Towards resorbable 3D-printed scaffolds for craniofacial bone regeneration.

This review will briefly examine the development of 3D-printed scaffolds for craniofacial bone regeneration. We will, in particular, highlight our work using Poly(L-lactic acid) (PLLA) and collagen-based bio-inks. This paper is a narrative review of the materials used for scaffold fabrication by 3D printing. We have also reviewed two types of scaffolds that we designed and fabricated. Poly(L-lactic acid) (PLLA) scaffolds were printed using fused deposition modelling technology. Collagen-based scaffolds were printed using a bioprinting technique. These scaffolds were tested for their physical properties and biocompatibility. Work in the emerging field of 3D-printed scaffolds for bone repair is briefly reviewed. Our work provides an example of PLLA scaffolds that were successfully 3D-printed with optimal porosity, pore size and fibre thickness. The compressive modulus was similar to, or better than, the trabecular bone of the mandible. PLLA scaffolds generated an electric potential upon cyclic/repeated loading. The crystallinity was reduced during the 3D printing. The hydrolytic degradation was relatively slow. Osteoblast-like cells did not attach to uncoated scaffolds but attached well and proliferated after coating the scaffold with fibrinogen. Collagen-based bio-ink scaffolds were also printed successfully. Osteoclast-like cells adhered, differentiated, and survived well on the scaffold. Efforts are underway to identify means to improve the structural stability of the collagen-based scaffolds, perhaps through mineralization by the polymer-induced liquid precursor process. 3D-printing technology is promising for constructing next-generation bone regeneration scaffolds. We describe our efforts to test PLLA and collagen scaffolds produced by 3D printing. The 3D-printed PLLA scaffolds showed promising properties akin to natural bone. Collagen scaffolds need further work to improve structural integrity. Ideally, such biological scaffolds will be mineralized to produce true bone biomimetics. These scaffolds warrant further investigation for bone regeneration.

Study of sacrificial ink-assisted embedded printing for 3D perfusable channel creation for biomedical applications.

For an engineered thick tissue construct to be alive and sustainable, it should be perfusable with respect to nutrients and oxygen. Embedded printing and then removing sacrificial inks in a cross-linkable yield-stress hydrogel matrix bath can serve as a valuable tool for fabricating perfusable tissue constructs. The objective of this study is to investigate the printability of sacrificial inks and the creation of perfusable channels in a cross-linkable yield-stress hydrogel matrix during embedded printing. Pluronic F-127, methylcellulose, and polyvinyl alcohol are selected as three representative sacrificial inks for their different physical and rheological properties. Their printability and removability performances have been evaluated during embedded printing in a gelatin microgel-based gelatin composite matrix bath, which is a cross-linkable yield-stress bath. The ink printability during embedded printing is different from that during printing in air due to the constraining effect of the matrix bath. Sacrificial inks with a shear-thinning property are capable of printing channels with a broad range of filaments by simply tuning the extrusion pressure. Bi-directional diffusion may happen between the sacrificial ink and matrix bath, which affects the sacrificial ink removal process and final channel diameter. As such, sacrificial inks with a low diffusion coefficient for gelatin precursor are desirable to minimize the diffusion from the gelatin precursor solution to minimize the post-printing channel diameter variation. For feasibility demonstration, a multi-channel perfusable alveolar mimic has been successfully designed, printed, and evaluated. The study results in the knowledge of the channel diameter controllability and sacrificial ink removability during embedded printing.

Effects of transglutaminase cross-linking process on printability of gelatin microgel-gelatin solution composite bioink.

Three-dimensional (3D) bioprinting has emerged as a powerful engineering approach for various tissue engineering applications, particularly for the development of 3D cellular structures with unique mechanical and/or biological properties. For the jammed gelatin microgel-gelatin solution composite bioink, comprising a discrete phase of microgels (enzymatically gelled gelatin microgels) and a cross-linkable continuous gelatin precursor solution-based phase containing transglutaminase (TG), its rheological properties and printability change gradually due to the TG enzyme-induced cross-linking process. The objective of this study is to establish a direct mapping between the printability of the gelatin microgel-gelatin solution based cross-linkable composite bioink and the TG concentration and cross-linking time, respectively. Due to the inclusion of TG in the composite bioink, the bioink starts cross-linking once prepared and is usually prepared right before a printing process. Herein, the bioink printability is evaluated based on the three metrics: injectability, feature formability, and process-induced cell injury. In this study, the rheological properties such as the storage modulus and viscosity have been first systematically investigated and predicted at different TG concentrations and times during the cross-linking process using the first-order cross-linking kinetics model. The storage modulus and viscosity have been satisfactorily modeled as exponential functions of the TG concentration and time with an experimentally calibrated cross-linking kinetic rate constant. Furthermore, the injectability, feature formability, and process-induced cell injury have been successfully correlated to the TG concentration and cross-linking time via the storage modulus, viscosity, and/or process-induced shear stress. By combing the good injectability, good feature formability, and satisfactory cell viability zones, a good printability zone (1.65, 0.61, and 0.31 h for the composite bioinks with 1.00, 2.00, and 4.00% w/v TG, respectively) has been established during the printing of mouse fibroblast-based 2% gelatin B microgel-3% gelatin B solution composite bioink. This printability zone approach can be extended to the use of other cross-linkable bioinks for bioprinting applications.

Injectable Gelatin Microgel-Based Composite Ink for 3D Bioprinting in Air.

Injectable hydrogels have attracted much attention in tissue engineering and regenerative medicine for their capability to replace implantation surgeries with a minimally invasive injection procedure and ability to fill irregular defects. The proposed composite ink is a gelatin microgel-based yield-stress and shear-thinning composite material that is injectable and solidifies quickly after injection at room temperature, which can be utilized for the creation of three-dimensional parts in air directly. The gelatin composite ink consists of a microgel solid phase (gelled gelatin microgels) and a cross-linkable solution phase (gelatin solution-based acellular or cellular suspension). The gelatin composite ink can be injected or printed directly in air and solidifies as physical cross-linking to hold printed structures at room temperature. The fabricated part further undergoes a chemical cross-linking process when immersed in a transglutaminase solution to enzymatically gel the gelatin solution, making a physiologically stable construct as needed. Lattice, tube-shaped, cup-shaped, and human anatomical (ear and nose) structures are printed to demonstrate the feasibility of the proposed composite ink for printing applications. The morphology and metabolic activity of cells cultured in the gelatin composite ink are further analyzed to confirm the suitability of the proposed composite ink to provide a beneficial physiological environment for bioprinting needs.

Cross-Linkable Microgel Composite Matrix Bath for Embedded Bioprinting of Perfusable Tissue Constructs and Sculpting of Solid Objects.

Tissue engineering is a rapidly growing field, which requires advanced fabrication technologies to generate cell-laden tissue analogues with a wide range of internal and external physical features including perfusable channels, cavities, custom shapes, and spatially varying material and/or cell compositions. A versatile embedded printing methodology is proposed in this work for creating custom biomedical acellular and cell-laden hydrogel constructs by utilizing a biocompatible microgel composite matrix bath. A sacrificial material is patterned within a biocompatible hydrogel precursor matrix bath using extrusion printing to create three-dimensional features; after printing, the matrix bath is cross-linked, and the sacrificial material is flushed away to create perfusable channels within the bulk composite hydrogel matrix. The composite matrix bath material consists of jammed cross-linked hydrogel microparticles (microgels) to control rheology during fabrication along with a fluid hydrogel precursor, which is cross-linked after fabrication to form the continuous phase of the composite hydrogel. For demonstration, gellan or enzymatically cross-linked gelatin microgels are utilized with a continuous gelatin hydrogel precursor solution to make the composite matrix bath herein; the composite hydrogel matrix is formed by cross-linking the continuous gelatin phase enzymatically after printing. A variety of features including discrete channels, junctions, networks, and external contours are fabricated in the proposed composite matrix bath using embedded printing. Cell-laden constructs with printed features are also evaluated; the microgel composite hydrogel matrices support cell activity, and printed channels enhance proliferation compared to solid constructs even in static culture. The proposed method can be expanded as a solid object sculpting method to sculpt external contours by printing a shell of sacrificial ink and further discarding excess composite hydrogel matrix after printing and cross-linking. While aqueous alginate solution is used as a sacrificial ink, more advanced sacrificial materials can be utilized for better printing resolution.

Printing of Hydrophobic Materials in Fumed Silica Nanoparticle Suspension.

Freeform three-dimensional (3D) printing of functional structures from liquid hydrophobic build materials is of great significance and widely used in various fields such as soft robotics and microfluidics. In particular, a yield-stress support bath-enabled 3D-printing methodology has been emerging to fabricate complex 3D structures. Unfortunately, the reported support bath materials are either hydrophobic or not versatile enough for the printing of a wide range of hydrophobic materials. The objective of this study is to propose a fumed silica nanoparticle-based yield-stress suspension as a hydrophobic support bath to enable 3D extrusion printing of various hydrophobic ink materials in a printing-then-solidification fashion. Hydrophobic ink is freeform-deposited in a hydrophobic fumed silica-mineral oil suspension and maintains its shape during printing; it is not cured until the whole structure is complete. Various hydrophobic inks including poly(dimethylsiloxane) (PDMS), SU-8 resin, and epoxy-based conductive ink are printed into complex 3D structures in the fumed silica-mineral oil bath and then cured using relevant cross-linking mechanisms, even at a temperature as high as 90 °C, to prove the feasibility and versatility of the proposed printing approach. In addition, the deposited feature can easily reach a much better resolution such as 30 µm for PDMS filaments due to the negligible interfacial tension effect.

Gellan Fluid Gel as a Versatile Support Bath Material for Fluid Extrusion Bioprinting.

Biomedical applications of three-dimensional (3D) printing demand complex hydrogel-based constructs laden with living cells. Advanced support materials facilitate the fabrication of such constructs. This work demonstrates the versatility and utility of a gellan fluid gel as a support bath material for fabricating freeform 3D hydrogel constructs from a variety of materials. Notably, the gellan fluid gel support bath can supply sensitive biological cross-linking agents such as enzymes to printed fluid hydrogel precursors for mild covalent hydrogel cross-linking. This mild fabrication approach is suitable for fabricating cell-laden gelatin-based constructs in which mammalian cells can form intercellular contacts within hours of fabrication; cellular activity is observed over several days within printed constructs. In addition, gellan is compatible with a wide range of ionic and thermal conditions, which makes it a suitable support material for ionically cross-linked structures generated by printing alginate-based ink formulations as well as thermosensitive hydrogel constructs formed from gelatin. Ultraviolet irradiation of printed structures within the support bath is also demonstrated for photoinitiated cross-linking of acrylated ink materials. Furthermore, gellan support material performance in terms of printed filament stability and residual support material on constructs is found to be comparable and superior, respectively, to previously reported support materials.