Periodic Reporting for period 2 - REBORN (Full human-based multi-scale constructs with jammed regenerative pockets for bone engineering)
Reporting period: 2023-05-01 to 2024-10-31
Engineered bone tissue has been viewed as a potential alternative to the traditional use of bone grafts, due to their limitless supply and reduced or no risk of disease transmission. Even though great advances have been achieved in bone tissue engineering (TE), those new platforms have not proceeded to clinical practice as they fail to fully recreate the suitable conditions to provide relevant vascularized grafts and, most importantly, enable their in vivo integration and remodeling. REBORN proposes rather unique toolboxes combining bioinstructive biomaterials fully based on human proteins obtained from the placenta (PC) and cells from the umbilical/blood cord. This project aims to offer ground-breaking advances in engineering totally time-self-regulated complex 3D devices, able to adjust the cascade of processes leading to faster high-quality and vascularized new bone tissue formation with minimum pre-processing of cells. Proteins from PC are chemically modified with bioorthogonal clickable moieties, enabling their selective association during the fabrication of liquified pockets or hydrogels. The liquified biofactories are being produced with a perm-selective PC protein membrane in order to confine all necessary ingredients for internal in vitro tissue development to recreate the bone niche, including (i) the correct cells’ ratio, (ii) hydrogel MicroBlocks that provides geometrical, mechanical and topographic cues to control cellular behavior and (iii) bioactive soluble factors. The developed human-based protein hydrogels are used to assemble the liquified pockets into a desired implantable device, with clinically relevant size, shape, and structural integrity, using non-conventional 3D bioprinting processing methodologies. With such an integrated multidisciplinary approach and given the expertise of the PI and the research conditions of our research group, we believe that the project REBORN will further advance the development of new strategies for bone TE. This ERC grant will contribute to the exploitation of ground-breaking materials, concepts, and methodologies that can be clinically explored to ultimately impact patients’ life quality.
For the development of the human-based tissue engineering devices proposed in the REBORN project, cells, and extracellular matrices have been isolated from perinatal tissues. In this context, mesenchymal stem cells (MSCs) have been isolated from the Wharton jelly of the umbilical cord, as a reliable alternative source of the bone marrow MSCs. Additionally, endothelial cells were isolated from the same tissue and used as a substitute for the outgrowth endothelial cells obtained from the peripheral blood. Such as the umbilical cord, the placenta is a widely available clinical waste obtained from a non-invasive procedure, that exhibits very interesting biochemical features for tissue engineering purposes. Therefore, the whole placenta and the individual amniotic and chorionic membranes were isolated, decellularized, enzymatically digested, and solubilized in order to be chemically modified. Of note, tissues from multiple donors were pooled to minimize the lot-to-lot variability and ensure high reproducible experiments. Diverse protein-targeting chemical groups were conjugated with these human-derived materials through cross-reactive chemical modifications using clickable and photoresponsive moieties, rendering high cell-friendly and viscoelastic hydrogels whose biochemical and mechanical properties perfectly match diverse tissues’ features. These innovative human-derived biomaterials were used to fabricate liquified pockets in which a robust PC protein shell was formed employing processing technologies based on an emulsion technique, including electrodynamic atomization and microfluidics. Leveraging the potential of these liquified pockets to confine biological materials in a semi-closed soft environment, we have been working on the development of a new generation of protein MicroBlocks with customizable size, shape, topography, and stiffness, to promote cell attachment and self-assembly, while mediating cell fate in a controllable way. Different in vitro assays demonstrated the successful encapsulation of cells and MicroBlocks in a liquified biofactory. We also incorporated magnetic nanoparticles into the MicroBlocks to regulate cell differentiation in a dynamic pseudo-3D environment controlled by a magnetic field, in which the biophysical stimulation improved the robustness of the microtissues and their osteogenic differentiation over the static culture. Interestingly, we were able to create an autonomous homeostatic bone microcompartment by encapsulating osteoblast- and osteoclast-like cells. Envisioning the fabrication of more complex 3D structures for bone remodeling, different bottom-up strategies are being studied, including unconventional 3D bioprinting and robot arm-mediated liquified pockets assembly. The results achieved so far under the scope of the REBORN project provide great indications of the potential of the developed hydrogel biomaterials and derivative microstructures to more efficiently direct stem cell differentiation towards a relevant 3D bone tissue construct.
Following up on the progress made, we are now focused on taking advantage of the best biomechanical properties of the hydrogel biomaterials produced so far for the development of mechanically tunable MicroBlocks. In the same line, we are also producing pseudo-3D microgels of human PC proteins with novel complex geometries to specifically modulate cell fate into the 3D bone-like microtissues. We are also combining the human proteins modified with different chemical moieties to obtain stable and viscoelastic hydrogels with enhanced mechanical properties. The liquified protein biofactories fabrication is currently being optimized using microfluidic technology, since this technique allows the production of small and monodispersed pockets with high yield. The combination of cells and design-specific MicroBlocks in this micro-compartmentalized liquified system enables the cell self-organization into a 3D microtissue where the cells can secrete bioactive mediators of bone tissue formation. These self-regulated systems are also intended to be used for disease modeling and drug screening. In parallel, we are also exploiting different bottom-up strategies to create highly complex 3D constructs. The developed human-derived biomaterials are being tested as bioinks to be used with unconventional 3D bioprinting techniques, which can ultimately incorporate the produced liquified biofactories to ensure a fully autonomous and self-regulated system. Additionally, the creation of hierarchical 3D structures is intended to be achieved using a controllable robotic arm.