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

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

3D printing of biphasic inks: beyond single-scale architectural control

To date, Additive Manufacturing (AM) has come to the fore as a major disruptive technology embodying two main research lines – developing increasingly sophisticated printing technologies and new processable materials. The latter has fostered a tremendous leap in AM technological advancement, allowing 3D printing to play a central role in dictating the tailorable settings for material design. In particular, the manufacturing of three-dimensional (3D) objects with functional hierarchical porous structure is of the utmost importance for numerous research areas, including tissue engineering, catalysis, aerospace, environmental science, electrochemistry, energy and sound absorption and light engineering materials. Biphasic inks such as emulsions, foams, and solid dispersions represent viable templating systems to realise multiscale porosity. The combination of AM techniques and biphasic inks provide pivotal control over multiple levels of material structure and function, enabling the use of advanced materials with unprecedented 3D architectures as well as physical, chemical, and mechanical properties. The related potential benefits are significant, with functional perspectives for a wide variety of research fields. In this concise review, we provide an updated overview of the employment of biphase inks and show how they are adapted to different AM technologies or vice versa

3D printing of inorganic-biopolymer composites for bone regeneration

In most cases, bone injuries heal without complications, however, there is an increasing number of instances where bone healing needs major clinical intervention. Available treatment options have severe drawbacks, such as donor site morbidity and limited availability for autografting. Bone graft substitutes containing growth factors would be a viable alternative, however they have been associated with dose-related safety concerns and lack control over spatial architecture to anatomically match bone defect sites. A 3D printing offers a solution to produce patient specific bone graft substitutes that are customized to the patient bone defect with temporal control over the incorporated therapeutics to maximize their efficacy. Inspired by the natural constitution of bone tissue, composites made of inorganic phases, such as nanosilicate particles, calcium phosphate, and bioactive glasses, combined with biopolymer matrices have been investigated as building blocks for the biofabrication of bone constructs. Besides capturing elements of the bone physiological structure, these inorganic/organic composites can be designed for specific cohesivity, rheological and mechanical properties, while both inorganic and organic constituents contribute to the composite bioactivity. This review provides an overview of 3D printed composite biomaterial-inks for bone tissue engineering. Furthermore, key aspects in biomaterial-ink design, 3D printing techniques, and the building blocks for composite biomaterial-inks are summarized.ISSN:1758-5082ISSN:1758-509

Printing bone in a gel: using nanocomposite bioink to print functionalised bone scaffolds

Free-form printing offers a novel biofabrication approach to generate complex shapes by depositing hydrogel materials within a temporary supportive environment. However, printed hydrogels typically lack the requisite mechanical properties and functionality of the desired tissue, limiting application and, more importantly, safety and efficacy of the implant. We have developed an innovative nanoclay-based bioink to print high shape fidelity functional constructs for potential skeletal application. Laponite® (LAP) nanoclay was combined with gellan gum (GG) to generate a printable hydrogel that was highly stable in vitro, displayed limited swelling ability compared to the silicate-free control and remained stable over time. An agarose fluid gel was found to provide the requisite support for the deposition of the material ink and preservation of the printed structure prior to crosslinking. Printed C2C12 myoblasts remained viable and displayed extensive proliferation over 21 days in culture. Cell-laden scaffolds demonstrated functionality within 1 day of culture in vitro and that was preserved over 3 weeks. Analysis of absorption and release mechanisms from LAP-GG using model proteins (lysozyme and bovine serum albumin (BSA)) demonstrated the retention capability of the clay-based materials for compound localisation and absence of burst release. Vascular endothelial growth factor (VEGF) was loaded within the agarose fluid gel and absorbed by the material ink via absorbtion during deposition. The 3D printed constructs was implanted on the chorioallantoic membrane of a 10-days old developing chick. Extensive and preferential vasculature infiltration was observed in LAP-GG loaded VEGF constructs compared to controls (p<0.01 and p<0.0001) after only 7 days of incubation. The current studies demonstrate, for the first time, the application of innovative LAP-GG 3D constructs in the generation of growth factor loaded 3D constructs for potential application in skeletal tissue repair

Biofabrication of nanocomposite-based scaffolds containing human bone extracellularmatrix 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