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See detailFluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates.
Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet ULg et al

in Biomaterials (2013), 34(4), 922-9

Molecular oxygen is a main regulator of various cell functions. Imaging methods designed as screening tools for fast, in situ, 3D and non-interfering measurement of oxygen tension in the cellular ... [more ▼]

Molecular oxygen is a main regulator of various cell functions. Imaging methods designed as screening tools for fast, in situ, 3D and non-interfering measurement of oxygen tension in the cellular microenvironment would serve great purpose in identifying and monitoring this vital and pivotal signalling molecule. We describe the use of dual luminophore oxygen sensitive microbeads to measure absolute oxygen concentrations in cellular aggregates. Stable microbead integration, a prerequisite for their practical application, was ensured by a site-specific delivery method that is based on the interactions between streptavidin and biotin. The spatial stability introduced by this method allowed for long term measurements of oxygen tension without interfering with the cell aggregation process. By making multiple calibration experiments we further demonstrated the potential of these sensors to measure local oxygen tension in optically dense cellular environments. [less ▲]

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See detailEctopic bone formation by 3D porous calcium phosphate-Ti6Al4V hybrids produced by perfusion electrodeposition.
Chai, Yoke Chin; Kerckhofs, Greet ULg; Roberts, Scott J. et al

in Biomaterials (2012), 33(16), 4044-58

Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised ... [more ▼]

Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised porous calcium phosphate-Ti6Al4V (CaP-Ti) hybrids were produced by perfusion electrodeposition, and the in vitro and in vivo biological performances were evaluated using human periosteum derived cells (hPDCs). By applying various current densities at the optimised deposition conditions, CaP coatings with sub-micrometer to nano-scale porous crystalline structures and different ion dissolution kinetics were deposited on the porous Ti6Al4V scaffolds. These distinctive physicochemical properties caused a significant impact on in vitro proliferation, osteogenic differentiation, and matrix mineralisation of hPDCs. This includes a potential role of hPDCs in mediating osteoclastogenesis for the resorption of CaP coatings, as indicated by a significant down-regulation of osteoprotegerin (OPG) gene expression and by the histological observation of abundant multi-nucleated giant cells near to the coatings. By subcutaneous implantation, the produced hybrids induced ectopic bone formation, which was highly dependent on the physicochemical properties of the CaP coating (including the Ca(2+) dissolution kinetics and coating surface topography), in a cell density-dependent manner. This study provided further insight on stem cell-CaP biomaterial interactions, and the feasibility to produced bone reparative units that are predictively osteoinductive in vivo by perfusion electrodeposition technology. [less ▲]

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See detailMechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers.
Chai, Y. C.; Roberts, S. J.; Desmet, E. et al

in Biomaterials (2012)

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for ... [more ▼]

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (muCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials. [less ▲]

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See detailThe combined bone forming capacity of human periosteal derived cells and calcium phosphates.
Roberts, Scott J; Geris, Liesbet ULg; Kerckhofs, Greet ULg et al

in Biomaterials (2011), 32(19), 4393-405

Current knowledge suggests that the periosteum, a fibrous tissue which covers the surface of all bones, contains a population of progenitor cells which mediate the repair of bone defects. In an effort to ... [more ▼]

Current knowledge suggests that the periosteum, a fibrous tissue which covers the surface of all bones, contains a population of progenitor cells which mediate the repair of bone defects. In an effort to optimise the utilisation of this source of cells for bone engineering, herein we describe the rational selection of calcium phosphate (CaP) containing materials, based on biomaterial properties, and evaluation of their combined bone forming capacity. Five different commercially available orthopaedic 3D matrices composed of CaP particles in an open collagen network (NuOss, CopiOs, Bio-Oss((R)), Collagraft and Vitoss((R))) were evaluated in vitro and in vivo with human periosteal derived cells (hPDCs). It was found that the cell-material combinations behaved quite differently in vivo, despite apparent in vitro similarities in gene expression profiles. Bone formation was highest within the NuOss/hPDC implant at 13.03%, which also contained the highest incidence of bone marrow formation. The bone formed in this implant was chimeric with approximately 65% originating from implanted cells. Upon analysis of human specific gene expression, although it was found that predominantly osteogenic differentiation was observed within NuOss/hPDC implants, a lesser induction of chondrogenic genes was also observed. The formation of a cartilage intermediate was confirmed by histology. Additionally the NuOss/hPDC implant integrated into the mouse environment with apparent active scaffold resorption. This study demonstrates the importance of matching a cell support/biological matrix with a cell type and subsequently has outlined parameters which can be used for the rational selection of biomaterials for bone engineering. [less ▲]

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See detailDevelopment of pH–responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery
Dehousse, Vincent ULg; Garbacki, Nancy ULg; Colige, Alain ULg et al

in Biomaterials (2010), 31

RNA interference-based therapies are dependent on intracellular delivery of siRNA. The release of siRNA from the endosomal compartment may be a rate limiting step in the transfection process. The purpose ... [more ▼]

RNA interference-based therapies are dependent on intracellular delivery of siRNA. The release of siRNA from the endosomal compartment may be a rate limiting step in the transfection process. The purpose of this study was to produce pH–responsive nanocarriers made of trimethylchitosan (TMC). To this end, pHsensitive methacrylic acid (MAA) copolymer was added to TMC–siRNA formulations. Four different TMCs associated or not with MAA were evaluated as siRNA carriers. Nanoparticles were characterized in terms of size, surface charge, morphology and interaction with siRNA. A swelling behaviour due to a decrease in pH was observed and was found to be dependent on MAA content in the complexes. In vitro experiments aimed at evaluating how the capacity of the nanocarriers to transfect siRNA in L929 cells was affected by MAA content. Confocal microscopy experiments showed that fluorescent MAA-containing particles exhibit a different distribution pattern inside the cells comparing to their counterpart without this pH-sensitive polymer. Transfection efficiency was investigated by RhoA mRNA expression inhibition. MAA–TMC–siRNA complexes were able to transfect L929 cells with greater efficiency than corresponding TMC–siRNA complexes. This study gives an insight into the opportunity of pH-sensitive nanocarriers for siRNA delivery. Such formulations may represent an attractive strategy to improve endosomal escape of siRNA. [less ▲]

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See detailTowards a quantitative understanding of oxygen tension and cell density evolution in fibrin hydrogels
Demol, Jan; Lambrechts, Dennis; Geris, Liesbet ULg et al

in Biomaterials (2010), 32(1), 107-118

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an ... [more ▼]

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cellseeded hydrogels. [less ▲]

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See detailPoly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord
Hurtado, Andres; Moon, Lawrence D F; Maquet, Véronique et al

in Biomaterials (2006), 27(3), 430-442

Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with ... [more ▼]

Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) Cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 It, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at I week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

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See detailPorous poly(α-hydroxyacid)/Bioglass® composite scaffolds for bone tissue engineering. I: preparation and in vitro characterisation
Maquet, Véronique; Boccaccini, Aldo R.; Pravata, Laurent et al

in Biomaterials (2004), 25(18), 4185-4194

Highly porous composites scaffolds of poly-D,L-lactide (PDLLA) and poly(lactide-co-glycolide) (PLGA) containing different amounts (10, 25 and 50wt%) of bioactive glass (45S5 bioglass®) were prepared by ... [more ▼]

Highly porous composites scaffolds of poly-D,L-lactide (PDLLA) and poly(lactide-co-glycolide) (PLGA) containing different amounts (10, 25 and 50wt%) of bioactive glass (45S5 bioglass®) were prepared by thermally induced solid-liquid phase separation (TIPS) and subsequent solvent sublimation. The addition of increasing amounts of bioglass® into the polymer foams decreased the pore volume. Conversely, the mechanical properties of the polymer materials were improved. The composites were incubated in phosphate buffer saline at 37°C to study the in vitro degradation of the polymer by measurement of water absorption, weight loss as well as changes in the average molecular weight of the polymer and in the pH of the incubation medium as a function of the incubation time. The addition of bioglass®to polymer foams increased the water absorption and weight loss compared to neat polymer foams. However, the polymer molecular weight, determined by size exclusion chromatography, was found to decrease more rapidly and to a larger extent in absence of bioglass®. The presence of the bioactive filler was therefore found to delay the degradation rate of the polymer as compared to the neat polymer foams. Formation of hydroxyapatite on the surface of composites, as an indication of their bioactivity, was recorded by EDXA, X-ray diffractometry and confirmed by Raman spectroscopy. [less ▲]

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See detailFreeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord
Patist, Carla M; Borgerhoff Mulder, Masha; Gautier, Sandrine et al

in Biomaterials (2004), 25(9), 1569-1582

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and ... [more ▼]

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response. [less ▲]

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See detailImage analysis of the axonal ingrowth into poly(D,L-lactide) porous scaffolds in relation to the 3-D porous structure
Blacher, Silvia ULg; Maquet, Véronique; Luyckx, Françoise ULg et al

in Biomaterials (2003), 24(6), 1033-1040

Porous polymer scaffolds are promising materials for neural tissue engineering because they offer valuable three-dimensional (3D) supports for the in vitro and in vivo axonal growth and tissue expansion ... [more ▼]

Porous polymer scaffolds are promising materials for neural tissue engineering because they offer valuable three-dimensional (3D) supports for the in vitro and in vivo axonal growth and tissue expansion. At the time being, how the in vivo neuronal cell development depends on the scaffold 3-D architecture is unknown. Therefore, scanning electron micrographs of longitudinal sections of porous polylactide scaffolds and immunohistological sections of these scaffolds after implantation and neurofilament staining have been studied by image analysis. Pore orientation and axonal ingrowth have been investigated by spectral analysis on gray level SEM images. Binary image processing has been carried out and the binary images have been studied by spectral analysis in order to estimate the possible effect of the image noise on the real pattern. In addition to axonal orientation, density and length distribution of the regenerated axons into the polymer scaffold have been measured. Dependence of the axonal ingrowth on the 3D-polymer scaffold has been discussed on the basis of the collected data. (C) 2002 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailDevelopment and in vitro characterisation of novel bioresorbable and bioactive composite materials based on polylactide foams and Bioglass (R) for tissue engineering applications
Roether, J. A.; Boccaccini, Aldo R.; Hench, L. L. et al

in Biomaterials (2002), 23(18), 3871-3878

Bioactive and bioresorbable composite materials were fabricated using macroporous poly(DL-lactide) (PDLLA) foams coated with and impregnated by bioactive glass (Bioglass®) particles. Stable and ... [more ▼]

Bioactive and bioresorbable composite materials were fabricated using macroporous poly(DL-lactide) (PDLLA) foams coated with and impregnated by bioactive glass (Bioglass®) particles. Stable and homogeneous Bioglasss coatings on the surface of PDLLA foams as well as infiltration of Bioglass® particles throughout the porous network were achieved using a slurry-dipping technique in conjunction with pre-treatment of the foams in ethanol. The quality of the bioactive glass coatings was reproducible in terms of thickness and microstructure. Additionally, electrophoretic deposition was investigated as an alternative method for the fabrication of PDLLA foam/Bioglass® composite materials. In vitro studies in simulated body fluid (SBF) were performed to study the formation of hydroxyapatite (HA) on the surface of PDLLA/Bioglass® composites. SEM analysis showed that the HA layer thickness rapidly increased with increasing time in SBF. The high bioactivity of the PDLLA foam/Bioglasss composites indicates the potential of the materials for use as bioactive, resorbable scaffolds in bone tissue engineering. [less ▲]

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See detailEffect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.
Kong, Xiangqing; Grabitz, R. G.; van Oeveren, W. et al

in Biomaterials (2002), 23(8), 1775-83

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this ... [more ▼]

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications. [less ▲]

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See detailPoly(D,L-lactide) foams modified by poly(ethylene oxide)-block-poly(D,L-lactide) copolymers and a-FGF: in vitro and in vivo evaluation for spinal cord regeneration
Maquet, Véronique; Martin, Didier ULg; Scholtes, Félix ULg et al

in Biomaterials (2001), 22(10), 1137-1146

The first goal of this study was to examine the influence that poly(ethylene oxide)-block-poly(D,L-lactide) (PELA) copolymer can have on the wettability, the in vitro controlled delivery capability, and ... [more ▼]

The first goal of this study was to examine the influence that poly(ethylene oxide)-block-poly(D,L-lactide) (PELA) copolymer can have on the wettability, the in vitro controlled delivery capability, and the degradation of poly(D,L-lactide) (PDLLA) foams. These foams were prepared by freeze-drying and contain micropores (10 μm) in addition of macropores (100 μm) organized longitudinally. Weight loss, water absorption, changes in molecular weight, polymolecularity (Mw/Mn) and glass transition temperature ( Tg) of PDLLA foams mixed with various amounts of PELA were followed with time. It was found that 10 wt% of PELA increased the wettability and the degradation rate of the polymer foams. The release of sulforhodamine (SR) was compared for PDLLA and PDLLA-PELA foams in relation with the foam porosity. An initial burst release was observed only in the case of the 90:10 PDLLA/PELA foam. The ability of the foam of this composition to be integrated and to promote tissue repair and axonal regeneration in the transected rat spinal cord was investigated. After implantation of ca. 20 polymer rods assembled with fibrin-glue, the polymer construct was able to bridge the cord stumps by forming a permissive support for cellular migration, angiogenesis and axonal regrowth. [less ▲]

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