Direct ink writing of multifunctional nanocellulose and allyl-modified gelatin biomaterial inks for the fabrication of mechanically and functionally graded constructs.

Recreating the intricate mechanical and functional gradients found in natural tissues through additive manufacturing poses significant challenges, including the need for precise control over time and space and the availability of versatile biomaterial inks. In this proof-of-concept study, we developed a new biomaterial ink for direct ink writing, allowing the creation of 3D structures with tailorable functional and mechanical gradients. Our ink formulation combined multifunctional cellulose nanofibrils (CNFs), allyl-functionalized gelatin (0.8-2.0 wt%), and polyethylene glycol dithiol (3.0-7.5 wt%). The CNF served as a rheology modifier, whereas a concentration of 1.8 w/v % in the inks was chosen for optimal printability and shape fidelity. In addition, CNFs were functionalized with azido groups, enabling the spatial distribution of functional moieties within a 3D structure. These functional groups were further modified using a spontaneous click chemistry reaction. Through additive manufacturing and a readily available static mixer, we successfully demonstrated the fabrication of mechanical gradients - ranging from 3 to 6 kPa in indentation strength - and functional gradients. Additionally, we introduced dual gradients by combining gradient printing with an anisotropic photocrosslinking step. The developed biomaterial ink opens up possibilities for printing intricate multigradient structures, resembling the complex hierarchical organization seen in living tissues.

3D Printed Solutions for Spheroid Engineering and Cancer Research.

In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)-was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.

Biobridge: An Outlook on Translational Bioinks for 3D Bioprinting.

3D-bioprinting (3DBP) possesses several elements necessary to overcome the deficiencies of conventional tissue engineering, such as defining tissue shape a priori, and serves as a bridge to clinical translation. This transformative potential of 3DBP hinges on the development of the next generation of bioinks that possess attributes for clinical use. Toward this end, in addition to physicochemical characteristics essential for printing, bioinks need to possess proregenerative attributes, while enabling printing of stable structures with a defined biological function that survives implantation and evolves in vivo into functional tissue. With a focus on bioinks for extrusion-based bioprinting, this perspective review advocates a rigorous biology-based approach to engineering bioinks, emphasizing efficiency, reproducibility, and a streamlined translation process that places the clinical endpoint front and center. A blueprint for engineering the next generation of bioinks that satisfy the aforementioned performance criteria for various translational levels (TRL1-5) and a characterization tool kit is presented.

Extrusion-Based 3D Bioprinting of Gradients of Stiffness, Cell Density, and Immobilized Peptide Using Thermogelling Hydrogels.

To study biological processes in vitro, biomaterials-based engineering solutions to reproduce the gradients observed in tissues are necessary. We present a platform for the 3D bioprinting of functionally graded biomaterials based on carboxylated agarose, a bioink amendable by extrusion bioprinting. Using this bioink, objects with a gradient of stiffness and gradient of cell concentration were printed. Functionalization of carboxylated agarose with maleimide moieties that react in minutes with a cysteine-terminated cell-adhesion peptide allowed us to print objects with a gradient of an immobilized peptide. This approach paves the way toward the development of tissue mimics with gradients.

Hydrogel-Forming Algae Polysaccharides: From Seaweed to Biomedical Applications.

With the increasing growth of the algae industry and the development of algae biorefinery, there is a growing need for high-value applications of algae-extracted biopolymers. The utilization of such biopolymers in the biomedical field can be considered as one of the most attractive applications but is challenging to implement. Historically, polysaccharides extracted from seaweed have been used for a long time in biomedical research, for example, agarose gels for electrophoresis and bacterial culture. To overcome the current challenges in polysaccharides and help further the development of high-added-value applications, an overview of the entire polysaccharide journey from seaweed to biomedical applications is needed. This encompasses algae culture, extraction, chemistry, characterization, processing, and an understanding of the interactions of soft matter with living organisms. In this review, we present algae polysaccharides that intrinsically form hydrogels: alginate, carrageenan, ulvan, starch, agarose, porphyran, and (nano)cellulose and classify these by their gelation mechanisms. The focus of this review further lays on the culture and extraction strategies to obtain pure polysaccharides, their structure-properties relationships, the current advances in chemical backbone modifications, and how these modifications can be used to tune the polysaccharide properties. The available techniques to characterize each organization scale of a polysaccharide hydrogel are presented, and the impact on their interactions with biological systems is discussed. Finally, a perspective of the anticipated development of the whole field and how the further utilization of hydrogel-forming polysaccharides extracted from algae can revolutionize the current algae industry are suggested.

Injectable biocompatible poly(2-oxazoline) hydrogels by strain promoted alkyne-azide cycloaddition.

Poly(2-alkyl-2-oxazoline) (PAOx) hydrogels are tailorable synthetic materials with demonstrated biomedical applications, thanks to their excellent biocompatibility and tunable properties. However, their use as injectable hydrogels is challenging as it requires invasive surgical procedures to insert the formed hydrogel into the body due to their nonsoluble 3D network structures. Herein, we introduce cyclooctyne and azide functional side chains to poly(2-oxazoline) copolymers to induce in situ gelation using strain promoted alkyne-azide cycloaddition. The gelation occurs rapidly, within 5 min, under physiological conditions when two polymer solutions are simply mixed. The influence of several parameters, such as temperature and different aqueous solutions, and stoichiometric ratios between the two polymers on the structural properties of the resultant hydrogels have been investigated. The gel formation within tissue samples was verified by subcutaneous injection of the polymer solution into an ex vivo model. The degradation study of the hydrogels in vitro showed that the degradation rate was highly dependent on the type of media, ranging from days to a month. This result opens up the potential uses of PAOx hydrogels in attempts to achieve optimal, injectable drug delivery systems and tissue engineering.

Advanced Bioink for 3D Bioprinting of Complex Free-Standing Structures with High Stiffness.

One of the challenges in 3D-bioprinting is the realization of complex, volumetrically defined structures, that are also anatomically accurate and relevant. Towards this end, in this study we report the development and validation of a carboxylated agarose (CA)-based bioink that is amenable to 3D printing of free-standing structures with high stiffness at physiological temperature using microextrusion printing without the need for a fugitive phase or post-processing or support material (FRESH). By blending CA with negligible amounts of native agarose (NA) a bioink formulation (CANA) which is suitable for printing with nozzles of varying internal diameters under ideal pneumatic pressure was developed. The ability of the CANA ink to exhibit reproducible sol-gel transition at physiological temperature of 37 °C was established through rigorous characterization of the thermal behavior, and rheological properties. Using a customized bioprinter equipped with temperature-controlled nozzle and print bed, high-aspect ratio objects possessing anatomically-relevant curvature and architecture have been printed with high print reproducibility and dimension fidelity. Objects printed with CANA bioink were found to be structurally stable over a wide temperature range of 4 °C to 37 °C, and exhibited robust layer-to-layer bonding and integration, with evenly stratified structures, and a porous interior that is conducive to fluid transport. This exceptional layer-to-layer fusion (bonding) afforded by the CANA bioink during the print obviated the need for post-processing to stabilize printed structures. As a result, this novel CANA bioink is capable of yielding large (5-10 mm tall) free-standing objects ranging from simple tall cylinders, hemispheres, bifurcated 'Y'-shaped and 'S'-shaped hollow tubes, and cylinders with compartments without the need for support and/or a fugitive phase. Studies with human nasal chondrocytes showed that the CANA bioink is amenable to the incorporation of high density of cells (30 million/mL) without impact on printability. Furthermore, printed cells showed high viability and underwent mitosis which is necessary for promoting remodeling processes. The ability to print complex structures with high cell densities, combined with excellent cell and tissue biocompatibility of CA bodes well for the exploitation of CANA bioinks as a versatile 3D-bioprinting platform for the clinical translation of regenerative paradigms.

Hydrogels with Cell Adhesion Peptide-Decorated Channel Walls for Cell Guidance.

A method is reported for making hollow channels within hydrogels decorated with cell-adhesion peptides exclusively at the channel surface. Sacrificial fibers of different diameters are used to introduce channels within poly(ethylene glycol) hydrogels crosslinked with maleimide-thiol chemistry, which are backfilled with a cysteine-containing peptide solution which is conjugated to the lumen with good spatial efficiency. This allows for peptide patterning in only the areas of the hydrogel where they are needed when used as cell-guides, reducing the amount of required peptide 20-fold when compared to bulk functionalization. The power of this approach is highlighted by successfully using these patterned hydrogels without active perfusion to guide fibroblasts and olfactory ensheathing cells-the latter having unique potential in neural repair therapies.

Going beyond RGD: screening of a cell-adhesion peptide library in 3D cell culture.

In tissue engineering, cell-adhesion peptides (CAPs) such as the ubiquitous arginine-glycine-aspartic acid (RGD) sequence have allowed the functionalization of synthetic materials to mimic macromolecules of the extracellular matrix (ECM). However, the variety of ECM macromolecules makes it challenging to reproduce all of the native tissue functions with only a limited variety of CAPs. Screening of libraries of CAPs, analogous to high-throughput drug discovery assays, can help to identify new sequences directing cell organization. However, challenges to this approach include the automation of cell seeding in three dimensions and characterization methods. Here, we report a method for robotically generating a library of 16 CAPs to identify a microenvironment capable of directing a chain-like morphology in olfactory ensheathing cells (OECs), a cell type of particular interest for guiding axon growth in spinal cord injury repair. This approach resulted in the identification of one CAP not previously reported to interact with OECs to direct their morphology into structures suitable for potential axon guidance. The same screening approach should be applicable to any range of cell types to discover new CAPs to direct cell fate or function.

Facile preparation of tissue engineering scaffolds with pore size gradients using the muesli effect and their application to cell spheroid encapsulation.

Porous biodegradable scaffolds have many applications in bioengineering, ranging from cell culture and transplantation, to support structures, to induce blood vessel and tissue formation in vivo. While numerous strategies have been developed for the manufacture of porous scaffolds, it remains challenging to control the spatial organization of the pores. In this study, we introduce the use of the granular convection effect, also known as the muesli or brazil nut effect, to rapidly engineer particulate templates with a vertical size gradient. These templates can then be used to prepare scaffolds with pore size gradients. To demonstrate this approach, we prepared templates with particle size gradients, which were then infused with a prepolymer solution consisting of the pentaerythritol ethoxylate (polyol), sebacoyl chloride (acid chloride), and poly(caprolactone). Following curing, the template was dissolved to yield biodegradable polyester-ether scaffolds with pore size gradients that could be tuned depending on the size range of the particulates used. The application of these scaffolds was demonstrated using pancreatic islets, which were loaded via centrifugation and retained within the scaffold's pores without a decrease in viability. The proposed strategy provides a facile approach to prepare templates with spatially organized pores that could potentially be used for cell transplantation, or guided tissue formation.

Biotin-Avidin-Mediated Capture of Microspheres on Polymer Fibers.

Systems for efficient and selective capture of micro-scale objects and structures have application in many areas and are of particular relevance for selective isolation of mammalian cells. Systems for the latter should also not interfere with the biology of the cells. This study demonstrates the capture of microspheres through orthogonal coupling using biotin (ligand) and (strept)avidin (receptor). Fibrous poly(ethylene terephthalate) (PET) meshes were hydrolyzed under controlled alkaline conditions to obtain activated surfaces with COOH groups allowing for the functionalization of the PET with biotin of various spacer length. The system capture efficiency was optimized by varying the length of spacer presenting the biotin against streptavidin. In a proof of concept experiment, avidin-functionalized microspheres were used as surrogates for cells, and their capture under dynamic conditions including virous mixing and high-flow rate perfusion is demonstrated. Functionalization of PET meshes with biotin conjugated to longest spacer yielded the most efficient capture of microspheres. These preliminary results lay the foundation for the development of biosystems for capture of specific cells under physiologically relevant conditions, using biorthogonal avidin-biotin interactions.

RGDSP functionalized carboxylated agarose as extrudable carriers for chondrocyte delivery.

The limited potential of cartilage to regenerate itself has led to development of new strategies and biomaterials for cartilage tissue engineering and regenerative medicine. Although de novo strategies for cartilage repair have been realized, extrudable hydrogels that can be administered in minimally invasive manner while simultaneously supporting chondrogenic differentiation could lead to development of new systems to deliver cells to cartilage lesions. In this work, we explored the suitability of thermo-reversible, extrudable gels derived from carboxylated agarose for maintaining human articular chondrocyte (HAC) phenotype. Towards this objective, we have investigated the impact of hydrogel stiffness and presence of integrin-binding peptide sequence GGGGRGDSP on HAC differentiation potential. We discovered that stiffer hydrogels (5.8 kPa) are more efficient than softer counterparts (0.6 kPa) in promoting chondrogenesis. Interestingly, in GGGGRGDSP modified gels, a synergy between stiffness and RGD signaling led to enhanced expression of chondrogenic related genes (aggrecan, collagen type II and sox9). These findings were also supported by quantitative analysis of sulfated glycosaminoglycans. Since carboxylated agarose are highly suitable as bioink for 3D bioprinting, we propose that extrudable GGGGRGDSP-linked stiff carboxylated agarose as a medium for direct printing of chondrocyte into cartilage lesion.

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature, Which Correlates with the Enrichment of a Novel Piezo-1+ Population of Circulating CD11b+ /CD115+ Monocytes.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

Discovering Cell-Adhesion Peptides in Tissue Engineering: Beyond RGD.

As an alternative to natural extracellular matrix (ECM) macromolecules, cell-adhesion peptides (CAPs) have had tremendous impact on the design of cell culture platforms, implants, and wound dressings. However, only a handful of CAPs have been utilized. The discrepancy in ECM composition strongly affects cell behavior, so it is paramount to reproduce such differences in synthetic systems. This Opinion article presents strategies inspired from high-throughput screening techniques implemented in drug discovery to exploit the potential of a growing CAP library. These strategies are expected to promote the use of a broader spectrum of CAPs, which in turn could lead to improved cell culture models, implants, and wound dressings.

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping.

Mechanically Tunable Bioink for 3D Bioprinting of Human Cells.

This study introduces a thermogelling bioink based on carboxylated agarose (CA) for bioprinting of mechanically defined microenvironments mimicking natural tissues. In CA system, by adjusting the degree of carboxylation, the elastic modulus of printed gels can be tuned over several orders of magnitudes (5-230 Pa) while ensuring almost no change to the shear viscosity (10-17 mPa) of the bioink solution; thus enabling the fabrication of 3D structures made of different mechanical domains under identical printing parameters and low nozzle shear stress. Human mesenchymal stem cells printed using CA as a bioink show significantly higher survival (95%) in comparison to when printed using native agarose (62%), a commonly used thermogelling hydrogel for 3D-bioprinting applications. This work paves the way toward the printing of complex tissue-like structures composed of a range of mechanically discrete microdomains that could potentially reproduce natural mechanical aspects of functional tissues.

Unravelling a Direct Role for Polysaccharide ß-Strands in the Higher Order Structure of Physical Hydrogels.

The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired ß-strands.

Oxygen-Releasing Coatings for Improved Tissue Preservation.

Current organ transplantation protocols require the rapid transport of freshly isolated donor tissue to the recipient patient at the site where the procedure is to be conducted. During transport, the tissue graft can quickly deteriorate as a result of oxygen starvation. In this study, we report the fabrication of oxygen-releasing coatings for improved tissue preservation. The coatings were prepared via the encapsulation of calcium peroxide or urea peroxide microparticles between layers of octadiene plasma polymer films. By varying the thickness of the plasma polymer coating and type of peroxide, formulations were obtained that generate oxygen upon contact with aqueous solutions, while at the same time limiting the amount of toxic reactive oxygen species produced. The optimized coatings were tested under hypoxic conditions using the MIN6 ß-cell line, which resulted in a 3-fold increase in the viability of cultured cells. These thin oxygen-releasing coatings can be deposited on a wide range of surfaces, creating a platform for oxygen delivery with the potential to extend the viability of transported tissues and increase the time frame available for graft transport.

Nonwoven Carboxylated Agarose-Based Fiber Meshes with Antimicrobial Properties.

Hydrogel forming polysaccharides, such as the seaweed derived agarose, are well suited for wound dressing applications as they have excellent cell and soft tissue compatibility. For wound dressings, fibrous structure is desirable as the high surface area can favor adsorption of wound exudate and promote drug delivery. Although electrospinning offers a straightforward means to produce nonwoven fibrous polymeric structures, processing agarose and its derivatives into fibers through electrospinning is challenging as it has limited solubility in solvents other than water. In this study we describe the processing of carboxylated agarose (CA) fibers with antibacterial properties by electrospinning from a solution of the ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([Bmim]+Cl-) possessing antimicrobial properties. The extent of carboxylation was found to impact fiber diameter, mesh elastic modulus, fiber swelling, and the loading and release of IL. IL-bearing CA fibers inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, bacteria commonly found in wound exudate. In sum, nonwoven CA fibers processed from IL are promising as biomaterials for wound dressing applications.