Final Report Summary - VascuPlug (Bioreactive composite scaffold design for improved vascular connexion of tissue-engineered products)
The field of tissue engineering holds enormous promise in regenerative medicine. However, engineered tissues today present generally lack appropriate connexion to the vascular system of the surrounding tissue at the implantation site. Hence the tissues implanted suffer from malnutrition and low gas exchange leading to necrosis. To overcome this bottleneck the project wanted to develop a novel three-dimensional 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) will 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 might be used to improve vascularisation. The signal for the stimulus-sensitive polymer to act is 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 migration and / or cell growth. The composite scaffold giving rise to evolving vessels is intended to promote the vascular connexion to the surrounding tissue in the course of wound healing.
The complexity of requirements for a functional vascularisation of tissue-engineered products calls for a strong inter- and transdisciplinary cooperation of chemists, biologists, engineers, physicists and physicians depending on a pronounced cross-frontier collaboration.
The project consortium is composed of seven participants from six European countries - three research centres with high expertise in the field of material development / evaluation and angiogenesis, and four institutes at universities that have high competence in materials characterisation, haemocompatibility, drug design and encapsulation.
The project comprised several workpackages.
Workpackage (WP) 1 was concerned with choice of polymers for the components (foam, tubular structures and pH sensitive gel) of the novel bioreactive composite scaffold in the further context also called VASCUPLUG unit. An assessment of mechanical and degradation behaviour of polymers to be used for the composite structure was made. As a result three commercial polymers were chosen for the fabrication of the foam and tubular structures. For the bioreactive part, pH sensitive hydrogels were prepared by free radical polymerisation and complexation, their pH dependent swelling behaviour was tested in buffer and under cell culture conditions in vitro.
An extensive functionality testing of the polymers, as offered from WP1 and of the composite structures from WP6, was performed in workpackage 2. This examination involved biocompatibility, haemocompatibility, degradation behaviour of unmodified and modified polymers before and after further processing steps (e.g. foaming) and sterilisation processes (WP7). The approach to identify the suitable polymeric materials for each component of the VASCUPLUG unit was accomplished with these biocompatibility tests since each further processing step changes the properties of the materials possibly influencing the reaction of biological material in contact with them.
Workpackage 3 involved the synthesis of bioactive substances and adequate delivery carriers. More specifically, the design of novel drugs to control angiogenesis (CRC) with focus on angiogenesis-promoting substances was envisaged. The second objective of this WP was to provide an adequate delivery system / carrier of selected pro-angiogenic compounds micro- and nanoparticles.
Workpackage 4 aims at definition of potent angiogenesis-promoting factors and/or combinations and at evaluating the action of the above mentioned drugs compared to commercially available substances in accepted and complex model systems.
The objectives of workpackage 5 are contributions to define conditions able to improve vascularisation in the three-dimensional tissue via the induction of angiogenic processes by the action of monocytes / macrophages, that can stimulate the process of neovascularisation. We have included this aspect 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 we established two in vitro models able to mimic different aspects of angiogenic processes.
In workpackage 6 investigations on processing methods (extrusion, CO2 foaming, thermally induced phase separation, non-solvent induced phase inversion, particle leaching) for the polymeric components and their consequences for the composite were performed. Decisions were made about the adequate foaming and processing methods for the complex structures in order to obtain fabricated structures with the desired dimensions, pore sizes and porosity.
Experimental work in workpackage 7 characterised the effect of sterilisation on degradation and mechanical performance of polymeric components of the VASCUPLUG unit. Details on optimum sterilisation and design rules for the degradation behaviour were developed.
The workpackage 8 was intended to represent the proof of concept and the implantation into an animal. A bioreactor system adaptable to the conditions / components as available during the various stages of the project was developed. Prolonged in vitro culturing tests of as many components of the VASCUPLUG unit as available have been performed to study cellular behaviour in the composite system. A complete and functional tissue could not be reproduced in vitro during project duration, therefore the final proof of concept by implantation into an animal model was not performed since this was intended to be realised with the complete composite device only (for ethical reasons).
Workpackage 9 describes the project coordination and management performed by the project coordinator according to the requirements of successful project outcome: meetings were organised, communication between the consortium partners and the Scientific Officer of the EC maintained, reports were prepared with strong contribution of the consortium, and presentations for a broader community were prepared.
Intentions for use and impact
The VASCUPLUG system to be developed in this project was intended to improve the success for implantation of tissues engineered outside the patient's body that have to be vascularised for ingrowth, healing and proper function. The need and market for these tissue-engineered products is large and it will grow in future due to increasing life expectancy of the population. An improvement of vascularisation of tissue-engineered products will raise the chance of these tissue replacement structures to grow in and to heal the wound. Successful replacing of tissues can offer benefits for the patients to recover and will save costs of the health system.
Dissemination and use
Novel methods for preparing scaffold materials from biodegradable polymers were established and optimised thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS) combined with and without porogen leaching procedures. Furthermore the biodegradable polymer poly(dioxanone), known for the small number of suitable solvents, was formed to tubular and foam-like structures from solution in a special temperature regime.
Knowledge was acquired with respect to methods for surface modification representing current state of the art. Surface modifications using deep ultraviolet irradiations, which were found to be effective on poly(styrene) surfaces, were not applicable to polymer foams. Oxygen and nitrogen radio frequency-plasma treatments resulted in polymer foam modifications but did not result in significant improvements of cell adhesion in the complex polymeric cavities. Also in this setting collagenisation turned out to be most effective method to improve cell attachment and tissue growth. The dehydration / rehydration vesicle (DRV) method using heparin-loaded liposomes developed improves the haemocompatibility of polymers. Applied to surfaces a significant improvement of the response in terms of activation of the coagulation system as well as platelet adhesion and activation was found, whereas no significant differences were observed in the case of complement activation.
A specific bioreactor suitable for controlled long-term in vitro culture has been designed and produced to meet the specific needs for testing the various composite elements developed in this project; bioreactor design was continuously adapted to the specific investigational challenges emerging in the course of the project and distributed to the partners reliant on them. The modular concept of this bioreactor design allows easy and fast adaptation to new challenges as already demonstrated having adapted the bioreactor to specific needs to be met in other projects of the group (allowing cartilage and bone cell cultivation). Modifications allowed pressure drop measurements and staining experiments to derive data on permeability and flow regimes respectively and assessment of the fabrication and characterisation of poly(-caprolactone) scaffolds for bone tissue engineering. In addition a flexible window and ceramic spacer allowed the scaffolds in the bioreactor to be subjected to oscillating mechanical strain by a purpose built actuator.
Three peptides modulating angiogenesis were designed and synthesised by CRC: two by direct approach and one by an indirect approach. Their biological activity could be demonstrated by in vitro cell proliferation assays using Karposi sarcoma cells. One of these novel synthetic peptides (SHA-2-22) was also tested in vitro in BCE-HT cell proliferation assays and on rat aortic explants after encapsulation in the mouse cornea assay by KI. These compounds developed in the course of this project were patented to protect IPR, because of their application potential since at present only high molecular weight factors are in clinical trials as pro-angiogenic factors. The development of such small molecular weight, enzyme resistant molecules might become important in the future as angiogenesis promoting agents. However, their clinical use might be limited based on their very short half life and rather high expenditure.
Three drug delivery devices for the delivery of growth factors and synthetic pro-angiogenic small molecular weight molecules have been designed. All these technologies have a great potential to provide medical benefits and to provide additional value to standard drug-based therapies from industry. We are fully confident that the company to which we have transferred the patents for these two drug delivery compositions - Advanced In Vitro Cell Technologies S. L. - will consider the advances we have made for this tissue engineering applications and will explore possible industrial uses in these fields.
Synergetic effects of growth factor combinations for angiogenic processes were found; intellectual property rights protected. Delivery of PDGF-AB or PDGF-BB (but not PDGF-AA) together with FGF-2 is able to significantly stimulate vascular stability and improve blood perfusion as demonstrated in different assay systems. Especially the PDGF-BB / FGF-2 combination, encapsulated into nanoparticles, has shown advantages for simultaneous application.
Patents, publications and presentations
First confidential results are patented to protect intellectual property rights. After filing patents the knowledge is disseminated through scientific publications, on conferences and in modern media.
Application of intelligent biomaterials (bioresorbable stimuli-sensitive polymers) and incorporation of bioactive substances (e.g. growth factors) will 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 might be used to improve vascularisation. The signal for the stimulus-sensitive polymer to act is 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 migration and / or cell growth. The composite scaffold giving rise to evolving vessels is intended to promote the vascular connexion to the surrounding tissue in the course of wound healing.
The complexity of requirements for a functional vascularisation of tissue-engineered products calls for a strong inter- and transdisciplinary cooperation of chemists, biologists, engineers, physicists and physicians depending on a pronounced cross-frontier collaboration.
The project consortium is composed of seven participants from six European countries - three research centres with high expertise in the field of material development / evaluation and angiogenesis, and four institutes at universities that have high competence in materials characterisation, haemocompatibility, drug design and encapsulation.
The project comprised several workpackages.
Workpackage (WP) 1 was concerned with choice of polymers for the components (foam, tubular structures and pH sensitive gel) of the novel bioreactive composite scaffold in the further context also called VASCUPLUG unit. An assessment of mechanical and degradation behaviour of polymers to be used for the composite structure was made. As a result three commercial polymers were chosen for the fabrication of the foam and tubular structures. For the bioreactive part, pH sensitive hydrogels were prepared by free radical polymerisation and complexation, their pH dependent swelling behaviour was tested in buffer and under cell culture conditions in vitro.
An extensive functionality testing of the polymers, as offered from WP1 and of the composite structures from WP6, was performed in workpackage 2. This examination involved biocompatibility, haemocompatibility, degradation behaviour of unmodified and modified polymers before and after further processing steps (e.g. foaming) and sterilisation processes (WP7). The approach to identify the suitable polymeric materials for each component of the VASCUPLUG unit was accomplished with these biocompatibility tests since each further processing step changes the properties of the materials possibly influencing the reaction of biological material in contact with them.
Workpackage 3 involved the synthesis of bioactive substances and adequate delivery carriers. More specifically, the design of novel drugs to control angiogenesis (CRC) with focus on angiogenesis-promoting substances was envisaged. The second objective of this WP was to provide an adequate delivery system / carrier of selected pro-angiogenic compounds micro- and nanoparticles.
Workpackage 4 aims at definition of potent angiogenesis-promoting factors and/or combinations and at evaluating the action of the above mentioned drugs compared to commercially available substances in accepted and complex model systems.
The objectives of workpackage 5 are contributions to define conditions able to improve vascularisation in the three-dimensional tissue via the induction of angiogenic processes by the action of monocytes / macrophages, that can stimulate the process of neovascularisation. We have included this aspect 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 we established two in vitro models able to mimic different aspects of angiogenic processes.
In workpackage 6 investigations on processing methods (extrusion, CO2 foaming, thermally induced phase separation, non-solvent induced phase inversion, particle leaching) for the polymeric components and their consequences for the composite were performed. Decisions were made about the adequate foaming and processing methods for the complex structures in order to obtain fabricated structures with the desired dimensions, pore sizes and porosity.
Experimental work in workpackage 7 characterised the effect of sterilisation on degradation and mechanical performance of polymeric components of the VASCUPLUG unit. Details on optimum sterilisation and design rules for the degradation behaviour were developed.
The workpackage 8 was intended to represent the proof of concept and the implantation into an animal. A bioreactor system adaptable to the conditions / components as available during the various stages of the project was developed. Prolonged in vitro culturing tests of as many components of the VASCUPLUG unit as available have been performed to study cellular behaviour in the composite system. A complete and functional tissue could not be reproduced in vitro during project duration, therefore the final proof of concept by implantation into an animal model was not performed since this was intended to be realised with the complete composite device only (for ethical reasons).
Workpackage 9 describes the project coordination and management performed by the project coordinator according to the requirements of successful project outcome: meetings were organised, communication between the consortium partners and the Scientific Officer of the EC maintained, reports were prepared with strong contribution of the consortium, and presentations for a broader community were prepared.
Intentions for use and impact
The VASCUPLUG system to be developed in this project was intended to improve the success for implantation of tissues engineered outside the patient's body that have to be vascularised for ingrowth, healing and proper function. The need and market for these tissue-engineered products is large and it will grow in future due to increasing life expectancy of the population. An improvement of vascularisation of tissue-engineered products will raise the chance of these tissue replacement structures to grow in and to heal the wound. Successful replacing of tissues can offer benefits for the patients to recover and will save costs of the health system.
Dissemination and use
Novel methods for preparing scaffold materials from biodegradable polymers were established and optimised thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS) combined with and without porogen leaching procedures. Furthermore the biodegradable polymer poly(dioxanone), known for the small number of suitable solvents, was formed to tubular and foam-like structures from solution in a special temperature regime.
Knowledge was acquired with respect to methods for surface modification representing current state of the art. Surface modifications using deep ultraviolet irradiations, which were found to be effective on poly(styrene) surfaces, were not applicable to polymer foams. Oxygen and nitrogen radio frequency-plasma treatments resulted in polymer foam modifications but did not result in significant improvements of cell adhesion in the complex polymeric cavities. Also in this setting collagenisation turned out to be most effective method to improve cell attachment and tissue growth. The dehydration / rehydration vesicle (DRV) method using heparin-loaded liposomes developed improves the haemocompatibility of polymers. Applied to surfaces a significant improvement of the response in terms of activation of the coagulation system as well as platelet adhesion and activation was found, whereas no significant differences were observed in the case of complement activation.
A specific bioreactor suitable for controlled long-term in vitro culture has been designed and produced to meet the specific needs for testing the various composite elements developed in this project; bioreactor design was continuously adapted to the specific investigational challenges emerging in the course of the project and distributed to the partners reliant on them. The modular concept of this bioreactor design allows easy and fast adaptation to new challenges as already demonstrated having adapted the bioreactor to specific needs to be met in other projects of the group (allowing cartilage and bone cell cultivation). Modifications allowed pressure drop measurements and staining experiments to derive data on permeability and flow regimes respectively and assessment of the fabrication and characterisation of poly(-caprolactone) scaffolds for bone tissue engineering. In addition a flexible window and ceramic spacer allowed the scaffolds in the bioreactor to be subjected to oscillating mechanical strain by a purpose built actuator.
Three peptides modulating angiogenesis were designed and synthesised by CRC: two by direct approach and one by an indirect approach. Their biological activity could be demonstrated by in vitro cell proliferation assays using Karposi sarcoma cells. One of these novel synthetic peptides (SHA-2-22) was also tested in vitro in BCE-HT cell proliferation assays and on rat aortic explants after encapsulation in the mouse cornea assay by KI. These compounds developed in the course of this project were patented to protect IPR, because of their application potential since at present only high molecular weight factors are in clinical trials as pro-angiogenic factors. The development of such small molecular weight, enzyme resistant molecules might become important in the future as angiogenesis promoting agents. However, their clinical use might be limited based on their very short half life and rather high expenditure.
Three drug delivery devices for the delivery of growth factors and synthetic pro-angiogenic small molecular weight molecules have been designed. All these technologies have a great potential to provide medical benefits and to provide additional value to standard drug-based therapies from industry. We are fully confident that the company to which we have transferred the patents for these two drug delivery compositions - Advanced In Vitro Cell Technologies S. L. - will consider the advances we have made for this tissue engineering applications and will explore possible industrial uses in these fields.
Synergetic effects of growth factor combinations for angiogenic processes were found; intellectual property rights protected. Delivery of PDGF-AB or PDGF-BB (but not PDGF-AA) together with FGF-2 is able to significantly stimulate vascular stability and improve blood perfusion as demonstrated in different assay systems. Especially the PDGF-BB / FGF-2 combination, encapsulated into nanoparticles, has shown advantages for simultaneous application.
Patents, publications and presentations
First confidential results are patented to protect intellectual property rights. After filing patents the knowledge is disseminated through scientific publications, on conferences and in modern media.