AN IN VIVO BIOENGINEERED CHIP AS A SMART INTRAVITAL MULTIPHOTON IMAGING WINDOW FOR NEW VALIDATION PROTOCOLS OF BIOMATERIALS

Project Information

IN2SIGHT

Grant agreement ID: 964481

DOI

10.3030/964481

Project closed

EC signature date 21 December 2020

Start date 1 March 2021

End date 31 August 2025

Funded under

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Total cost

€ 3 438 959,00

EU contribution

€ 3 438 959,00

Coordinated by

UNIVERSITA' DEGLI STUDI DI MILANO-BICOCCA
Italy

Periodic Reporting for period 3 - IN2SIGHT (AN IN VIVO BIOENGINEERED CHIP AS A SMART INTRAVITAL MULTIPHOTON IMAGING WINDOW FOR NEW VALIDATION PROTOCOLS OF BIOMATERIALS)

Reporting period: 2023-09-01 to 2025-08-31

Summary of the context and overall objectives of the project

Routine clinical use of biomaterials must comply with the biocompatibility tests stated by the ISO10993 EU norms that require a wide use of laboratory animals with an excessive economic burden, particularly for SMEs, and with heavy ethical costs for the society. IN2SIGHT wants to renovate the way the tests are made in order to reduce the number of animals used and to increase the statistical reliability of the analyses.

The objectives are to:
1. develop a new technology, based on non-linear optical microscopy, capable to observe the immune reaction to the implant of a biomaterial for weeks, during the evolution of the possible reaction, without the need to sacrifice the animal;
2. formulate a novel statistical approach to analyze the data relative to the immune reaction;
3. make the IN2SIGHT technology known among the stakeholders and initiate the process for an update of the norms through contacts with the relevant regulatory bodies in Europe and USA;
3. make the society aware of the possibility to reduce the number of animals in the tests and of alternative methods for the biomaterial and drugs testing;
4. develop an innovative numerical simulation method for the immune reaction to the implant of a biomaterial based on agent based techniques with the aim to enhance our interpretation of the immune reaction observed experimentally.

The project reaches, after 54 months of coordinated R&D, all the milestones foreseen. It paves the way to the use of an alternative method to the study of the immune-reaction induced by the implant of biomaterial. Although, the contacts with the relevant regulatory bodies put into evidence a lack of regulatory indications at the EU level, for devices to be used for testing biomaterial and biomedical devices, the technology developed here has a clear possibility to induce a change in the attitude to use lab animals to test biomaterial and to stimulate a more objective evaluation of the immunogenicity of biomaterials.
The Consortium has filed a patent (WO2023218340) on May 2022 which is now at the PCT stage.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project lasted 54 months and achieved all the foreseen milestones. At the end of the project the Consortium has reached the following results.
1. development of protocols for the fabrication of the device by means of 2-photon polymerization and nano imprint lithography;
2. fabrication of microlenses for in vivo imanging of biological samples coupled to a regular lattice for longitudinal observation of the immune reaction;
3. a device, IN2SIGHT, composed of microlenses coupled to a regular atlas for the extended longitudinal studies of the reaction to biomaterials;
4. the prototype of a device to couple the biomaterials to be tested (in the form of thin slabs) to the IN2SIGHT device ;
6. In-vivo imaging on implanted In2Sight devices
7. a protocol for the comparison of the IN2SIGHT approach to the study of the immuno-genicoity of biomaterials to the standard ISO10993-6 norm.
8. a simulation platform based on MatLab and a propetary agent-based platform (CellStudio) for the simulation of the immune reaction to the biomaterial in contact with the device;
9. contacts with regulatory bodies for the presentation of the project;
10. activation and use of social media channels, X and linkedin, and of a dedicated website;
11. organization of Dissemination and Communication events for PhD students, junior researchers and high school pupils.

Part of the above results have been published (21 publications open access, 6 proceedings) and presented to congresses as talks (more than 30 talks ).
The Consortium has been present at the following events:
- Eusaat Congress 2022 and 2024;
- SPIE Optical Metrology 2023;
- EOS- European optical society annual meeting;
- 28th Congress of the European Society of Biomechanics 2023 (ESB2023);
- Tissue Engineering and Regenerative Medicine International Society (TERMIS) 2023;
- CLEO2023, Muniche June 2023;
- EOSAM2023, Porto Sept 2023;
- FOM2022 and 2024;
- 3R’ Italian center congress, Milano, September 2023;
- Icton, Bucharest, July 2023;
- Photonica 2023, Belgrade, August 2023;
- Optica (OSA) Imaging Congress 2023, Boston .
- FisMat 23 Congress, Milano, 4-8 September 2023.
- Optica (OSA) congress 2024, Toulouse (F)
- Optica (OSA), Frontiers in Optics, 2024 (Denver, USA)
- SPIE, Photonics west, 2025 San Francisco (USA)
- Light Conference, 2025 ChangChun (China)

Additional information can be found on the project web site: www.in2sight.eu

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The consortium R&D actions has already produced innovative results in the fields of the laser micro-structuring of resins based on silica and Zirconium, the test of their biocompatibility and of their rigidity for long term implants.

Moreover, additional improvement beyond the state of the art are in the field of the optical setup for in-vivo imaging. These encompass the micro-fabrication with resins engineered for high index of refraction and low auto-fluorescence. The major leap forward in R&D of the second period of the project is the possibility to use the IN2SIGHT device to obtain in vivo images when implanted. The Consortium has started the exploitation by obtaining the first patent (WO2023218340) on May 2022 which is now at the PCT stage.

We have developed and tested on literature and project data a modular and effective framework for the simulation of the immune reaction in a confined, well defined environment constituted by the micro-structured niche that is the exact replica of the one actually embedded in the IN2SIGHT device. This framework is developed both in MatLab and ina propretary agent based framework (CellStudio). In the MatLab version it is available on a dedicated github site (https://github.com/lucaxx85/Fibro_ABM).

We have developed a statistical approach to quantify the difference, if any, between the IN2SIGHT protocol and the ISO10993-6 norm for the evaluation of the immune reaction to biomaterial. This method, which is at the core of the main result of the IN2SIGHT project, is described in the deliverable 4.3 and in the dedicated github site: https://github.com/Gchirico63/IN2SIGHT_DigitalPathology

The economic impact of the project comes from the integration of the technologies developed in the laser fabrication facilities of the consortium to the level of microfabrication through nano-imprinting lithography. Apart from the parabolic shape of the microlenses used in the IN2SIGHT device, we are currently exploring the possibility to fabricate metalenses and kinoform lenses with the same technology. From the societal point of view, our impact can be judged from our effort to communicate (at congresses and workshops in the field of regulatory actions, beyond the field of photonics research, see for example our presence at the EUSAAT 2022 conference and at the 3R's summer school held in Genoa (I) in July 2025) and to disseminate the results of the project among stakeholders. The Consortium put a large effort in organizing a doctorate school in May 2025 on "Shaping light in space and time to advance bioimaging" (Erice, Italy) that gathered together 40 students and 12 lecturers from alla over the world. We keep constantly updated the web site for the project and make use of the twitter and linkedin pages. The IN2SIGHT consortium has also organized workshops and dedicated sessions at international congresses and was presented at international conference also for invited talks.

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