Harnessing biofabrication strategies to re-surface osteochondral defects. Repair, enhance, and regenerate

Osteochondral tissue (OC) is a complex and multiphasic system comprising cartilage and subchondral bone. The discrete OC architecture is layered with specific zones characterized by different compositions, morphology, collagen orientation, and chondrocyte phenotypes. To date, the treatment of osteochondral defects (OCD) remains a major clinical challenge due to the low self-regenerative capacity of damaged skeletal tissue, as well as the critical lack of functional tissue substitutes. Current clinical approaches fail to fully regenerate damaged OC recapitulating the zonal structure while granting long-term stability. Thus, the development of new biomimetic treatment strategies for the functional repair of OCDs is urgently needed. Here, we review recent developments in the preclinical investigation of novel functional approaches for the resurfacing of skeletal defects. The most recent studies on preclinical augmentation of OCDs and highlights on novel studies for the in vivo replacement of diseased cartilage are presented

Microfluidic fiber spinning for 3D bioprinting: Harnessing microchannels to build macrotissues

Microfluidics is rapidly revolutionizing the scientific panorama, providing unmatched high-throughput platforms that find application in numerous areas of physics, chemistry, biology, and materials science. Recently, microfluidic chips have been proposed, in combination with bioactive materials, as promising tools for spinning cell-laden fibers with on-demand characteristics. However, cells encapsulated in filaments produced via microfluidic spinning technology are confined in a quasi-three-dimensional (3D) environment that fails to replicate the intricate 3D architecture of biological tissues. Thanks to the recent synergistic combination of microfluidic devices with 3D bioprinting technologies that enable the production of sophisticated microfibers serving as the backbone of 3D structures, a new age of tissue engineering is emerging. This review looks at how combining microfluidics with 3D printing is contributing to the biofabrication of relevant human substitutes and implants. This paper also describes the whole manufacturing process from the production of the microfluidic tool to the printing of tissue models, focusing on cutting-edge fabrication technologies and emphasizing the most noticeable achievements for microfluidic spinning technology. A theoretical insight for thixotropic hydrogels is also proposed to predict the fiber size and shear stress developing within microfluidic channels. The potential of using microfluidic chips as bio-printheads for multi-material and multi-cellular bioprinting is discussed, highlighting the challenges that microfluidic bioprinting still faces in advancing the field of biofabrication for tissue engineering and regenerative medicine purposes

Rapid Production of Nanoscale Liposomes Using a 3D-Printed Reactor-In-A-Centrifuge: Formulation, Characterisation, and Super-Resolution Imaging

Nanoscale liposomes have been extensively researched and employed clinically for the delivery of biologically active compounds, including chemotherapy drugs and vaccines, offering improved pharmacokinetic behaviour and therapeutic outcomes. Traditional laboratory-scale production methods often suffer from limited control over liposome properties (e.g., size and lamellarity) and rely on laborious multistep procedures, which may limit pre-clinical research developments and innovation in this area. The widespread adoption of alternative, more controllable microfluidic-based methods is often hindered by complexities and costs associated with device manufacturing and operation, as well as the short device lifetime and the relatively low liposome production rates in some cases. In this study, we demonstrated the production of liposomes comprising therapeutically relevant lipid formulations, using a cost-effective 3D-printed reactor-in-a-centrifuge (RIAC) device. By adjusting formulation- and production-related parameters, including the concentration of polyethylene glycol (PEG), temperature, centrifugation time and speed, and lipid concentration, the mean size of the produced liposomes could be tuned in the range of 140 to 200 nm. By combining selected experimental parameters, the method was capable of producing liposomes with a therapeutically relevant mean size of ~174 nm with narrow size distribution (polydispersity index, PDI ~0.1) at a production rate of >8 mg/min. The flow-through method proposed in this study has potential to become an effective and versatile laboratory-scale approach to simplify the synthesis of therapeutic liposomal formulations

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite® ) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone- ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair

Differences in the organization of interface residues tunes the stability of the SARS-CoV-2 spike-ACE2 complex

The continuous emergence of novel variants represents one of the major problems in dealing with the SARS-CoV-2 virus. Indeed, also due to its prolonged circulation, more than ten variants of concern emerged, each time rapidly overgrowing the current viral version due to improved spreading features. As, up to now, all variants carry at least one mutation on the spike Receptor Binding Domain, the stability of the binding between the SARS-CoV-2 spike protein and the human ACE2 receptor seems one of the molecular determinants behind the viral spreading potential. In this framework, a better understanding of the interplay between spike mutations and complex stability can help to assess the impact of novel variants. Here, we characterize the peculiarities of the most representative variants of concern in terms of the molecular interactions taking place between the residues of the spike RBD and those of the ACE2 receptor. To do so, we performed molecular dynamics simulations of the RBD-ACE2 complexes of the seven variants of concern in comparison with a large set of complexes with different single mutations taking place on the RBD solvent-exposed residues and for which the experimental binding affinity was available. Analyzing the strength and spatial organization of the intermolecular interactions of the binding region residues, we found that (i) mutations producing an increase of the complex stability mainly rely on instaurating more favorable van der Waals optimization at the cost of Coulombic ones. In particular, (ii) an anti-correlation is observed between the shape and electrostatic complementarities of the binding regions. Finally, (iii) we showed that combining a set of dynamical descriptors is possible to estimate the outcome of point mutations on the complex binding region with a performance of 0.7. Overall, our results introduce a set of dynamical observables that can be rapidly evaluated to probe the effects of novel isolated variants or different molecular systems

De Novo Design of Functional Co-Assembling Organic-Inorganic Hydrogels for Hierarchical Mineralization and Neovascularization

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite™, Lap) and peptide amphiphiles (PAs, PAH3) rationally designed to co-assemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessedto confirm that PAH3 nanofibers electrostatically adsorbed and conformed to the surface of Lapnanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface areaof PAH3nanofibers after co-assembly with Lap. Dynamic oscillatory rheology revealed that the co-assembled PAH3-Laphydrogels displayed high stiffness and robust self-healing behaviour while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional matrix(3D) provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3Dmatrix. Applicability of the organic-inorganic PAH3-Laphydrogels was assessed in vitrousing human bone marrow-derived stromal cells(hBMSCs) and ex vivousing a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganic PAH3-Laphydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls

Engineering next-generation bioinks with nanoparticles: moving from reinforcement fillers to multifunctional nanoelements

The application of additive manufacturing in the biomedical field has become a hot topic in the last decade owing to its potential to provide personalized solutions for patients. Different bioinks have been designed trying to obtain a unique concoction that addresses all the needs for tissue engineering and drug delivery purposes, among others. Despite the remarkable progress made, the development of suitable bioinks which combine printability, cytocompatibility, and biofunctionality is still a challenge. In this sense, the well-established synthetic and functionalization routes to prepare nanoparticles with different functionalities make them excellent candidates to be combined with polymeric systems in order to generate suitable multi-functional bioinks. In this review, we briefly discuss the most recent advances in the design of functional nanocomposite hydrogels considering their already evaluated or potential use as bioinks. The scientific development over the last few years is reviewed, focusing the discussion on the wide range of functionalities that can be incorporated into 3D bioprinted constructs through the addition of multifunctional nanoparticles in order to increase their regenerative potential in the field of tissue engineering.Authors acknowledge financial support from the ERC Grant CoG MagTendon nr 772817; FCT – Fundação para a Ciência e a Tecnologia for the PhD grant of SMB (PD/BD/129403/2017), for the contract to MGF (CEECIND/01375/2017); and for project SmarTendon (PTDC/NAN-MAT/30595/2017). AP is grateful to Xunta de Galicia for his postdoctoral grant ED481B2019/025. Some figures were created with BioRender.com

(Photo-)crosslinkable gelatin derivatives for biofabrication applications

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for bio-fabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs

Novel 3D scaffolds for bone formation and cell printing

Current approaches to treat bone fractures typically use: i) autologous bone graft harvested from the patient, which can be proved painful, and ii) non-degradable metal implants that provide the mechanical support needed, but can require numerous revisions and replacement. Biofabrication has come to the fore to target the unmet clinical needs in orthopaedic regenerative medicine aiming to produce degradable tissue-like structures in an automated fashion by the simultaneous extrusion of biomaterials (bioinks) and living cells. Such an approach contains a number of challenges including post-printing cell damage and limited functionality. The biofabrication paradigm involves the use of high polymeric content bioinks to ensure shape fidelity which often impacts on cell viability. Biopolymer-silicate nanocomposite hydrogels at low polymer fractions present remarkable shear-thinning and tuneable viscoelastic properties, ideal for bioprinting purposes.  This study has examined a library of clay-based bioinks for the fabrication of threedimensional functional constructs for skeletal regeneration. This thesis investigates the hypothesis that a clay nanomaterial (Laponite, LAP) can be used to enhance printability and functionality of i) alginate-methylcellulose, ii) gellan gum and iii) gelatin methacryloyl bioinks.  Laponite-alginate-methylcellulose bioink (named 3-3-3 after the 3 % w/v concentration of each component) was fully characterised in vitro and the behaviour of printed cells investigated during 21 days of culture. Cells displayed evidence of proliferation (p<0.0001) after 7, 14 and 21 days in clay-based bioinks compared to silicate-free constructs. Skeletal stem cells (SSCs) were encapsulated and printed with the bioink to create viable and functional 3D scaffolds cultured in vitro for 21 days. Scaffolds implanted in a chick chorioallantoic membrane (CAM) model displayed excellent integration and vascular infiltration. SSCs-laden 3-3-3 printed scaffolds implanted subcutaneously in a mouse model induced significant (p<0.0001) new bone formation compared to acellular scaffolds and bulk controls.  Addition of Laponite to gellan gum (GG) significantly (p<0.0001) modulated the swelling kinetics. LAP-GG nanocomposite, printed in an agarose fluid gel, was found to sustain cell viability over 21 days in vitro, and support the functionality of printed cells evidenced by the significant (p<0.0001) alkaline phosphatase expression at 7 and 21 days compared to GG alone. LAP-GG scaffolds displayed functionality in a CAM model when absorbed in VEGF-agarose solution during printing evidenced by enhanced angiogenesis.  Laponite and GelMA (LAP-GelMA) bioink was observed to be printable with the application of a visible-light crosslinking technology during extrusion, producing scaffolds with significant (p<0.0001) shape fidelity. SSCs remained viable and functional in LAPGelMA constructs. Drugs were demonstrated to be absorbable in cast discs which displayed enhanced (p<0.0001) angiogenesis and integration when implanted in CAM model following VEGF absorption.  Overall, the results presented in this thesis auger well for the generation of innovative approaches to deliver skeletal populations and bioactive agents for orthopaedic application using 3D printing technologies and clay-based bioinks

The cell in the ink: improving biofabrication by printing stem cells for skeletal regenerative medicine

Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i) post-printing cell damage, (ii) proliferation impairment and, (iii) poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro, clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population