Periodic Reporting for period 2 - LAF-GRAFT (An Investigation into the viability of employing lipoaspirate fluid as a cellular source in the production of small diameter tissue engineered vascular grafts)
Reporting period: 2018-09-01 to 2019-08-31
Tissue engineered vascular grafts (TEVGs) are biodegradable scaffolds intended to replace autologous and synthetic grafts that suffer from suboptimal performance. The majority of TEVG approaches require the incorporation of therapeutic cells to prevent acute thrombosis and promote endogenous cell infiltration followed by tissue remodelling. This project sought to investigate a novel autologous cell source for TEVGs: liposuction aspirate fluid (LAF). LAF would represent a significant advancement as an autologous cell base for TEVGs as LAF requires minimal manipulation, therefore bypassing numerous regulatory issues and presenting with low processing costs. The viability of LAF to act as a cell source for TEVGs was to be investigated by (1) characterisation of LAF cells, (2) modification of the scaffold to best promote positive vascular remodelling, (3) in vivo assessment of the LAF functionalised TEVGs in a small animal model, and (4) scaling of the technology for assessment in a large animal model.
New knowledge generated at the beginning of this action has meant that revisions to objectives 1 and 2 were required. Work conducted by the secondment host lab (Vorp Lab) immediately prior to the commencement of this action identified that adipose stem cells (the primary therapeutic cell found in LAF) derived from elderly and/or diabetic patients are not capable of acting as an effective therapeutic base for TEVGs as they have reduced anti-thrombotic and cell-signalling capabilities. As these patient groups represent the vast majority of patients requiring TEVGs, an autologous cell based approach for TEVGs is not a viable option. These points are addressed in our review article. A further obstacle encountered was the difficulty in obtaining quality PEUU scaffolds. For this reason, an alternate scaffold type has been investigated. Specifically, Bombex mori silk. The action has therefore revised the first 2 objectives of the action to investigate (1) an alternative method of scaffold fabrication and (2) the use of allogeneic adipose derived mesenchymal stem cells (ADMSCs) and their secreted extracellular vesicles (EVs) as a therapeutic base for TEVGs. Furthermore, objective 4 has been abbreviated due to the prohibitive cost of acquiring and maintaining large animals. This study therefore reduced the number of large animal implants from 8 to 1.
Objective 1 (Revised): Design a silk based scaffold for use as a TEVG that can be fabricated in-house in a repeatable manner. This work has been completed and a silk based scaffold has been developed in collaboration with Prof Biman Mandal of the Indian Institute of Technology Guwahati.
Objective 2 (Revised): Investigate the viability of employing commercially available ADMSCs and ADMSC derived EVs as a therapeutic base for TEVGs via in vitro cell-based assays. This work has been completed and demonstrated the positive therapeutic effects of administering ADMSC conditioned media or EVs to the vascular cells necessary for TEVG remodelling.
Objective 3: In vivo assessment of a novel functionalised TEVG in a small animal model. Specifically, incorporate ADMSCs and ADMSC-EVs into the silk based scaffold and perform an in vivo assessment of remodelling in an aortic interposition murine model. This work has been completed and demonstrated that incorporating EVs into silk based scaffolds improves graft patency and remodeling compared to blank and ADMSC seeded scaffolds.
Objective 4 (Abbreviated): Scale the technology to a size appropriate for use in humans and perform an in vivo assessment of the functionalised scaffolds in a large animal model. The technology to incorporate cells into silk based scaffolds has been successfully scaled and allows for even seeding of a human sized scaffold. An ADMSC seeded scaffold was implanted as a carotid interposition graft in a sheep for 10 weeks. The graft remained patent and demonstrated positive matrix remodelling.
New knowledge generated at the beginning of this action has meant that revisions to objectives 1 and 2 were required. Work conducted by the secondment host lab (Vorp Lab) immediately prior to the commencement of this action identified that adipose stem cells (the primary therapeutic cell found in LAF) derived from elderly and/or diabetic patients are not capable of acting as an effective therapeutic base for TEVGs as they have reduced anti-thrombotic and cell-signalling capabilities. As these patient groups represent the vast majority of patients requiring TEVGs, an autologous cell based approach for TEVGs is not a viable option. These points are addressed in our review article. A further obstacle encountered was the difficulty in obtaining quality PEUU scaffolds. For this reason, an alternate scaffold type has been investigated. Specifically, Bombex mori silk. The action has therefore revised the first 2 objectives of the action to investigate (1) an alternative method of scaffold fabrication and (2) the use of allogeneic adipose derived mesenchymal stem cells (ADMSCs) and their secreted extracellular vesicles (EVs) as a therapeutic base for TEVGs. Furthermore, objective 4 has been abbreviated due to the prohibitive cost of acquiring and maintaining large animals. This study therefore reduced the number of large animal implants from 8 to 1.
Objective 1 (Revised): Design a silk based scaffold for use as a TEVG that can be fabricated in-house in a repeatable manner. This work has been completed and a silk based scaffold has been developed in collaboration with Prof Biman Mandal of the Indian Institute of Technology Guwahati.
Objective 2 (Revised): Investigate the viability of employing commercially available ADMSCs and ADMSC derived EVs as a therapeutic base for TEVGs via in vitro cell-based assays. This work has been completed and demonstrated the positive therapeutic effects of administering ADMSC conditioned media or EVs to the vascular cells necessary for TEVG remodelling.
Objective 3: In vivo assessment of a novel functionalised TEVG in a small animal model. Specifically, incorporate ADMSCs and ADMSC-EVs into the silk based scaffold and perform an in vivo assessment of remodelling in an aortic interposition murine model. This work has been completed and demonstrated that incorporating EVs into silk based scaffolds improves graft patency and remodeling compared to blank and ADMSC seeded scaffolds.
Objective 4 (Abbreviated): Scale the technology to a size appropriate for use in humans and perform an in vivo assessment of the functionalised scaffolds in a large animal model. The technology to incorporate cells into silk based scaffolds has been successfully scaled and allows for even seeding of a human sized scaffold. An ADMSC seeded scaffold was implanted as a carotid interposition graft in a sheep for 10 weeks. The graft remained patent and demonstrated positive matrix remodelling.
WP1
Bombex mori (BM) silk fibroin has been established as an effective protein for forming silk based scaffolds. The fabrication, mechanical properties and degradation of BM silk scaffolds have been characterised and the findings of this study are being prepared for publication. A significant milestone of the project has therefore been achieved as appropriate scaffolds can be fabricated in-house in a repeatable manner. The deliverables of this objective have been achieved: (1) Silk based scaffolds can be fabricated and (2) Obtained the technical skillset required to fabricate scaffolds that will form the basis of subsequent research within the action. These scaffolds can be functionalised with ADMSCs or EVs and therefore utilised in the subsequent WPs of the action.
WP2
The viability of employing commercial ADMSCs and EVs as a therapeutic base for TEVGs has been assessed using in vitro cell-based assays. A significant milestone has been completed as the effect of ADMSC conditioned media and EVs on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) has been examined using in vitro proliferation and migration studies. The deliverables of this WP have been achieved as results for SMCs and ECs demonstrate that ADMSC conditioned media and EVs increase both proliferation and migration relative to controls. These results suggest that commercial ADMSC and EVs can be used as a therapeutic base for TEVGs as they will trigger re-population of the implanted scaffold with endogenous SMCs and ECs. These results will be exploited to fabricate a silk-based scaffold functionalised with ADMSCs or EVs.
WP3
ADMSCs and EVs have been incorporated into silk scaffolds using established rotational-vacuum technology. Implants of blank, ADMSC seeded and EV seeded silk scaffolds in an established murine model have performed. The deliverables of this WP has been achieved as results show that commercial ADMSC and EVs can be incorporated into silk based scaffolds and that EV seeded grafts demonstrate improved patency and matrix remodelling compared to blank and ADMSC seeded scaffolds. These results have been exploited in the form of an invention disclosure and as a manuscript intended for publication.
WP4
The technology required to incorporate cells and into porous tubular scaffolds using a rotational-vacuum approach has successfully been scaled to allow for seeding of human sized scaffolds. The deliverables of this WP has been achieved as results demonstrate that the scaled technology is capable of evenly seeding cells throughout a human-sized scaffold. An ADMSC seeded scaffold was implanted as a sheep carotid interposition graft. The graft remained patent and demonstrated positive matrix remodelling after 10 weeks. The findings of this WP will be exploited in the form of a further invention disclosure submission and as a manuscript intended for publication.
Bombex mori (BM) silk fibroin has been established as an effective protein for forming silk based scaffolds. The fabrication, mechanical properties and degradation of BM silk scaffolds have been characterised and the findings of this study are being prepared for publication. A significant milestone of the project has therefore been achieved as appropriate scaffolds can be fabricated in-house in a repeatable manner. The deliverables of this objective have been achieved: (1) Silk based scaffolds can be fabricated and (2) Obtained the technical skillset required to fabricate scaffolds that will form the basis of subsequent research within the action. These scaffolds can be functionalised with ADMSCs or EVs and therefore utilised in the subsequent WPs of the action.
WP2
The viability of employing commercial ADMSCs and EVs as a therapeutic base for TEVGs has been assessed using in vitro cell-based assays. A significant milestone has been completed as the effect of ADMSC conditioned media and EVs on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) has been examined using in vitro proliferation and migration studies. The deliverables of this WP have been achieved as results for SMCs and ECs demonstrate that ADMSC conditioned media and EVs increase both proliferation and migration relative to controls. These results suggest that commercial ADMSC and EVs can be used as a therapeutic base for TEVGs as they will trigger re-population of the implanted scaffold with endogenous SMCs and ECs. These results will be exploited to fabricate a silk-based scaffold functionalised with ADMSCs or EVs.
WP3
ADMSCs and EVs have been incorporated into silk scaffolds using established rotational-vacuum technology. Implants of blank, ADMSC seeded and EV seeded silk scaffolds in an established murine model have performed. The deliverables of this WP has been achieved as results show that commercial ADMSC and EVs can be incorporated into silk based scaffolds and that EV seeded grafts demonstrate improved patency and matrix remodelling compared to blank and ADMSC seeded scaffolds. These results have been exploited in the form of an invention disclosure and as a manuscript intended for publication.
WP4
The technology required to incorporate cells and into porous tubular scaffolds using a rotational-vacuum approach has successfully been scaled to allow for seeding of human sized scaffolds. The deliverables of this WP has been achieved as results demonstrate that the scaled technology is capable of evenly seeding cells throughout a human-sized scaffold. An ADMSC seeded scaffold was implanted as a sheep carotid interposition graft. The graft remained patent and demonstrated positive matrix remodelling after 10 weeks. The findings of this WP will be exploited in the form of a further invention disclosure submission and as a manuscript intended for publication.
The use of commercial ADMSCs or ADMSC derived EVs as a therapeutic base for TEVGs holds great potential in the field of vascular tissue engineering and could potentially decrease the cost, time and regulatory issues associated with current cell/therapeutic sourcing methods used in tissue engineering. The in vitro and in vivo evidence, performed in both small and large animal models, has led to the submission of intellectual property and could lead to the commercialisation of a clinically viable approach to TEVG.