Véronique Maquet

Development and in vitro characterisation of novel bioresorbable and bioactive composite materials based on polylactide foams and Bioglass® for tissue engineering applications

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 Bioglass 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/Bioglass composites indicates the potential of the materials for use as bioactive, resorbable scaffolds in bone tissue engineering.

Polylactide macroporous biodegradable implants for cell transplantation. II. Preparation of polylactide foams by liquid-liquid phase separation

Potential of thermally induced phase separation as a porogen technique has been studied in an effort to produce a surgical implant suitable for cell transplantation. Emphasis has been placed on the liquid-liquid phase separation of solutions of amorphous poly DL-lactide and semicrystalline poly L-lactide in an 87/13 dioxane/water mixture. The related temperature/composition phase diagrams have been set up by turbidimetry, and the possible occurrence of a gel has been discussed. Freeze-drying of some phase-separated polylactide solutions can produce flexible and tough foams with an isotropic morphology. Interconnected pores of 1-10 microns in diameter are expected to result from the spinodal decomposition of the polylactide solutions with formation of co-continuous phases. Thermodynamics of the polymer/solvent pair has a decisive effect on the final macroporous foams, as shown by the dependence of their porosity, density, porous morphology, and mechanical behavior on molecular weight and crystallinity of polylactide and concentration of the original solutions. On the basis of the foam characteristics, potential of the liquid-liquid phase separation (spinodal decomposition) has been compared with the solid/liquid phase separation (solvent crystallization) as a porogen technique.

Bioresorbable and bioactive polymer/Bioglass® composites with tailored pore structure for tissue engineering applications

An overview about the development of porous bioresorbable composite materials for applications as scaffolds in tissue engineering is presented. A thermally induced phase separation method was developed to fabricate porous foam-like structures of poly(lactide-co-glycolide) (PLGA) containing bioactive glass particle additions (up to 50 wt.%) and exhibiting well-defined, oriented and interconnected porosity. The in vitro bioactivity and the degradability of the composite foams were investigated in contact with phosphate buffer saline (PBS). Weight loss, water absorption and molecular weight measurements were used to monitor the polymer degradation after incubation periods of up to 7 weeks in PBS. It was found that the presence of bioactive glass retards the polymer degradation rate for the time period investigated. The present results show a way of controlling the in vitro degradation behaviour of PLGA porous composite scaffolds by tailoring the concentration of bioactive glass.

Biodegradable and macroporous polylactide implants for cell transplantation: 1. Preparation of macroporous polylactide supports by solid-liquid phase separation

Abstract Freeze-drying of polylactide solutions in 1,4-dioxane has been studied as a way to produce microcellular foams. The thermally induced phase separation has been studied in relation to several processing and formulation parameters. The effects of polymer concentration, chain stereoregularity, polymer molecular weight and cooling rate have been investigated in connection with the porous morphology and the physicomechanical characteristics of the final foams. As a rule, bundles of channels are formed with a diameter of ≈100 μm. They have a preferential orientation that fits the cooling direction. A porous substructure (≈10 μm) is observed in the internal walls of the tubular macropores. Variations in this general porous morphology—and particularly in the porosity, density, solvent residue, mechanical resistance and degree of regularity in the spatial organization of pores—have been observed when polymer concentration in 1,4-dioxane and polylactide stereoregularity are changed. As expected, cooling rate has a strong effect on the foam morphology, which is essentially controlled by the solvent crystallization. Pores are nothing but the fingerprints of 1,4-dioxane crystallites.

Freeze-dried poly(d,l-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord

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.

Peripheral nerve regeneration using bioresorbable macroporous polylactide scaffolds

The ability of DRG-derived neurons to survive and attach onto macroporous polylactide (PLA) foams was assessed in vitro. The foams were fabricated using a thermally induced polymer-solvent phase separation. Two types of pore structures, namely oriented or interconnected pores, can be produced, depending on the mechanism of phase separation, which in turn can be predicted by the thermodynamics of the polymer-solvent pair. Coating of the porous foams with polyvinylalcohol (PVA) considerably improved the wettability of the foams and allowed for cell culture. The in vitro biocompatibility of the PVA-coated supports was demonstrated by measuring cell viability and neuritogenesis. Microscopic observations of the cells seeded onto the polymer foams showed that the interconnected pore networks were more favorable to cell attachment than the anisotropic ones. The capacity of highly oriented foams to support in vivo peripheral nerve regeneration was studied in rats. A sciatic nerve gap of 5-mm length was bridged with a polymer implant showing macrotubes of 100 microm diameter. At 4 weeks postoperatively, the polymer implant was still present. It was well integrated and had restored an anatomic continuity. An abundant cell migration was observed at the outer surface of the polymer implant, but not within the macrotubes. This dense cellular microenvironment was found to be favorable for axogenesis.

Novel bioresorbable and bioactive composites based on bioactive glass and polylactide foams for bone tissue engineering

Bioresorbable and bioactive tissue engineering scaffolds based on bioactive glass (45S5 Bioglass®) particles and macroporous poly(DL-lactide) (PDLLA) foams were fabricated. A slurry dipping technique in conjunction with pretreatment in ethanol was used to achieve reproducible and well adhering bioactive glass coatings of uniform thickness on the internal and external surfaces of the foams. In vitro studies in simulated body fluid (SBF) demonstrated rapid hydroxyapatite (HA) formation on the surface of the composites, indicating their bioactivity. For comparison, composite foams containing Bioglass® particles as filler for the polymer matrix (in concentration of up to 40 wt %) were prepared by freeze-drying, enabling homogenous glass particle distribution in the polymer matrix. The formation of HA on the composite surfaces after immersion in phosphate buffer saline (PBS) was investigated to confirm the bioactivity of the composites. Human osteoblasts (HOBs) were seeded onto as-fabricated PDLLA foams and onto PDLLA foams coated with Bioglass® particles to determine early cell attachment and spreading. Cells were observed to attach and spread on all surfaces after the first 90 min in culture. The results of this study indicate that the fabricated composite materials have potential as scaffolds for guided bone regeneration.

Bioresorbable and bioactive composite materials based on polylactide foams filled with and coated by Bioglass® particles for tissue engineering applications

Poly(DL-lactide) (PDLLA) foams and bioactive glass (Bioglass®) particles were used to form bioresorbable and bioactive composite scaffolds for applications in bone tissue engineering. A thermally induced phase separation process was applied to prepare highly porous PDLLA foams filled with 10 wt % Bioglass® particles. Stable and homogeneous layers of Bioglass® particles on the surface of the PDLLA/Bioglass® composite foams as well as infiltration of Bioglass® particles throughout the porous network were achieved using a slurry-dipping technique. The quality of the bioactive glass coatings was reproducible in terms of thickness and microstructure. In vitro studies in simulated body fluid (SBF) were performed to study the formation of hydroxyapatite (HA) on the surface of the PDLLA/Bioglass® composites, as an indication of the bioactivity of the materials. Formation of the HA layer after immersion in SBF was confirmed by X-ray diffraction and Raman spectroscopy measurements. The rate of HA formation in Bioglass®-coated samples was higher than that observed in non-coated samples. SEM analysis showed that the HA layer thickness rapidly increased with increasing time in SBF in the Bioglass®-coated samples. The high bioactivity of the developed composites suggests that the materials are attractive for use as bioactive, resorbable scaffolds in bone tissue engineering.

Poly(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.

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 microm) in addition of macropores (100 microm) 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 10wt% 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.

Olfactory ensheathing cells, olfactory nerve fibroblasts and biomatrices to promote long-distance axon regrowth and functional recovery in the dorsally hemisected adult rat spinal cord.

Cellular transplantation, including olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF), after experimental spinal cord injury in the rat has previously resulted in regrowth of severed corticospinal (CS) axons across small lesion gaps and partial functional recovery. In order to stimulate CS axon regrowth across large lesion gaps, we used a multifactorial transplantation strategy to create an OEC/ONF continuum in spinal cords with a 2-mm-long dorsal hemisection lesion gap. This strategy involved the use of aligned OEC/ONF-poly(D,L)-lactide biomatrix bridges within the lesion gap and OEC/ONF injections at 1 mm rostral and caudal to the lesion gap. In order to test the effects of this complete strategy, control animals only received injections with culture medium rostral and caudal to the lesion gap. Anatomically, our multifactorial intervention resulted in an enhanced presence of injured CS axons directly rostral to the lesion gap (65.0 +/- 12.8% in transplanted animals versus 13.1 +/- 3.9% in control animals). No regrowth of these axons was observed through the lesion site, which may be related to a lack of OEC/ONF survival on the biomatrices. Furthermore, a 10-fold increase of neurofilament-positive axon ingrowth into the lesion site as compared to untreated control animals was observed. With the use of quantitative gait analysis, a modest recovery in stride length and swing speed of the hind limbs was observed. Although multifactorial strategies may be needed to stimulate repair of large spinal lesion gaps, we conclude that the combined use of OEC/ONF and poly(D,L)-lactide biomatrices is rather limited.