Extrusion 3D printing of a multiphase collagen-based material: An optimized strategy to obtain biomimetic scaffolds with high shape fidelity.

Extrusion printing represents one of the leading additive manufacturing techniques for tissue engineering purposes due to the possibility of achieving accurate control of the final shape and porosity of the scaffold. Despite many polymeric materials having already been optimized for this application, the processing of biopolymer-based systems still presents several limitations mainly ascribed to their poor rheological properties. Moreover, the introduction of inorganic components into the biomaterial formulation may introduce further difficulties related to system homogeneity, finally compromising its extrudability. In this context, the present study aimed at developing a new multi-phase biomaterial ink able to mimic the native composition of bone extracellular matrix, combining type-I-collagen with nano-hydroxyapatite and mesoporous bioactive glass nanoparticles. Starting from a comprehensive rheological assessment, computational-fluid-dynamics-based models were exploited to describe the material flow regime and define the optimal printing process planning. During printing, a gelatin-based bath was exploited to support the deposition of the material, while the gelation of collagen and its further chemical crosslinking with genipin enabled the stabilization of the printed structure, characterized by high shape fidelity. The developed strategy enables the extrusion printing of complex multi-phase systems and the design of high-precision biomimetic scaffolds with great potential for bone tissue engineering.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Cholecalciferol as Bioactive Plasticizer of High Molecular Weight Poly(D,L-Lactic Acid) Scaffolds for Bone Regeneration.

Synthetic thermoplastic polymers are a widespread choice as material candidates for scaffolds for tissue engineering (TE), thanks to their ease of processing and tunable properties with respect to biological polymers. These features made them largely employed in melt-extrusion-based additive manufacturing, with particular application in hard-TE. In this field, high molecular weight (Mw) polymers ensuring entanglement network strength are often favorable candidates as scaffold materials because of their enhanced mechanical properties compared with lower Mw grades. However, this is accompanied by high viscosities once processed in molten conditions, which requires driving forces not always accessible technically or compatible with often chemically nonstabilized biomedical grades. When possible, this is circumvented by increasing the operating temperature, which often results in polymer chain scission and consequent degradation of properties. In addition, synthetic polymers are mostly considered bioinert compared with biological materials, and additional processing steps are often required to make them favorable for tissue regeneration. In this study, we report the plasticization of a common thermoplastic polymer with cholecalciferol, the metabolically inactive form of vitamin D3 (VD3). Plasticization of the polymer allowed us to reduce its melt viscosity, and therefore the energy requirements (mechanical [torque] and heat [temperature]) for extrusion, limiting ultimately polymer degradation. In addition, we evaluated the effect of cholecalciferol, which is more easily available than its active counterpart, on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Results indicated that cholecalciferol supported osteogenic differentiation more than the osteogenic culture medium, suggesting that hMSCs possess the enzymatic toolbox for VD3 metabolism. Impact statement Limitations in mechanical and biological performances of scaffolds manufactured through melt deposition may result from material thermal degradation during processing and inherent bioinertness of synthetic polymers. Current approaches involve the incorporation of chemical additives to reduce the extent of thermal degradation, which are often nonbiocompatible or may lead to uncontrolled modifications to the polymer structure. Lack of polymer bioactivity is tackled by postfunctionalization methods that often involve extra processes extending scaffold production time. Therefore, new methods to improve scaffolds performances should consider preserving the integrity of the molecular structure and improving biological responsiveness of the material while keeping the process as straightforward as possible.

Additive Manufacturing Using Melt Extruded Thermoplastics for Tissue Engineering.

Melt extrusion of thermoplastic materials is an important technique for fabricating tissue engineering scaffolds by additive manufacturing methods. Scaffold manufacturing is commonly achieved by one of the following extrusion-based techniques: fused deposition modelling (FDM), 3D-fiber deposition (3DF), and bioextrusion. FDM needs the input material to be strictly in the form of a filament, whereas 3DF and bioextrusion can be used to process input material in several forms, such as pellets or powder. This chapter outlines a common workflow for all these methods, going from the material to a scaffold, while highlighting the special requirements of particular methods. A few ways of characterizing the scaffolds are also briefly described.

Additive Manufactured Scaffolds for Bone Tissue Engineering: Physical Characterization of Thermoplastic Composites with Functional Fillers.

Thermoplastic polymer-filler composites are excellent materials for bone tissue engineering (TE) scaffolds, combining the functionality of fillers with suitable load-bearing ability, biodegradability, and additive manufacturing (AM) compatibility of the polymer. Two key determinants of their utility are their rheological behavior in the molten state, determining AM processability and their mechanical load-bearing properties. We report here the characterization of both these physical properties for four bone TE relevant composite formulations with poly(ethylene oxide terephthalate)/poly(butylene terephthalate (PEOT/PBT) as a base polymer, which is often used to fabricate TE scaffolds. The fillers used were reduced graphene oxide (rGO), hydroxyapatite (HA), gentamicin intercalated in zirconium phosphate (ZrP-GTM) and ciprofloxacin intercalated in MgAl layered double hydroxide (MgAl-CFX). The rheological assessment showed that generally the viscous behavior dominated the elastic behavior (G″ > G') for the studied composites, at empirically determined extrusion temperatures. Coupled rheological-thermal characterization of ZrP-GTM and HA composites showed that the fillers increased the solidification temperatures of the polymer melts during cooling. Both these findings have implications for the required extrusion temperatures and bonding between layers. Mechanical tests showed that the fillers generally not only made the polymer stiffer but more brittle in proportion to the filler fractions. Furthermore, the elastic moduli of scaffolds did not directly correlate with the corresponding bulk material properties, implying composite-specific AM processing effects on the mechanical properties. Finally, we show computational models to predict multimaterial scaffold elastic moduli using measured single material scaffold and bulk moduli. The reported characterizations are essential for assessing the AM processability and ultimately the suitability of the manufactured scaffolds for the envisioned bone regeneration application.