Novel 3D scaffolds for bone formation and cell printing

Abstract

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.

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Identifiers

ORCID for Gianluca Cidonio: orcid.org/0000-0002-9445-6994

ORCID for Shoufeng Yang: orcid.org/0000-0002-3888-3211

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