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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ATLAS (Bioengineered autonomous cell-biomaterials devices for generating humanised micro-tissues for regenerative medicine)

Teaser

Summary

New generations of devices for tissue engineering (TE) should rationalize better the physical and biochemical cues operating in tandem during native regeneration, in particular at the scale/organizational-level of the stem cell niche. The understanding and the deconstruction of these factors (e.g. multiple cell types exchanging both paracrine and direct signals, structural and chemical arrangement of the extra-cellular matrix, mechanical signalsâ€¦) should be then incorporated into the design of truly biomimetic biomaterials. ATLAS proposes rather unique toolboxes combining smart biomaterials and cells for the ground-breaking advances of engineering fully time-self-regulated complex 2D and 3D devices, able to adjust the cascade of processes leading to faster high quality new tissue formation with minimum pre-processing of cells. Versatile biomaterials based on marine-origin macromolecules are being used, namely in the supramolecular assembly of instructive multilayers as nanostratified building-blocks for engineer such structures. The backbone of these biopolymers are equipped with a variety of (bio)chemical elements permitting: post-processing chemistry and micro-patterning, specific/nonspecific cell attachment, and cell-controlled degradation. Aiming at being applied in bone TE, ATLAS integrates cells from different units of tissue physiology, namely bone and hematopoietic basic elements and consider the interactions between the immune and skeletal systems. These ingredients permit to architect innovative films with high-level dialogue control with cells, but in particular sophisticated quasi-closed 3D capsules able to compartmentalise such components in a â€œglobe-likeâ€ organization, providing local and long-range order for in vitro microtissue development and function. Bone is used as the model system but it is expected that the developed methodologies can be used for an all range of other tissues at a later stage, as ATLAS concepts are mounted onto rather fundamental bases. Besides the potential in regenerative medicine, we envisage to apply the developed capsules in two other important areas: bioengineered cell niches for basic biological research and in vitro platforms for modelling disease and drug screening. We believe that ATLAS, with its integrated multidisciplinary approach and the unique research environment found at our research group and the expertises of the PI, will for sure allow for a major step further in the development of radically new bone TE strategies. We trust that under this ERC grant framework, knowledge will be created, but simultaneously we will be able to optimize procedures and devices that might be exploited clinically. This is really applied research, with a very strong basic and fundamental support, and with the use of ground-breaking concepts and methodologies, that can create value and in the long-term an impact in the economy and quality of life of patients.

Work performed

As an alternative source of bone marrow mesenchymal stem cells (MSCs) we have been encapsulating MSCs isolated from the Wharton jelly of the umbilical cord. Additionally, we are also using endothelial cells using the same tissue, and thus the same donor, as an alternative source of outgrowth endothelial cells isolated from peripheral blood. Therefore, umbilical cord represents an appealing alternative source from which all the required cell phenotypes could be obtained from the same donor. Isolating cells from umbilical cord also avoids the inherent risks of invasive procedures to collect MSCs from bone marrow and peripheral blood. We also obtained new conjugates of a marine-derived polysaccharide by incorporating molecules with specific properties. The new conjugates were tested as potential candidates to develop new hydrogels and to multilayer assemblies performed using the layer-by-layer technology (LbL), as the polymers and the new groups inserted have different charges, allowing this way to assemble them into multilayers. The polysaccharides identified were then used to produce multilayer coatings and films. Besides the coatings on the hydrogel templates to produce liquefied and multilayered capsules, we are also employing the technology to coat nano and micro spherical templates that could have applications in the development of drug delivery devices. We have also developed a tailor-made device comprising (i) a perforated platform accommodating miniaturized three-dimensional (3D) biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable and easy-to-use perfusion chamber. The device was assembled using widely available low-cost plastic labware, and could be upscaled to parallelizable setups, increasing the number of analyzed platforms per experiment. For the specific case of the ATLAS project the results achieved so far are extremely useful to develop surface modified microparticles that could direct stem cell behaviour in a more efficient way, in particular, it can be implemented in the design experiment to produce surface modified microparticles responsible to provide cell adhesion sites in the liquefied environment of capsules. We also functionalized the multilayered membrane of capsules with a peptide motif known to favour cell attachment in order to produce an indirect co-culture system with pre-osteoblastic (core) and endothelial (membrane) cells. While the encapsulated cells proliferated and deposited a mineralized matrix forming aggregates with surface modified microparticles, cells on the outside of the membrane adhered and aggregated the liquified and multilayered capsules. Different in vitro assays were performed to demonstrate the success of the bioencapsulation system proposed. Additionally, after implantation in a chick chorioallantoic membrane (CAM), the proposed capsules were able to recruit new vessels evidencing their angiogenic potential. Afterwards, we tested the developed capsules under a dynamic cell culture environment. Interestingly, we observed that the dynamic environment led to the development of larger aggregates of cells and microparticles, providing biophysical stimulation and robust improvements in microtissues formation and in the osteogenic differentiation fate of the encapsulated cells over static culture. Ultimately, we also produced microplatforms as bottom-up strategies to improve the anchoring sites dispersed in the liquefied environment of capsules. For that, the surface of the microfilms was modified to present micron-sized grooves, aiming to deliver cues for differentiation of cells into the osteogenic lineage. Results show that cells are able to attach to microfilms and microspheres and produce their own 3D culture system by creating aggregates. The larger surface area provided by microfilms comparing to microspheres led to an improved biological outcome in terms of metabolic activity, proliferation, and osteogenic diff

Final results

We are now focused in exploring new complex geometries besides spherical systems to produce highly organized encapsulation systems that better mimic the complex hierarchical organization of native tissues. We are also testing the application of harsh conditions, such as high pressure, on the biological outcome of the encapsulated cells, namely in their osteogenic differentiation fate. The compartmentalized capsules are currently being employed as a magneto-responsive living bioreactor that aims to dynamically stimulate encapsulated key cells and promote the secretion of bioactive mediators of bone tissue formation upon implantation. Additionally, the proposed bioreactor that enables the incorporation of the biomaterial-containing array into a disposable and easy-to-use perfusion chamber represents a great output of the project, since it allows to test several conditions in 3D, rather than in the 2D conditions that we have previously envisage. We also intend to explore the applicability of the liquefied and multilayered capsules as external bioreactors to produce microtissues in vitro that could also be used as drug testing platforms. Since one of the disadvantages of the LbL process is its inherent time-consuming, which can have an impact on cell viability, we are developing a protocol to reduce the required immersion time and thus, to reduce significantly the overall process of the multilayered membrane build-up. In parallel, we are also developing an one-step method by using an aqueous biphasic system with oppositely charged polyelectrolytes dissolved in each phase. We are also developing a hermetically sealed encapsulation system which will be able to, in the absence of in vitro manipulation, provide all the required conditions to assure the survival of cells in an fully autonomously and self-regulated fashion.