Suspension bath bioprinting and maturation of anisotropic meniscal constructs.

Due to limited intrinsic healing capacity of the meniscus, meniscal injuries pose a significant clinical challenge. The most common method for treatment of damaged meniscal tissues, meniscectomy, leads to improper loading within the knee joint, which can increase the risk of osteoarthritis. Thus, there is a clinical need for the development of constructs for meniscal repair that better replicate meniscal tissue organization to improve load distributions and function over time. Advanced three-dimensional bioprinting technologies such as suspension bath bioprinting provide some key advantages, such as the ability to support the fabrication of complex structures using non-viscous bioinks. In this work, the suspension bath printing process is utilized to print anisotropic constructs with a unique bioink that contains embedded hydrogel fibers that align via shear stresses during printing. Constructs with and without fibers are printed and then cultured for up to 56 din vitroin a custom clamping system. Printed constructs with fibers demonstrate increased cell and collagen alignment, as well as enhanced tensile moduli when compared to constructs printed without fibers. This work advances the use of biofabrication to develop anisotropic constructs that can be utilized for the repair of meniscal tissue.

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., "microgels"). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

Computational Modeling and Experimental Characterization of Extrusion Printing into Suspension Baths.

The extrusion printing of inks into suspension baths is an exciting tool, as it allows the printing of diverse and soft hydrogel inks into 3D space without the need for layer-by-layer fabrication. However, this printing process is complex and there have been limited studies to experimentally and computationally characterize the process. In this work, hydrogel inks (i.e., gelatin methacrylamide (GelMA)), suspension baths (i.e., agarose, Carbopol), and the printing process are examined via rheological, computational, and experimental analyses. Rheological data on various hydrogel inks and suspension baths is utilized to develop computational printing simulations based on Carreau constitutive viscosity models of the printing of inks within suspension baths. These results are then compared to experimental outcomes using custom print designs where features such as needle translation speed, defined in this work as print speed, are varied and printed filament resolution is quantified. Results are then used to identify print parameters for the printing of a GelMA ink into a unique guest-host hyaluronic acid suspension bath. This work emphasizes the importance of key rheological properties and print parameters for suspension bath printing and provides a computational model and experimental tools that can be used to inform the selection of print settings.

Programmable and contractile materials through cell encapsulation in fibrous hydrogel assemblies.

The natural extracellular matrix (ECM) within tissues is physically contracted and remodeled by cells, allowing the collective shaping of functional tissue architectures. Synthetic materials that facilitate self-assembly similar to natural ECM are needed for cell culture, tissue engineering, and in vitro models of development and disease. To address this need, we develop fibrous hydrogel assemblies that are stabilized with photocrosslinking and display fiber density­dependent strain-responsive properties (strain stiffening and alignment). Encapsulated mesenchymal stromal cells locally contract low fiber density assemblies, resulting in macroscopic volumetric changes with increased cell densities and moduli. Because of properties such as shear-thinning and self-healing, assemblies can be processed into microtissues with aligned ECM deposition or through extrusion bioprinting and photopatterning to fabricate constructs with programmed shape changes due to cell contraction. These materials provide a synthetic approach to mimic features of natural ECM, which can now be processed for applications in biofabrication and tissue engineering.

A biofabrication method to align cells within bioprinted photocrosslinkable and cell-degradable hydrogel constructs via embedded fibers.

The extracellular matrix (ECM) is composed of biochemical and biophysical cues that control cell behaviors and bulk mechanical properties. For example, anisotropy of the ECM and cell alignment are essential in the directional properties of tissues such as myocardium, tendon, and the knee meniscus. Technologies are needed to introduce anisotropic behavior into biomaterial constructs that can be used for the engineering of tissues as models and towards translational therapies. To address this, we developed an approach to align hydrogel fibers within cell-degradable bioink filaments with extrusion printing, where shear stresses during printing align fibers and photocrosslinking stabilizes the fiber orientation. Suspensions of hydrogel fibers were produced through the mechanical fragmentation of electrospun scaffolds of norbornene-modified hyaluronic acid, which were then encapsulated with meniscal fibrochondrocytes, mesenchymal stromal cells, or cardiac fibroblasts within gelatin-methacrylamide bioinks during extrusion printing into agarose suspension baths. Bioprinting parameters such as the needle diameter and the bioink flow rate influenced shear profiles, whereas the suspension bath properties and needle translation speed influenced filament diameters and uniformity. When optimized, filaments were formed with high levels of fiber alignment, which resulted in directional cell spreading during culture over one week. Controls that included bioprinted filaments without fibers or non-printed hydrogels of the same compositions either with or without fibers resulted in random cell spreading during culture. Further, constructs were printed with variable fiber and resulting cell alignment by varying print direction or using multi-material printing with and without fibers. This biofabrication technology advances our ability to fabricate constructs containing aligned cells towards tissue repair and the development of physiological tissue models.

Bioprinting for the Biologist.

Building tissues from scratch to explore entirely new cell configurations could revolutionize fundamental understanding in biology. Bioprinting is an emerging technology to do this. Although typically applied to engineer tissues for therapeutic tissue repair or drug screening, there are many opportunities for bioprinting within biology, such as for exploring cellular crosstalk or cellular morphogenesis. The overall goals of this Primer are to provide an overview of bioprinting with the biologist in mind, outline the steps in extrusion bioprinting (the most widely used and accessible technology), and discuss alternative bioprinting technologies and future opportunities for bioprinting in biology.

Recent Advances in Enabling Technologies in 3D Printing for Precision Medicine.

Advances in areas such as data analytics, genomics, and imaging have revealed individual patient complexities and exposed the inherent limitations of generic therapies for patient treatment. These observations have also fueled the development of precision medicine approaches, where therapies are tailored for the individual rather than the broad patient population. 3D printing is a field that intersects with precision medicine through the design of precision implants with patient-directed shapes, structures, and materials or for the development of patient-specific in vitro models that can be used for screening precision therapeutics. Toward their success, advances in 3D printing and biofabrication technologies are needed with enhanced resolution, complexity, reproducibility, and speed and that encompass a broad range of cells and materials. The overall goal of this progress report is to highlight recent advances in 3D printing technologies that are helping to enable advances important in precision medicine.

Influence of 3D printed porous architecture on mesenchymal stem cell enrichment and differentiation.

The interactions between cells and an underlying biomaterial are important for the promotion of cell adhesion, proliferation, and function. Mesenchymal stem cells (MSCs) have great clinical potential as they are an adult stem cell population capable of multilineage differentiation. The relationship between MSC behavior and several material properties including substrate stiffness and pore size are well investigated, but there has been little research on the influence of porous architecture in a three-dimensional scaffold with a well-controlled architecture. Here, we investigate the impact of two different three-dimensionally printed, pore geometries on the enrichment and differentiation of MSCs. 3D printed scaffolds with ordered cubic pore geometry were supportive of MSC enrichment from unprocessed bone marrow, resulting in cell surface marker expression that was comparable to typical adhesion to tissue culture polystyrene, the gold standard for MSC culture. Results also show that scaffolds fabricated with ordered cubic pores significantly increase the gene expression of MSCs undergoing adipogenesis and chondrogenesis, when compared to scaffolds with ordered cylindrical pores. However, at the protein expression level, these differences were modest. For MSCs undergoing osteogenesis, gene expression results suggest that cylindrical pores may initially increase early osteogenic marker expression, while protein level expression at later timepoints is increased for scaffolds with ordered cubic pores. Taken together, these results suggest that 3D printed scaffolds with ordered cubic pores could be a suitable culture system for single-step MSC enrichment and differentiation. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) have great therapeutic potential, as they are capable of multilineage differentiation. MSC behavior, including lineage commitment, may be influenced by biomaterial properties including substrate stiffness and pore size. With three-dimensional (3D) printing, we can investigate these relationships in 3D culture systems. Here, we fabricated scaffolds with two different well-controlled pore geometries, and investigated the impact on MSC enrichment and differentiation. Results show that scaffolds with ordered cubic pore geometry were supportive of both MSC enrichment from unprocessed bone marrow as well as MSC differentiation, resulting in increased gene expression during adipogenesis and chondrogenesis. These results suggest that 3D printed scaffolds with ordered cubic pores could be a suitable culture system for single-step MSC enrichment and differentiation.

A supramolecular helix that disregards chirality.

The functions of complex crystalline systems derived from supramolecular biological and non-biological assemblies typically emerge from homochiral programmed primary structures via first principles involving secondary, tertiary and quaternary structures. In contrast, heterochiral and racemic compounds yield disordered crystals, amorphous solids or liquids. Here, we report the self-assembly of perylene bisimide derivatives in a supramolecular helix that in turn self-organizes in columnar hexagonal crystalline domains regardless of the enantiomeric purity of the perylene bisimide. We show that both homochiral and racemic perylene bisimide compounds, including a mixture of 21 diastereomers that cannot be deracemized at the molecular level, self-organize to form single-handed helical assemblies with identical single-crystal-like order. We propose that this high crystalline order is generated via a cogwheel mechanism that disregards the chirality of the self-assembling building blocks. We anticipate that this mechanism will facilitate access to previously inaccessible complex crystalline systems from racemic and homochiral building blocks.

Homochiral columns constructed by chiral self-sorting during supramolecular helical organization of hat-shaped molecules.

A library of dendronized cyclotriveratrylene (CTV) crowns substituted with chiral, racemic, or achiral peripheral alkyl chains, including enantiopure R and S branched alkyls, "racemic by mixture", "racemic by synthesis", n-octyl, and n-dodecyl groups was synthesized. In solvophobic solvents and in bulk they self-assemble in helical columns. Their solution and bulk shape-persistent supramolecular structures were determined by a complementary combination of circular dichroism (CD) and UV in solution and thin film, microspot CD in thin film, differential scanning calorimetry combined with fiber X-ray diffraction, computer simulation, and molecular models. In solution, self-assembly via a cooperative mechanism generates single-handed columns from enantiopure CTVs and mixtures of right- and left-handed columns from racemic by mixture, racemic by synthesis, other combinations of R and S, and even from achiral compounds. In bulk state all supramolecular columns form a 3D hexagonal crystalline phase, Φ(h)(k) (P63 symmetry), that can be obtained only from single-handed columns and a columnar hexagonal 2D liquid crystal, Φ(h). The highest order Φ(h)(k) consists of enantiopure single-handed columns that are slightly distorted 12-fold triple helices. The "hat-shaped" dendronized CTV assembles in bent-branch pine-tree columns that allow interdigitation of alkyl groups in adjacent columns regardless of their direction. Enantiomerically rich, racemic, and achiral compositions undergo deracemization in the crystal state by transfer of the transient disc-like conformer of dendronized CTV from column to column during crown inversion. Solid state NMR experiments identified motional processes that allow such transfer. This unprecedented supramolecular chiral self-sorting will impact the creation of functions in complex systems.

Development of a dynamic stem cell culture platform for mesenchymal stem cell adhesion and evaluation.

The importance of providing a physiologically relevant environment for cell culture is well recognized. The combination of proper environmental cues which are provided in vivo by the bloodstream and extracellular matrix must be reproduced to properly examine cell response in vitro, and cannot be recapitulated using traditional culture on polystyrene. Here, we have developed a device, the dynamic stem cell culture platform (DSCCP), consisting of a biomimetic scaffold cultured within the dynamic environment of a perfusion bioreactor. By varying scaffold parameters including stiffness and protein inclusion at the material surface, we found that human mesenchymal stem cells (hMSCs) were able to adhere to modified substrates, while still maintaining multipotency. Culture in a perfusion bioreactor showed cell survival and proliferation, particularly on modified substrates. The DSCCP represents a complete platform for cell adhesion and subsequent evaluation, including the response of a cell population to drug treatment.