Periodic Reporting for period 1 - SMART-BONE (Smart electroactive 3D models of osteoregeneration)
Reporting period: 2016-09-01 to 2018-08-31
In the European Union alone, the annual number of bone fractures is projected to reach 4.5 million in 2025.1 In the EU in 2010 the costs of treating incident fractures connected solely with osteoporosis amounted to about €24 billion. Despite the advances in implant technology, grafts prepared using bone extracted from the patient to fill the bone defect are the only material reported so far to retain any significant efficacy in sustaining new bone formation.2 However, the aging worldwide population and increased life expectancy call for a different answer to the growing need for effective implants. The possibility to have easily available and effective bone regeneration implants that are biocompatible, and with low inflammatory response is a crucial goal for the research community, both in material science and orthopedics, translating to major benefits for future patients.
The three main objectives of the SMART-BONE project are to:
- Develop electroactive 3D-scaffolds as substrate for hosting BM-MScs and NCSCs culture and stimulation,
- Implement an OECT specifically recognizing analytes related to osteogenesis and vasculogenesis,
- Monitor stem cells fate upon electrical stimulation with the OECT integrating the 3D-scaffolds.
The three main objectives of the SMART-BONE project are to:
- Develop electroactive 3D-scaffolds as substrate for hosting BM-MScs and NCSCs culture and stimulation,
- Implement an OECT specifically recognizing analytes related to osteogenesis and vasculogenesis,
- Monitor stem cells fate upon electrical stimulation with the OECT integrating the 3D-scaffolds.
The project was divided into three work packages.
WP 1. Electroactive porous scaffold development. The first step has therefore been the establishment of the 3D matrices. The 3D substrates mimicking bone tissue were developed using the conjugated conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and a matrix polymer, determining the mechanical properties of the scaffold. The polymer used to tune the mechanical properties of the scaffolds was collagen Type 1. This polymer was selected because it represents the biological component of bones. Collagen Type 1, indeed, represents the master used by cells to deposit Hydroxyapatite, the mineral counterpart. Blends of the two polymers were prepared and characterized for their mechanical and electrical properties.
Human adipose derived stem cells and Neural crest derived stem cells were used to assess both scaffolds cytocompatibility and their ability to influence stem cells differentiation.
WP 2. OECT fabrication and 3D scaffolds integration. Organic electrochemical transistors were developed targeting BMP-2, an earlier marker of differentiation. A protocol was established to functionalize the electrodes and a thorough characterization was performed to assess each functionalization step. Electrical characterization of the devices was performed to assess devices response to the specific analyte.
WP 3. Monitoring stem cells differentiation upon electrical stimulation with the developed OECT.
In collaboration with Prof Sarah Cartmell, from the University of Manchester capactive coupling experiments were performed in order to evaluate the effect on stem cells differentiation. A protocol was defined involving a bioreactor, previously developed in Prof Cartmell laboratories, the electroactive scaffolds and human adipose derived stem cells. The developed OECTs were tested to evaluate their ability to detect BMP-2 from a real sample.
As a result of the research activities of the fellow a number of publications are in preparation or have already been accepted for publication, as reported t=in the list below.
Publications:
1. Iandolo, D., Pitsalidis, C, Santoro, F., Cui, B., Widera, D., Owens, R. M., Collagen-enriched 3D electroactive scaffolds for human stem cells growth and osteogenic differentiation. Manuscript in preparation.
2. Pitsalidis, C., Pappa, A.M. Moysidou, C.M. Iandolo, D. and Owens, R. M. “Conducting and Conjugated Polymers for Biosensing Applications” book chapter in the Fourth Edition of the Handbook of Conducting Polymers, publisher CRC Press/Taylor & Francis (accepted for publication).
3. Iandolo, D., Pennacchio, F. A., Mollo, V., Rossi, D., Dannhauser, D., Cui, B., Owens, M. R., Santoro, F.. Electron microscopy for 3D scaffolds-cell biointerface characterization. Adv. Biosys. 2018, 1800103.
4. Pitsalidis, C., Ferro M., Iandolo, D., Tzounis, L., Inal, S., Owens, R. M. Transistor in a tube: a route to three-dimensional bioelectronics. Science Advances, 2018.
5. Owens, M.R. Iandolo, D., Wittmann, K., A (bio) materials approach to three-dimensional cell biology. MRS Communications, 2017, 7, 287–288.
WP 1. Electroactive porous scaffold development. The first step has therefore been the establishment of the 3D matrices. The 3D substrates mimicking bone tissue were developed using the conjugated conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and a matrix polymer, determining the mechanical properties of the scaffold. The polymer used to tune the mechanical properties of the scaffolds was collagen Type 1. This polymer was selected because it represents the biological component of bones. Collagen Type 1, indeed, represents the master used by cells to deposit Hydroxyapatite, the mineral counterpart. Blends of the two polymers were prepared and characterized for their mechanical and electrical properties.
Human adipose derived stem cells and Neural crest derived stem cells were used to assess both scaffolds cytocompatibility and their ability to influence stem cells differentiation.
WP 2. OECT fabrication and 3D scaffolds integration. Organic electrochemical transistors were developed targeting BMP-2, an earlier marker of differentiation. A protocol was established to functionalize the electrodes and a thorough characterization was performed to assess each functionalization step. Electrical characterization of the devices was performed to assess devices response to the specific analyte.
WP 3. Monitoring stem cells differentiation upon electrical stimulation with the developed OECT.
In collaboration with Prof Sarah Cartmell, from the University of Manchester capactive coupling experiments were performed in order to evaluate the effect on stem cells differentiation. A protocol was defined involving a bioreactor, previously developed in Prof Cartmell laboratories, the electroactive scaffolds and human adipose derived stem cells. The developed OECTs were tested to evaluate their ability to detect BMP-2 from a real sample.
As a result of the research activities of the fellow a number of publications are in preparation or have already been accepted for publication, as reported t=in the list below.
Publications:
1. Iandolo, D., Pitsalidis, C, Santoro, F., Cui, B., Widera, D., Owens, R. M., Collagen-enriched 3D electroactive scaffolds for human stem cells growth and osteogenic differentiation. Manuscript in preparation.
2. Pitsalidis, C., Pappa, A.M. Moysidou, C.M. Iandolo, D. and Owens, R. M. “Conducting and Conjugated Polymers for Biosensing Applications” book chapter in the Fourth Edition of the Handbook of Conducting Polymers, publisher CRC Press/Taylor & Francis (accepted for publication).
3. Iandolo, D., Pennacchio, F. A., Mollo, V., Rossi, D., Dannhauser, D., Cui, B., Owens, M. R., Santoro, F.. Electron microscopy for 3D scaffolds-cell biointerface characterization. Adv. Biosys. 2018, 1800103.
4. Pitsalidis, C., Ferro M., Iandolo, D., Tzounis, L., Inal, S., Owens, R. M. Transistor in a tube: a route to three-dimensional bioelectronics. Science Advances, 2018.
5. Owens, M.R. Iandolo, D., Wittmann, K., A (bio) materials approach to three-dimensional cell biology. MRS Communications, 2017, 7, 287–288.
A procedure was established to prepare scaffolds that can be used to culture (human adipose derived and neural crest derived) stem cells. The composition of the solution used to prepare the blends enabled us to control the scaffolds mechanical properties. Indeed, including Collagen Type 1 within the solution allowed us to tune the elastic properties of the scaffolds. Neural crest derived stem cells responded differently from human adipose derived stem cells, with one of the composition inducing a much higher response by the cells in terms of osteogenic differentiation. A protocol has been established that, by combining a specific scaffold composition and electrical stimulation protocol, seems to positively affect cells ability to differentiate into bone forming cells. Further analyses must be performed to confirm and fine tune the procedure but these preliminary data set the ground for further developments.