbits were evaluated using biochemical and immunohistochemical morphologic techniques. The nucleus pulposus from adult rabbits contained cell clusters embedded in proteoglycan-collagen matrix. The cells exhibited a well-defined Golgi system, an extensive endoplasmic reticulum, and a complex vesicular system filled with beaded structures (proteoglycans). Neither necrotic nor apoptotic cells were evident. There was a lack of mitochondria. The extensive extracellular matrix contained amorphous, beaded, and fibrillar components. The fibrillar banding was indicative of Type VI collagen. The nucleus pulposus of adult rabbits expressed aggrecan, collagen Type I and Type II, and CD44, but not collagen Type X and displayed low alkaline phosphatase activity.

Animals↗

PubMed2003

Intervertebral disc tissue engineering II: cultures of nucleus pulposus cells.

The main objective of the current investigation was to regenerate cells of the nucleus pulposus without loss of phenotype. Nucleus pulposus cells were isolated from intervertebral discs from adult rabbits, grown in monolayer culture, and then maintained as a micromass pellet in tube culture. The specimens were evaluated by transmission and light microscopy, reverse transcriptase polymerase chain reaction, and immunohistochemistry. Nucleus pulposus cells proliferated in monolayer culture. When almost confluent, the cells were transferred to a tube and sedimented to form a pellet. The cells reverted to a rounded configuration and formed cell nests surrounded by extensive extracellular matrix, similar to that seen in vivo. These cells did not proliferate. Similar to that observed in situ, cells in pellet culture also expressed aggrecan, CD44, collagen Type II, and collagen Type I, but not collagen Type X, and had low alkaline phosphatase activity. The results of the investigation indicated that nucleus pulposus cells grown in monolayer culture might revert to their original characteristics when transferred to an environment that allows three-dimensional growth, such as upon implantation, a one-step approach. The results also indicated that the two-stage culture procedure might provide an expedient technique to regenerate nucleus pulposus tissue for disc repair.

Animals↗

PubMed2002

Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2.

Attempts to study the biology of the nucleus pulposus have been limited in scope due to the low rates of cell proliferation, difficulties in maintaining viable disc cells in culture and the absence of a clearly defined phenotype. The major objective of this communication is to construct a phenotypic signature for cells of the nucleus pulposus that is based on the hypothesis that in response to restriction on oxygen and nutrient flux, there is expression of HIF-1, GLUT-1 and MMP-2. Nucleus pulposus, as well as annulus fibrosus and cartilage of the vertebral end plates, was collected from rat spinal units. Western blot analysis and immunohistochemistry clearly showed that there was a significant level of expression of the HIF-1 beta isoform in the nucleus pulposus; HIF-1 beta was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1 beta, HIF-1 alpha was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1 alpha immunohistochemically in the nucleus pulposus; however, the staining was light and diffuse. Cells of the nucleus pulposus expressed GLUT-1; in contrast, when probed by Western blot analysis the annulus and cartilage were negative for this protein. Western blot analysis also showed that in the nucleus pulposus the level of MMP-2 was high when compared to the adjacent tissues. We suggest that the differential expression of the two HIF isoforms, and GLUT-1 and MMP-2, provides a phenotypic signature that permits cells of the nucleus pulposus to be distinguished from neighboring tissues. Moreover, the presence of these isoforms provides evidence that cells of the disc respond to hypoxia and nutrient stress by upregulating stress-responsive genes.

Animals↗

PubMed2002

Silicon excretion from bioactive glass implanted in rabbit bone.

Bioactive glass granules were implanted in the tibiae of rabbits in order to determine the pathway of the silicon released from bioactive glass in vivo. We traced and quantified the silicon released by obtaining 24-h urine samples and blood samples for up to 7 months after implantation. Bone tissue as well as the following organs were resected for chemical and histopathological analyses: kidney, liver, lung, lymph nodes, and spleen. The urinary silicon of the implanted group was significantly higher than in the control group. From the data, the calculated average excretion rate was approximately 1.8 mg/day, and as such, the amount of implanted silicon was excreted within statistical bounds in 24 weeks. At this point, only elevated concentrations of silicon were found at the implant site and not in the other organs. The concentrations of silicon measured in the urine were well below saturation. Since no significant increase in silicon was found in any of the organs including the kidney, the increased silicon excretion rate was within the physiological capacity of rabbits. Therefore, it can be concluded that the resorbed silica gel is harmlessly excreted in soluble form through the urine.

Animals↗

PubMed2002

In vitro bioactivity and degradation behavior of silica xerogels intended as controlled release materials.

Room temperature-processed silica-based sol-gel, termed silica xerogel, is a novel type of controlled release material. As shown previously, these materials are porous, degradable and can release biologically functional molecules in a controlled manner. It was also demonstrated that these materials are biocompatible in vivo. Herein we report on the ability of silica-based xerogels to form a bioactive, apatite-like (AP) surface and the effect of AP surface on the xerogel stability in vitro. Formation of a crystalline, carbonated AP (c-AP) was found on all silica xerogels studied, with or without Ca- and P-oxides. Calcium and phosphate (Ca-P) free xerogels showed long times to Ca-P precipitation and to formation of a detectable AP-layer (up to 2 weeks). In contrast, the times to precipitation were reduced by 2-3 orders of magnitude, and the c-AP layer was formed within 24 h on all Ca-P containing xerogels. Mechanisms of the c-AP formation on these xerogels were similar to those typical for Ca-P based ceramics: dissolution of calcium and phosphate ions, solution oversaturation with respect to AP and subsequent precipitation of bone-like minerals. The presence of the c-AP surface film produced a remarkable surface stabilizing effect: the rates and the amounts of Si release were significantly reduced in comparison to those for xerogels without the film. This evidence of in vitro bioactivity and controlled degradation, combined with previous in vitro and in vivo reports, suggests that silica xerogel is a promising controlled release material.

Apatites↗

PubMed2002

Bioactive, degradable composite microspheres: effect of filler material on surface reactivity.

Composite microspheres with two different fillers were developed using a solid-in-oil-in-water (s/o/w) emulsion solvent removal method. Two types of bioactive ceramic powders, specifically calcium hydroxyapatite (HA) and modified bioactive glass (MBG), were incorporated into degradable poly(lactic acid) (PLA) polymer matrix to form composite microspheres. For each filler material, microspheres with three different weight ratios of filler material to polymer, namely, 1: 1, 1: 3, and 1: 9, were synthesized. In vitro immersion using simulated physiological fluid (SPF) was employed to evaluate the surface reactivity of the microspheres. SEM analysis revealed that after a 14-day immersion the surface of the microspheres containing 50% MBG was fully transformed into a bone-like apatite. In contrast, a limited number of mineral nodules were present on the surface of microspheres containing HA. The solution chemical analyses performed to determine changes of Ca, P, and Si concentrations as a function of the immersion time showed that the ion concentration profiles were similar for all microspheres, except the [Si] profile. A higher Si concentration was detected in the SPF immersed with MBG-containing microspheres. These data show that the MBG filler significantly enhances the surface reactivity of the composite microspheres. This observation enables us to conclude that the composite MBG-containing microspheres are the preferable microspheres for three-dimensional bone tissue engineering.

Biocompatible Materials↗