Periodic Reporting for period 1 - SYNERGIES (Smart bioreactor-based platform for the multi-phYsical stimulatioN of Engineered osteochondRal tissues towards functional reGeneratIon and advanced diseasE modelS)
Reporting period: 2024-05-16 to 2026-05-15
The incidence and socioeconomic burden of osteochondral (OC) defects and degenerative diseases are growing worldwide, representing a major challenge for the healthcare systems. Tissue Engineering (TE) emerged as a promising alternative to the current ineffective OC disease treatments. However, state-of-the-art OCTE strategies are still unable to create functional engineered tissues, mainly due to their inability to replicate in vitro the complex physicochemical environment of the native OC tissue within the articular joint, making OC regeneration a critical unmet medical need.
The ambitious objective of the SYNERGIES project was to fill this gap by developing a pioneering smart biomimetic bioreactor-based platform able to culture OC constructs under physiologically and clinically relevant multi-physical stimulations in a controlled manner. Cutting-edge in silico modelling and machine learning (ML) tools were employed for the optimization of stimulation parameters and automated control of the bioreactor platform. In vitro cell culture experiments were performed to validate both the new OC tissue construct and the bioreactor developed. Overall, the developed SYNERGIES platform contributed to enhance the knowledge of OC tissue mechanobiology beyond the state-of-the-art and guide the improvement of protocols towards the production of functional OC substitutes, suitable to be used as in vitro models for OC disease research and drug screening, paving the way for new effective therapies.
The ambitious objective of the SYNERGIES project was to fill this gap by developing a pioneering smart biomimetic bioreactor-based platform able to culture OC constructs under physiologically and clinically relevant multi-physical stimulations in a controlled manner. Cutting-edge in silico modelling and machine learning (ML) tools were employed for the optimization of stimulation parameters and automated control of the bioreactor platform. In vitro cell culture experiments were performed to validate both the new OC tissue construct and the bioreactor developed. Overall, the developed SYNERGIES platform contributed to enhance the knowledge of OC tissue mechanobiology beyond the state-of-the-art and guide the improvement of protocols towards the production of functional OC substitutes, suitable to be used as in vitro models for OC disease research and drug screening, paving the way for new effective therapies.
Staring from an existing proof-of-concept prototype, the SYNERGIES bioreactor developed to assure (1) full compatibility of the system with good laboratory and manufacturing practices (GLP/GMP) standards and to (2) allow the culture of osteochondral (OC) tissue constructs under controlled and physiologically relevant stimulation culture conditions. The bioreactor culture chamber was designed and developed to house the OC construct, based on a 3D printed bi-layered scaffold, press-fit into a tailored flexible holder. When the OC construct is inserted, the chamber becomes separated into two independent compartments for the bone and the cartilage parts, supplied by specific differentiation media and exposed to tissue-specific physical stimulation. The bioreactor developed was based on a modular architecture consisting of: i) a culture chamber with individualized bone and cartilage compartments; ii) a perfusion circuit for the bone compartment; iii) a HP unit based on pinch valves for the cartilage compartment iv); a recirculation circuit for the cartilage compartment; v) a pulsed electromagnetic fields (PEMF) stimulator provided by the company IGEA; vi) a monitoring unit; and vii) a ML-driven control unit. The culture chamber and the required sample holders and bioreactor parts were designed using the SolidWorks software and fabricated by additive manufacturing by stereolithography (SLA). All the bioreactor culture chamber parts were fabricated using biocompatible and autoclavable resin materials to avoid any cytotoxicity issues and promote the efficient sterilization of the system prior to the in vitro cell culture studies performance. A transparent material (Biomed Clear) was selected to facilitate the visualization of the internal structure, allowing for the rapid detection of any media leakage event. The bilayer OC scaffold construct developed to integrate the bioreactor platform was characterized in terms of its structural/architectural, physico-chemical and mechanical properties. To obtain a highly biomimetic OC model, the developed 3D bilayer scaffolds were seeded with tissue-specific human mesenchymal stem/stromal cells (MSC) (derived from synovium membrane (hSMSC) for the cartilage part and from bone marrow (hBMSC) for the bone part). Multiphysics in silico modelling approaches were employed to study/characterize the physical environment (hydrodynamics, fluid-induced shear stress, pressure, deformation and electromagnetic fields) within the bioreactor culture chamber and across the OC scaffold structure. This knowledge was critical to optimize the bioreactor operational parameters (i.e. stimuli magnitude, frequency, duration). Finally, in vitro cell culture studies were followed for the biological validation of the new bilayer OC tissue construct and of the bioreactor platform as well as to allow the progressive optimization of the OCTE culture protocols.
Overall, we expect that the advanced biomimetic platform developed in SYNERGIES will be crucial to improve the current understanding of OC tissue mechanobiology and of how cells/tissues respond to different native-like physical stimuli. Such knowledge gain will be essential to develop OC tissue substitutes with proper structural/mechanical properties and functions, making them suitable: i) to be used as reliable in vitro 3D models of OC tissues in healthy/disease (e.g. for OA research) state as well as for drug screening; and ii) for future clinical translation to treat OC defects.
A detailed description of the work performed during the project and the related scientific publications can be found in the Technical Report.
The SYNERGIES achievements were disseminated to the scientific community through high quality scientific publications and by participating in highly relevant international conferences focused on bioengineering, biomaterials, biomechanics and tissue engineering research. The prepared dissemination plan allowed the researcher to share the SYNERGIES results with several leading researchers and bioreactor companies, supporting this MSCA action impact on European scientific excellence and competitiveness.
The SYNERGIES project results were disseminated in the following publications:
1. Masante B, Gabetti S, Silva JC, Putame G, Israel, S, Bignardi, C, Massai, D. Insights into Bone and Cartilage Responses to Pulsed Electromagnetic Field Stimulation: A Review with Quantitative Comparisons. Frontiers in Bioengineering and Biotechnology 13, 1557572. (2025) DOI: open access(opens in new window)
2. Silva JC, Masante B, Gabetti S, Israel S, Tosini M, Marcelino P, Miguel F, Serino G, Deriu M, di Benedetto G, Massai D. Bioreactor-based investigation platform to study the effects of multi-physical stimuli on the osteochondral differentiation of human mesenchymal stem/stromal cells in 3D printed bilayer scaffolds. Conference abstract published in Tissue Engineering Part A 31, No 11-12 (2025) DOI: open access(opens in new window)
3. Domingues MF, Silva JC, Sanjuan-Alberte, P. From Spheroids to Bioprinting: A Literature Review on Biomanufacturing Strategies of 3D In Vitro Osteosarcoma Models. Advanced Therapeutics 7(11), 2400047. (2024) DOI: https://doi.org/10.1002/adtp.202400047(opens in new window)
4. Domingues MF, Carvalho MS, Sanjuan-Alberte, P., Silva JC. Synthetic scaffolds functionalized with mesenchymal stem/stromal cells-derived extracellular matrix for bone tissue engineering: a review. RSC Advances (2025) (Accepted, in press, open access)
The SYNERGIES project outputs were also disseminated in the following international conference meetings:
1. 29th Congress of the European Society of Biomechanics (ESB 2024), 30th June-3rd July 2024, Edinburgh, Scotland. Oral Presentation: Silva JC, Garrudo FF, Marcelino P, Meneses J, Barbosa F, Alves NM, Massai D, Pascoal-Faria P, Ferreira FC. “3D Printed Conductive Scaffolds Combined with Electrical Stimuli for Improved Bone Tissue Engineering Strategies”
2. International Materials Science and Engineering Congress – MSE 2024, 24th-26th September 2024, Darmstadt, Germany. Presentation of an Invited Highlight Lecture: Silva JC “Additive manufactured electroactive scaffolds combined with biophysical stimuli towards improved bone tissue engineering strategies”
3. SupraLife EU (Supramolecular Multifunctional Biomaterials) Third Conference and Training School, 09th – 14th March 2025, University of Aveiro, Portugal. Poster Presentation: “Novel Piezoelectric 3D-Printed Scaffolds combined with Ultrasound Stimulation for Bone Tissue Engineering”
4. TERMIS-EU 2025 – European Chapter Meeting of the Tissue Engineering and Regenerative Medicine International Society, 20th – 23rd May 2025, Freiburg, Germany. (Organization and chairing of the Special Symposium “Advances in Electroactive Biomaterials for Cell Fate Modulation and Tissue Regeneration” Poster Presentation "Silva JC, Masante B, Gabetti S, Israel S, Tosini M, Marcelino P, Miguel F, Serino G, Deriu M, di Benedetto G, Massai D. “Bioreactor-based investigation platform to study the effects of multi-physical stimuli on the osteochondral differentiation of human mesenchymal stem/stromal cells in 3D printed bilayer scaffolds”
5. Microphysiological Systems (MPS) World Summit 2025, 09th – 13th June 2025, Brussels, Belgium. Poster Presentation: Schuler K, Masante B, Gabetti S, Massai D, Sanjuan-Alberte P, Silva JC, Ferreira FC. “A novel bio-instructive scaffold for biomimetic collagen deposition aiming at cartilage tissue engineering and in vitro disease modelling of osteoarthritis”
Overall, we expect that the advanced biomimetic platform developed in SYNERGIES will be crucial to improve the current understanding of OC tissue mechanobiology and of how cells/tissues respond to different native-like physical stimuli. Such knowledge gain will be essential to develop OC tissue substitutes with proper structural/mechanical properties and functions, making them suitable: i) to be used as reliable in vitro 3D models of OC tissues in healthy/disease (e.g. for OA research) state as well as for drug screening; and ii) for future clinical translation to treat OC defects.
A detailed description of the work performed during the project and the related scientific publications can be found in the Technical Report.
The SYNERGIES achievements were disseminated to the scientific community through high quality scientific publications and by participating in highly relevant international conferences focused on bioengineering, biomaterials, biomechanics and tissue engineering research. The prepared dissemination plan allowed the researcher to share the SYNERGIES results with several leading researchers and bioreactor companies, supporting this MSCA action impact on European scientific excellence and competitiveness.
The SYNERGIES project results were disseminated in the following publications:
1. Masante B, Gabetti S, Silva JC, Putame G, Israel, S, Bignardi, C, Massai, D. Insights into Bone and Cartilage Responses to Pulsed Electromagnetic Field Stimulation: A Review with Quantitative Comparisons. Frontiers in Bioengineering and Biotechnology 13, 1557572. (2025) DOI: open access(opens in new window)
2. Silva JC, Masante B, Gabetti S, Israel S, Tosini M, Marcelino P, Miguel F, Serino G, Deriu M, di Benedetto G, Massai D. Bioreactor-based investigation platform to study the effects of multi-physical stimuli on the osteochondral differentiation of human mesenchymal stem/stromal cells in 3D printed bilayer scaffolds. Conference abstract published in Tissue Engineering Part A 31, No 11-12 (2025) DOI: open access(opens in new window)
3. Domingues MF, Silva JC, Sanjuan-Alberte, P. From Spheroids to Bioprinting: A Literature Review on Biomanufacturing Strategies of 3D In Vitro Osteosarcoma Models. Advanced Therapeutics 7(11), 2400047. (2024) DOI: https://doi.org/10.1002/adtp.202400047(opens in new window)
4. Domingues MF, Carvalho MS, Sanjuan-Alberte, P., Silva JC. Synthetic scaffolds functionalized with mesenchymal stem/stromal cells-derived extracellular matrix for bone tissue engineering: a review. RSC Advances (2025) (Accepted, in press, open access)
The SYNERGIES project outputs were also disseminated in the following international conference meetings:
1. 29th Congress of the European Society of Biomechanics (ESB 2024), 30th June-3rd July 2024, Edinburgh, Scotland. Oral Presentation: Silva JC, Garrudo FF, Marcelino P, Meneses J, Barbosa F, Alves NM, Massai D, Pascoal-Faria P, Ferreira FC. “3D Printed Conductive Scaffolds Combined with Electrical Stimuli for Improved Bone Tissue Engineering Strategies”
2. International Materials Science and Engineering Congress – MSE 2024, 24th-26th September 2024, Darmstadt, Germany. Presentation of an Invited Highlight Lecture: Silva JC “Additive manufactured electroactive scaffolds combined with biophysical stimuli towards improved bone tissue engineering strategies”
3. SupraLife EU (Supramolecular Multifunctional Biomaterials) Third Conference and Training School, 09th – 14th March 2025, University of Aveiro, Portugal. Poster Presentation: “Novel Piezoelectric 3D-Printed Scaffolds combined with Ultrasound Stimulation for Bone Tissue Engineering”
4. TERMIS-EU 2025 – European Chapter Meeting of the Tissue Engineering and Regenerative Medicine International Society, 20th – 23rd May 2025, Freiburg, Germany. (Organization and chairing of the Special Symposium “Advances in Electroactive Biomaterials for Cell Fate Modulation and Tissue Regeneration” Poster Presentation "Silva JC, Masante B, Gabetti S, Israel S, Tosini M, Marcelino P, Miguel F, Serino G, Deriu M, di Benedetto G, Massai D. “Bioreactor-based investigation platform to study the effects of multi-physical stimuli on the osteochondral differentiation of human mesenchymal stem/stromal cells in 3D printed bilayer scaffolds”
5. Microphysiological Systems (MPS) World Summit 2025, 09th – 13th June 2025, Brussels, Belgium. Poster Presentation: Schuler K, Masante B, Gabetti S, Massai D, Sanjuan-Alberte P, Silva JC, Ferreira FC. “A novel bio-instructive scaffold for biomimetic collagen deposition aiming at cartilage tissue engineering and in vitro disease modelling of osteoarthritis”
The SYNERGIES knowledge and outcomes can positively impact on the European biomedical, bioengineering, and regenerative medicine research and innovation ecosystem. In particular, the new smart biomimetic platform has the potential to advance fundamental knowledge in the field of osteochondral (OC) tissue engineering, specifically by enabling the investigation of how different forms of physical stimulation influence OC tissue regeneration, addressing a critical knowledge gap in regenerative medicine. The automated bioreactor-based platform designed within the project provides significant advantages over conventional manual culture systems. By minimizing operator intervention, the platform improves reproducibility, supports protocol standardization, and facilitates scalability, key factors for regulatory compliance and to enable the cost-effective production of engineered OC tissues intended for clinical use.
The technological innovations generated within the SYNERGIES project will be made available for further research through collaborations with academic and clinical partners. Potential future applications of the developed bioreactor platform include: i) Investigating the pathophysiological effects of mechanical stimulation during tissue development; ii) Supporting personalized in vitro disease models for osteoarthritis (OA); iii) Enabling drug screening assays under controlled culture conditions, contributing to the reduction of animal testing in accordance with the EU 3Rs principles (Replacement, Reduction, and Refinement). Moreover, the in silico models/AI tools under development will allow the in silico optimization of the procedures, reducing the number of experiments and saving costs.
In terms of knowledge translation, the potential for commercial exploitation of the SYNERGIES bioreactor platform is actively being explored, representing a promising opportunity for technology transfer and innovation beyond the academic context.
This SYNERGIES MSCA action also enabled the establishment of highly multidisciplinary international scientific network between the Fellow and the Host institution, already fostering joint grant applications, the exchange of MSc/PhD students, and the performance of other collaborative research projects.
The technological innovations generated within the SYNERGIES project will be made available for further research through collaborations with academic and clinical partners. Potential future applications of the developed bioreactor platform include: i) Investigating the pathophysiological effects of mechanical stimulation during tissue development; ii) Supporting personalized in vitro disease models for osteoarthritis (OA); iii) Enabling drug screening assays under controlled culture conditions, contributing to the reduction of animal testing in accordance with the EU 3Rs principles (Replacement, Reduction, and Refinement). Moreover, the in silico models/AI tools under development will allow the in silico optimization of the procedures, reducing the number of experiments and saving costs.
In terms of knowledge translation, the potential for commercial exploitation of the SYNERGIES bioreactor platform is actively being explored, representing a promising opportunity for technology transfer and innovation beyond the academic context.
This SYNERGIES MSCA action also enabled the establishment of highly multidisciplinary international scientific network between the Fellow and the Host institution, already fostering joint grant applications, the exchange of MSc/PhD students, and the performance of other collaborative research projects.