Periodic Reporting for period 4 - THIRST (Third Strategy in Tissue Engineering – Functional microfabricated multicellular spheroid carriers for tissue engineering and regeneration)
Período documentado: 2022-11-01 hasta 2023-10-31
Tissue engineering (TE) is a highly interdisciplinary research field with the long-term goal to restore and/or replace defective tissues. Taking into account increases in life expectancy and ageing population, this research field is more relevant than ever. Despite the enormous amount of new knowledge and undisputable progress achieved during the last few decades, current clinical advances in TE are largely represented by fragmented solutions restricted to a particular disease or tissue type. This situation is partially conditioned by the technical limitations of available methods and the inability to apply them universally for treatment of different target tissues and / or organs.
The overarching aim of the project is to develop a new technological platform sufficiently versatile to potentially address a wide range of current TE challenges. The concept of this third tissue engineering strategy (THIRST) relies on a directed tissue assembly from multicellular spheroids encaged within robust 3D printed microscaffolds. In contrast to the two most widespread approaches, namely scaffold-based and a scaffold-free approach, this project developed a radically new strategy combining the advantages of the two approaches.
The project results demonstrated several substantial advantages of using scaffolded spheroids. Microscaffolds allow to prepare large quantities of TE building blocks containing high cell densities, highly reproducible in terms of shape, size, but also the cell number. The presence of microscaffolds facilitates enhanced fusion of these building blocks, resulting in rapid formation of cohesive tissue constructs by bottom-up self-assembly. Furthermore, supported by the microscaffolds these TE constructs preserve their overall volume throughout the spheroid fusion and tissue maturation process, crucial for stable filling of tissue defects. The first in vivo studies conducted using a drill-hole model in rodents and osteochondral defects in rabbits confirmed these conclusions. Finally, the scaffolds can be used to deliver and release bioactive molecules, such as growth factors, as was demonstrated on the example of vascular endothelial growth factor (VEGF).
The overarching aim of the project is to develop a new technological platform sufficiently versatile to potentially address a wide range of current TE challenges. The concept of this third tissue engineering strategy (THIRST) relies on a directed tissue assembly from multicellular spheroids encaged within robust 3D printed microscaffolds. In contrast to the two most widespread approaches, namely scaffold-based and a scaffold-free approach, this project developed a radically new strategy combining the advantages of the two approaches.
The project results demonstrated several substantial advantages of using scaffolded spheroids. Microscaffolds allow to prepare large quantities of TE building blocks containing high cell densities, highly reproducible in terms of shape, size, but also the cell number. The presence of microscaffolds facilitates enhanced fusion of these building blocks, resulting in rapid formation of cohesive tissue constructs by bottom-up self-assembly. Furthermore, supported by the microscaffolds these TE constructs preserve their overall volume throughout the spheroid fusion and tissue maturation process, crucial for stable filling of tissue defects. The first in vivo studies conducted using a drill-hole model in rodents and osteochondral defects in rabbits confirmed these conclusions. Finally, the scaffolds can be used to deliver and release bioactive molecules, such as growth factors, as was demonstrated on the example of vascular endothelial growth factor (VEGF).
One of the main technological aims of the project was to develop the means for a drastic upscaling of microscaffold production and their handling. This was achieved by developing a prototype of a next generation two-photon polymerization (2PP) system, based on a resonant scanner technology, allowing to increase the laser scanning speed to over 60 m/s, which is two orders of magnitude faster than any currently available commercial device. An according publication is currently under revision in the Elsevier journal of Additive Manufacturing. The systematic studies allowed to develop material formulations uniquely suitable for high-resolution 2PP of highly porous microscaffolds. Thorough characterization of these materials, including biocompatibility evaluation using extract protocols, in accordance to ISO 10993-5 standard, along with in vitro degradation study and 2PP-processing results, served as a basis for a manuscript published in a highly cited Elsevier journal Materials Today in 2021. The screening and selection of appropriate photoinitiators allowed to reduce the unwanted autofluorescence for these materials, which can be quite bothersome during fluorescence imaging of THIRST tissue constructs. Furthermore, a dedicated microfluidic system, capable of handling of these microscaffolds in a high-throughput manner, was successfully developed.
Apart from the capability of microscaffolds to dramatically improve the quality of the TE building blocks based on spheroids, it was demonstrated that the presence of the microscaffolds supports differentiation of spheroids formed from stem cells into osteogenic and chondrogenic lineages, evidenced the comparable level of gene expression markers and extra cellular matrix deposition. These results were published in the Acta Biomaterialia journal in 2023. It is known that fusion of multiple spheroids leads to compaction and an according reduction of overall volume of the tissue self-assembled in this way. The TE constructs produced from scaffolded-spheroids preserve their overall volume throughout the spheroid fusion and tissue maturation process, a crucial characteristic for stable filling of tissue defects. Furthermore, on the example of scaffolded-spheroids pre-differentiated into the chondrogenic lineage it was shown that the presence of microscaffold substantially improved the fusion process. A novel method was developed to quantify this. The results were published in the Acta Biomaterialia in 2024.
Apart from the capability of microscaffolds to dramatically improve the quality of the TE building blocks based on spheroids, it was demonstrated that the presence of the microscaffolds supports differentiation of spheroids formed from stem cells into osteogenic and chondrogenic lineages, evidenced the comparable level of gene expression markers and extra cellular matrix deposition. These results were published in the Acta Biomaterialia journal in 2023. It is known that fusion of multiple spheroids leads to compaction and an according reduction of overall volume of the tissue self-assembled in this way. The TE constructs produced from scaffolded-spheroids preserve their overall volume throughout the spheroid fusion and tissue maturation process, a crucial characteristic for stable filling of tissue defects. Furthermore, on the example of scaffolded-spheroids pre-differentiated into the chondrogenic lineage it was shown that the presence of microscaffold substantially improved the fusion process. A novel method was developed to quantify this. The results were published in the Acta Biomaterialia in 2024.
The project developments on the technological side, enabled a substantial increase in the 2PP throughput, an order of magnitude over the current state of the art. Furthermore, the new biodegradable material concept allowed to overcome limitations associated with the 2PP-based high-resolution structuring, setting the new standard for the biomedical applications of this technology. The developments enabled for the first time to produce microscaffolds and scaffolded-spheroids in quantities that can be used for systematic TE studies. Multiple advantages were demonstrated using these microscaffolds, from the supreme TE block quality, to improved fusion of spheroids and possibility to preserve the intended construct volume. Furthermore, a novel method to measure the force necessary to separate the pairs of fused spheroids was developed, allowing to quantify the fusion strength. This new method is very versatile and can be used to study the effects of spheroid composition, treatment, fusion protocols etc, extending its applicability far beyond THIRST project and TE area in general.