Emulsion Inks for 3D Printing of High Porosity Materials.

Photocurable emulsion inks for use with solid freeform fabrication (SFF) to generate constructs with hierarchical porosity are presented. A high internal phase emulsion (HIPE) templating technique was utilized to prepare water-in-oil emulsions from a hydrophobic photopolymer, surfactant, and water. These HIPEs displayed strong shear thinning behavior that permitted layer-by-layer deposition into complex shapes and adequately high viscosity at low shear for shape retention after extrusion. Each layer was actively polymerized with an ultraviolet cure-on-dispense (CoD) technique and compositions with sufficient viscosity were able to produce tall, complex scaffolds with an internal lattice structure and microscale porosity. Evaluation of the rheological and cure properties indicated that the viscosity and cure rate both played an important role in print fidelity. These 3D printed polyHIPE constructs benefit from the tunable pore structure of emulsion templated material and the designed architecture of 3D printing. As such, these emulsion inks can be used to create ultra high porosity constructs with complex geometries and internal lattice structures not possible with traditional manufacturing techniques.

Injectable polymerized high internal phase emulsions with rapid in situ curing.

Polymerized high internal phase emulsions (polyHIPEs) have been utilized in the creation of injectable scaffolds that cure in situ to fill irregular bone defects and potentially improve tissue healing. Previously, thermally initiated scaffolds required hours to cure, which diminished the potential for clinical translation. Here, a double-barrel syringe system for fabricating redox-initiated polyHIPEs with dramatically shortened cure times upon injection was demonstrated with three methacrylated macromers. The polyHIPE cure time, compressive properties, and pore architecture were investigated with respect to redox initiator chemistry and concentration. Increased concentrations of redox initiators reduced cure times from hours to minutes and increased the compressive modulus and strength without compromising the pore architecture. Additionally, storage of the uncured emulsion at reduced temperatures for 6 months was shown to have minimal effects on the resulting graft properties. These studies indicate that the uncured emulsions can be stored in the clinic until they are needed and then rapidly cured after injection to rigid, high-porosity scaffolds. In summary, we have improved upon current methods of generating injectable polyHIPE grafts to meet translational design goals of long storage times and rapid curing (<15 min) without sacrificing porosity or mechanical properties.

Hydroxyapatite nanoparticle-modified porous bone grafts with improved cell attachment.

Emulsion-templated foams have displayed promise as injectable bone grafts; however, the use of a surfactant as an emulsifier resulted in relatively small pores and impedes cell attachment. Hydroxyapatite nanoparticles were explored as an alternative stabilizer to address these limitations. To this end, hydroxyapatite nanoparticles were first modified with myristic acid to generate the appropriate balance of hydrophobicity to stabilize a water-in-oil emulsion of neopentyl glycol diacrylate and 1,4-butanedithiol. In situ surface modification of the resulting foam with hydroxyapatite was confirmed with elemental mapping and transmission electron microscopy. Nanoparticle-stabilized foams displayed improved human mesenchymal stem cell viability (91 ± 5%) over surfactant-stabilized foams (23 ± 11%). Although the pore size was appropriate for bone grafting applications (115 ± 71 µm), the foams lacked the interconnected architecture necessary for cell infiltration. We hypothesized that a co-stabilization approach with both surfactant and nanoparticles could be used to achieve interconnected pores while maintaining improved cell attachment and larger pore sizes. A range of hydroxyapatite nanoparticle and surfactant concentrations were investigated to determine the effects on microarchitecture and cell behavior. By balancing these interactions, a co-stabilized foam was identified that possessed large, interconnected pores (108 ± 67 µm) and improved cell viability and attachment. The co-stabilized foam was then evaluated as an injectable bone graft including network formation, microscale integration with bone, push out strength, and compressive properties. Overall, this work demonstrated that in situ surface modification with nHA improved cell attachment while retaining desirable bone grafting features and injectability.

Engineering Toolbox for Systematic Design of PolyHIPE Architecture.

Polymerization of high internal phase emulsions (polyHIPEs) is a well-established method for the production of high porosity foams. Researchers are often regulated to using a time-intensive trial and error approach to achieve target pore architectures. In this work, we performed a systematic study to identify the relative effects of common emulsion parameters on pore architecture (mixing speed, surfactant concentration, organic phase viscosity, molecular hydrophobicity). Across different macromer chemistries, the largest magnitude of change in pore size was observed across surfactant concentration (~6 fold, 5-20 wt%), whereas changing mixing speeds (~4 fold, 500-2000 RPM) displayed a reduced effect. Furthermore, it was observed that organic phase viscosity had a marked effect on pore size (~4 fold, 6-170 cP) with no clear trend observed with molecular hydrophobicity in this range (logP = 1.9-4.4). The efficacy of 1,4-butanedithiol as a reactive diluent was demonstrated and provides a means to reduce organic phase viscosity and increase pore size without affecting polymer fraction of the resulting foam. Overall, this systematic study of the microarchitectural effects of these macromers and processing variables provides a framework for the rational design of polyHIPE architectures that can be used to accelerate design and meet application needs across many sectors.

Review of Integrin-Targeting Biomaterials in Tissue Engineering.

The ability to direct cell behavior has been central to the success of numerous therapeutics to regenerate tissue or facilitate device integration. Biomaterial scientists are challenged to understand and modulate the interactions of biomaterials with biological systems in order to achieve effective tissue repair. One key area of research investigates the use of extracellular matrix-derived ligands to target specific integrin interactions and induce cellular responses, such as increased cell migration, proliferation, and differentiation of mesenchymal stem cells. These integrin-targeting proteins and peptides have been implemented in a variety of different polymeric scaffolds and devices to enhance tissue regeneration and integration. This review first presents an overview of integrin-mediated cellular processes that have been identified in angiogenesis, wound healing, and bone regeneration. Then, research utilizing biomaterials are highlighted with integrin-targeting motifs as a means to direct these cellular processes to enhance tissue regeneration. In addition to providing improved materials for tissue repair and device integration, these innovative biomaterials provide new tools to probe the complex processes of tissue remodeling in order to enhance the rational design of biomaterial scaffolds and guide tissue regeneration strategies.

Improved in situ seeding of 3D printed scaffolds using cell-releasing hydrogels.

The design of tissue engineered scaffolds based on polymerized high internal phase emulsions (polyHIPEs) has emerged as a promising bone grafting strategy. We previously reported the ability to 3D print emulsion inks to better mimic the structure and mechanical properties of native bone while precisely matching defect geometry. In the current study, redox-initiated hydrogel carriers were investigated for in situ delivery of human mesenchymal stem cells (hMSCs) utilizing the biodegradable macromer, poly(ethylene glycol)-dithiothreitol. Hydrogel carrier properties including network formation time, sol-gel fraction, and swelling ratio were modulated to achieve rapid cure without external stimuli and a target cell-release period of 5-7 days. These in situ carriers enabled improved distribution of hMSCs in 3D printed polyHIPE grafts over standard suspension seeding. Additionally, carrier-loaded polyHIPEs supported sustained cell viability and osteogenic differentiation of hMSCs post-release. In summary, these findings demonstrate the potential of this in situ curing hydrogel carrier to enhance the cell distribution and retention of hMSCs in bone grafts. Although initially focused on improving bone regeneration, the ability to encapsulate cells in a hydrogel carrier without relying on external stimuli that can be attenuated in large grafts or tissues is expected to have a wide range of applications in tissue engineering.

Fabrication of biomimetic bone grafts with multi-material 3D printing.

Extrusion deposition is a versatile method for the 3D printing of biomaterials such as hydrogels, ceramics, and suspensions. Recently, a new class of emulsion inks were developed that can be used to create tunable, hierarchically porous materials with a cure-on-dispense method. Propylene fumarate dimethacrylate (PFDMA) was selected to fabricate bone grafts using this technology due to its established biocompatibility, osteoconductivity, and good compressive properties. Scaffolds fabricated from PFDMA emulsion inks displayed compressive modulus and yield strength of approximately 15 and 1 MPa, respectively. A decrease in infill (from 100% to 70%) resulted in a six-fold increase in permeability; however, there was also a corollary decrease in mechanical properties. In order to generate scaffolds with increased permeability without sacrificing mechanical strength, a biomimetic approach to scaffold design was used to reinforce the highly porous emulsion inks with a dense cortical shell of thermoplastic polyester. Herein, we present an open source method for printing multi-material bone grafts based on PFDMA polyHIPEs with hierarchical porosity and reinforced with a dense shell of poly(ε-caprolactone) (PCL) or poly(lactic acid) (PLA). A multi-modal printing setup was first developed that combined paste extrusion and high temperature thermoplastic extrusion with high positional accuracy in dual deposition. Scaffolds printed with a PCL shell displayed compressive modulus and yield strength of approximately 30 and 3 MPa, respectively. Scaffolds printed with a PLA shell showed compressive modulus and yield strength of approximately 100 and 10 MPa, respectively. By combining this new paste extrusion of emulsion inks with traditional thermoplastic extrusion printing, we have created scaffolds with superior strength that promote cell viability and proliferation of human mesenchymal stem cells. The development of this technique shows great promise for the fabrication of a myriad of other complex tissue engineered scaffolds.

A Review of Three-Dimensional Printing in Tissue Engineering.

Recent advances in three-dimensional (3D) printing technologies have led to a rapid expansion of applications from the creation of anatomical training models for complex surgical procedures to the printing of tissue engineering constructs. In addition to achieving the macroscale geometry of organs and tissues, a print layer thickness as small as 20 µm allows for reproduction of the microarchitectures of bone and other tissues. Techniques with even higher precision are currently being investigated to enable reproduction of smaller tissue features such as hepatic lobules. Current research in tissue engineering focuses on the development of compatible methods (printers) and materials (bioinks) that are capable of producing biomimetic scaffolds. In this review, an overview of current 3D printing techniques used in tissue engineering is provided with an emphasis on the printing mechanism and the resultant scaffold characteristics. Current practical challenges and technical limitations are emphasized and future trends of bioprinting are discussed.