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VASCUPLUG Résumé de rapport

Project ID: 13811
Financé au titre de: FP6-NMP
Pays: Germany

Periodic Report Summary - VASCUPLUG (Bioreactive composite scaffold design for improved vascular connexion of tissue-engineered products)

The ultimate aim of the VASCUPLUG project was to develop a novel Three-dimensional (3D) scaffold structure for improved vascularisation of tissue-engineered products. Application of intelligent biomaterials (bioresorbable stimuli-sensitive polymers) and incorporation of bioactive substances (e.g. growth factors) would enhance a structured vascularisation of the tissue-engineered constructs by gradually opening inserted microchannels for vessel ingrowth into cell-seeded polymeric scaffolds. Furthermore, the mechanism of induction of secondary angiogenesis by monocytes could be used to promote vascularisation. The signal for the stimulus-sensitive polymer to act was intended to be a change in pH associated with malnutrition of cells. The use of angiogenic factors in promoting vascularisation of tissue-engineered constructs so far was performed by a rather isotropic distribution of factors in the scaffolds preventing the build-up of a gradient of bioactive substances for directed cell growth (angiogenesis). The composite scaffold giving rise to evolving vessels intended to allow the vascular connexion to the surrounding tissue in the course of wound healing.

Work package (WP) one concerned the designing components (foam, tubular structures and pH sensitive gel) for the novel bioreactive composite scaffold. For the fabrication of the foam and tubular structures poly-(L-lactide-co-glycolide) (LGA) with 15 % by weight glycolide from Boehringer Ingelheim was used due to the sufficient degradation in short studies and no cytotoxicity in agar overlay experiments. The pH sensitive hydrogels were prepared by free radical polymerisation and their pH dependent swelling behaviour was tested under cell culture conditions in vitro. Since polysaccharides were already used as biocompatible and biodegradable material, the synthesis of the hydrogels based on sodium alginate and chitosan was established. For the construction of the composite scaffold the tubular structure would be formed by dip coating a hydrogel fibre. Then a highly porous structure would be foamed around the filled fibre by TIPS.

The work performed in WP2 focused on the biocompatibility examination of polymers processed in WP1. After tuning biocompatibility test methods to the requirement of the project evaluations on flat and 3D materials were undertaken. Furthermore, a bioreactor system was developed to test the functional components of the VASCUPLUG unit.

WP3 involved the synthesis of bioactive substances and adequate delivery carriers. More specifically, it involved the design of novel drugs to control angiogenesis, with main focus on angiogenesis-promoting substances, to be included in the final composite structure. These drugs were intended to promote angiogenesis and vascularisation of tissue. An additional objective of this WP was to provide an adequate delivery carrier of selected proangiogenic compounds. On the other hand, WP4 aimed to evaluate the action of these drugs compared to commercially available substances.

The objectives of WP5 could be described as being contributions to improve vascularisation in the 3D tissue via the induction of angiogenic processes by the action of monocytes / macrophages. It is well established, that monocytes / macrophages can stimulate the process of neovascularisation. This aspect was included in order to further improve vascular morphogenesis by the addition of monocytes in the composite structure taking into account the physiological conditions during the in vitro period of culturing, as well as during implantation. To study these types of processes two in vitro models able to mimic different stages of angiogenic processes were established.

In WP6 investigations on processing methods (extrusion, CO2 foaming, thermally induced phase separation, non-solvent induced phase inversion) for the polymeric components of the composite were performed and a decision was made about the implemented foaming method and processing of the other structures in order to obtain fabricated structures with the desired dimensions, pore sizes and porosity.

Experimental work in WP7 characterised the effect of sterilisation on degradation and mechanical performance of one polymeric component of the VASCUPLUG unit. Details on optimum sterilisation and design rules for the degradation behaviour were developed.

WP8 was intended to represent the proof of concept and the implantation into an animal. A bioreactor system adapted to the conditions/components that were developed at that time. Preparatory in vitro culturing tests are possible. The final proof of concept by implantation was intended to be realised with the complete composite device only.

In WP9, the project coordination and management was performed by the project coordinator from GKSS according to the requirements of successful project outcome: meetings were organised, communication between the consortium partners and the scientific officer of the European Commission (EC) maintained, reports were prepared with strong contribution of the consortium.

Starting from a decision made by the consortium concerning the choice of polymer materials for foam and tubular structures, as well as the parameters of the structures, such as mechanical behaviour, porosity, pore size, diameter, interconnectivity of pores and degradation, behaviour samples of polymers for the bulk components of the composite were processed into test specimen and characterised mechanically and with respect to degradation and in vitro biocompatibility, also under influence of several sterilisation procedures.

A special stimuli-sensitive polymeric material was developed to be used as component of the VASCUPLUG unit reacting on a signal from the patient's body. Principal feasibility was shown, as well as temperature-dependence.

A number of relevant bioactive substances, angiogenesis modifying agents, was designed, synthesised, characterised and tested for their angiogenic activities. These new molecules fell into two groups, peptide or peptidomimetic agonists of Vascular endothelial growth factor (VEGF) family and peptides or peptidomimetics stabilising Hypoxia inducing factor (HIF) by prolyl hydroxilase inhibitors.

In this context CRC developed a VEGF agonistic small peptide and a presumable HIF stabilising small molecular weight compound (prolyl hydroxylase domain-containing enzymes - PHD - inhibitor). Both compounds stimulate angiogenesis in vitro and in vivo. The VEGF-ENZ-7-fragment (SHA-1-78) which comprises in one molecule two of the loops responsible for the angiogenic activity of VEGF-ENZ-7 molecule, showed 3 and 7 fold new vessel formation compared to control in 10 and 100 mg/kg doses, respectively. The small protected peptide fragment (SHA-2-22) which has structural similarities to factors (e.g. oxoglutarate) which are able to influence the HIF pathway through the iron co-ordination motif, showed 3 and 10 fold new vessel formation compared to control in 1 and 10 mg/kg doses, respectively. This new compounds have the potential as new angiogenesis influencing agents. A patent application was filed comprising these results and preliminary data were also published at the 43JPS/PEM4.

Two different families of nanoparticles, based on Poly lactic-co-glycolic acid (PLGA) or polysaccharides, were designed and characterised with regard to their capacity for the encapsulation and delivery of commercially available growth factors and also a novel drug synthesised by CRC. Additionally, these nanoparticles exhibited a very low cytotoxicity and an adequate haemocompatibility.

Establishment of functional and stable collaterals in the ischemic myocardium is crucial to restore cardiac function after myocardial infarction. It was shown that only dual delivery of a combination of angiogenic and arteriogenic factors to the ischemic myocardium could significantly re-establish stable collateral networks and improve myocardial perfusion and function. A combination of fibroblast growth factor (FGF)-2 with PDGF-BB, two factors primarily targeting endothelial cells and vascular smooth muscle cells, remarkably promotes myocardial collateral growth and stabilises the newly formed collateral networks, which significantly restores myocardial perfusion and function. Using various members of the PDGF family together with FGF-2 in an angiogenesis assay, KI demonstrated that PDGF-receptor-alpha (PDGFR) is mainly involved in angiogenic synergism, whereas PDGFR-beta mediates vessel stability signals. The findings provided conceptual guidelines for the clinical development of proangiogenic/arteriogenic factors for the treatment of ischemic heart disease.

The parameters for monocyte's promotion of angiogenesis to be used in the VASCUPLUG device like the adequate cell ratio (human microvascular endothelial cells/human peripheral blood monocytes) were studied in vitro for their effects on the induction of vascular morphogenesis. The results indicated that the presence of monocytes / macrophages indeed affect the onset of angiogenesis in the in vitro setting. To mimic physiological conditions during the in vitro culturing period the ratio of endothelial cells / monocytes should not exceed 1/5 to 1/10 to result in angiogenic effects.

Experimental work in WP7 characterised the effect of sterilisation on degradation and mechanical performance of porous structures developed in WP6. Optimum sterilisation method for the polymers considered was selected. Dry and wet DMA techniques and associated design rules for assessing degradation and the mechanical performance of these isotropic anisotropic solid and porous structures were developed. Acceptable degradation profile of the degradable structures was completed. Additional work included fabricating porous isotropic structures of a different polymer with optimum window sizes for initial studies in WP2 and supplying phosphate glass fibres to WP6.

Informations connexes

Reported by

Max-Planck-Strasse, 1
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