Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage.

Fluorescent polymeric hydrogels are promising soft and wet media for information storage that are desirable for lifelike biomaterials and flexible electronics. Hydrogels based on engineered proteins have attracted considerable interest. However, their potential utility as information storage media has remained largely unexplored. Here, we report a protein-based hydrogel that can serve as an information storage medium. Using LOVTRAP, which consists of protein LOV2 and its binding partner ZDark1, we developed a novel strategy to decorate/release fluorescent proteins onto/from a blank protein hydrogel slate in light-controlled and spatially defined fashions, reversibly generating fluorescent patterns such as quick response codes. To increase the information storage capacity, we further developed grayscale patterning to generate pseudo-colored multi-dimensional fluorescent images. Results of this new method demonstrate a novel reversible information storage approach in soft and wet materials and open a new avenue toward developing next-generation protein-based smart materials for information storage and anti-counterfeit applications.

Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP.

Protein-based hydrogels can mimic many aspects of native extracellular matrices (ECMs) and are promising biomedical materials that find various applications in cell proliferation, drug/cell delivery, and tissue engineering. To be adapted for different tasks, it is important that the mechanical and/or biochemical properties of protein-based hydrogels can be regulated by external stimuli. Light as a regulation stimulus is of advantage because it can be easily applied in demanded spatiotemporal manners. The noncovalent binding between the light-oxygen-voltage-sensing domain 2 (LOV2) and its binding partner ZDark1 (zdk1), named as LOVTRAP, is a light-responsive interaction. The binding affinity of LOVTRAP is much higher in dark than that under blue light irradiation. Taking advantage of these light-responsive interactions, herein we endeavored to use LOVTRAP as a crosslinking mechanism to engineer light-responsive protein hydrogels. Using LOV2-containing and zdk1-containing multifunctional protein building blocks, we successfully engineered a light-responsive protein hydrogel whose viscoelastic properties can change in response to light: in the dark, the hydrogel showed higher storage modulus; under blue light irradiation, the storage modulus decreased. Due to the noncovalent nature of the LOVTRAP, the engineered LOVTRAP protein hydrogels displayed shear-thinning and self-healing properties and served as an excellent injectable protein hydrogel. We anticipated that this new class of light-responsive protein hydrogels will broaden the scope of dynamic protein hydrogels and help develop other light-responsive protein hydrogels for biomedical applications.

Generation of Cost-Effective Paper-Based Tissue Models through Matrix-Assisted Sacrificial 3D Printing.

Due to the combined advantages of cellulose and nanoscale (diameter 20-60 nm), bacterial cellulose possesses a series of attractive features including its natural origin, moderate biosynthesis process, good biocompatibility, and cost-effectiveness. Moreover, bacterial cellulose nanofibers can be conveniently processed into three-dimensional (3D) intertwined structures and form stable paper devices after simple drying. These advantages make it suitable as the material for construction of organ-on-a-chip devices using matrix-assisted sacrificial 3D printing. We successfully fabricated various microchannel structures embedded in the bulk bacterial cellulose hydrogels and retained their integrity after the drying process. Interestingly, these paper-based devices containing hollow microchannels could be rehydrated and populated with relevant cells to form vascularized tissue models. As a proof-of-concept demonstration, we seeded human umbilical vein endothelial cells (HUVECs) into the microchannels to obtain the vasculature and inoculated the MCF-7 cells onto the surrounding matrix of the paper device to build a 3D paper-based vascularized breast tumor model. The results showed that the microchannels were perfusable, and both HUVECs and MCF-7 cells exhibited favorable proliferation behaviors. This study may provide a new strategy for constructing simple and low-cost in vitro tissue models, which may find potential applications in drug screening and personalized medicine.

Engineering Protein-Clay Nanosheets Composite Hydrogels with Designed Arginine-Rich Proteins.

Clay nanosheets (CNSs) have been widely used in the design of nanocomposite biomaterials. CNSs display a disk-like morphology with strong negatively charged surfaces. It has been shown that guanidinium-containing molecules can bind CNSs through noncovalent salt-bridge interactions and thus serve as "molecular glues" for CNSs. Making use of the guanidinium side chain in arginine, here, we designed novel arginine-rich elastomeric proteins to engineer protein-CNS nanocomposite hydrogels. Our results showed that these arginine-rich proteins can interact with CNSs effectively and can cross-link CNSs into hydrogels. Rheological measurements showed that mechanical properties of the resultant hydrogels depended on the arginine content in the arginine-rich proteins as well as CNS/protein concentration. Compared with hydrogels constructed from CNSs or proteins alone, the novel protein-CNS nanocomposite hydrogels show much improved mechanical properties. Our work opens up a new avenue to engineer functional protein hydrogels for various applications.

An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response.

Hydrogels with dynamic mechanical properties are of special interest in the field of tissue engineering and drug delivery. However, it remains challenging to tailor the dynamic mechanical response of hydrogels to simultaneously meet diverse application needs. Here, we report a hetero-coiled-coil complex cross-linked protein hydrogel exhibiting unusual multiple energy dissipation modes and tunable dynamic response. Such unique features confer on the hydrogel responsiveness to mechanical stimuli in a broad range of frequencies. Therefore, the hydrogels are injectable due to their shearing-thinning properties at low shear rates of 0.8 rad s-1 and can fully recover their mechanical properties within a few seconds due to the intrinsic fast dynamics of the cross-linkers. Moreover, the dynamic response of these hydrogels can be fine-tuned by the temperature and the hydrogel network structures. We anticipate that these hydrogels are promising candidates for delivering therapeutic drugs, biological molecules, and cells in a broad spectrum of biomedical applications.

Proteomic Landscape Has Revealed Small Rubber Particles Are Crucial Rubber Biosynthetic Machines for Ethylene-Stimulation in Natural Rubber Production.

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.

Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass Taraxacum Kok-saghyz.

The rubber grass Taraxacum kok-saghyz (TKS) contains large amounts of natural rubber (cis-1,4-polyisoprene) in its enlarged roots and it is an alternative crop source of natural rubber. Natural rubber biosynthesis (NRB) and storage in the mature roots of TKS is a cascade process involving many genes, proteins and their cofactors. The TKS genome has just been annotated and many NRB-related genes have been determined. However, there is limited knowledge about the protein regulation mechanism for NRB in TKS roots. We identified 371 protein species from the mature roots of TKS by combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Meanwhile, a large-scale shotgun analysis of proteins in TKS roots at the enlargement stage was performed, and 3545 individual proteins were determined. Subsequently, all identified proteins from 2-DE gel and shotgun MS in TKS roots were subject to gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and most proteins were involved in carbon metabolic process with catalytic activity in membrane-bounded organelles, followed by proteins with binding ability, transportation and phenylpropanoid biosynthesis activities. Fifty-eight NRB-related proteins, including eight small rubber particle protein (SRPP) and two rubber elongation factor(REF) members, were identified from the TKS roots, and these proteins were involved in both mevalonate acid (MVA) and methylerythritol phosphate (MEP) pathways. To our best knowledge, it is the first high-resolution draft proteome map of the mature TKS roots. Our proteomics of TKS roots revealed both MVA and MEP pathways are important for NRB, and SRPP might be more important than REF for NRB in TKS roots. These findings would not only deepen our understanding of the TKS root proteome, but also provide new evidence on the roles of these NRB-related proteins in the mature TKS roots.

Decorating a Blank Slate Protein Hydrogel: A General and Robust Approach for Functionalizing Protein Hydrogels.

Protein hydrogels constructed from recombinant proteins have attracted increasing interests for fundamental biological studies as well as applications in biomedical engineering field. In such protein hydrogels, biochemical and physical properties of protein hydrogels are often coupled to each other, making it challenging to investigate the individual effect of chemical and physical cues on cells. Moreover, laborious engineering is often required to incorporate different protein ligands into such hydrogels. To address these challenges, functionalizing a blank slate protein hydrogel is an attractive approach. However, conjugating ligands to such a blank slate protein hydrogel is challenging, as existing bioconjugation methods developed in synthetic polymer hydrogels cannot be readily adapted for protein hydrogels, significantly impeding the use of this approach in the field. Here we report a facile, general, and robust method, which is based on the SpyCatcher-SpyTag chemistry, to covalently functionalize the "blank slate" of protein hydrogels using genetically encoded interacting partners. We demonstrate that this novel method enables covalent conjugation of a wide variety of ligands, including full-length intact folded proteins, to a blank slate protein hydrogel, and allows for the decoupling of biochemical and physical properties of hydrogels from each other and investigating the individual effect of biochemical and mechanical cues on cell behaviors. To our best knowledge, this is the first general approach enabling functionalization of protein hydrogels, and we anticipate that this novel approach will find a broad range of uses in protein-based biomaterials for applications in biomedical engineering.

Designed biomaterials to mimic the mechanical properties of muscles.

The passive elasticity of muscle is largely governed by the I-band part of the giant muscle protein titin, a complex molecular spring composed of a series of individually folded immunoglobulin-like domains as well as largely unstructured unique sequences. These mechanical elements have distinct mechanical properties, and when combined, they provide the desired passive elastic properties of muscle, which are a unique combination of strength, extensibility and resilience. Single-molecule atomic force microscopy (AFM) studies demonstrated that the macroscopic behaviour of titin in intact myofibrils can be reconstituted by combining the mechanical properties of these mechanical elements measured at the single-molecule level. Here we report artificial elastomeric proteins that mimic the molecular architecture of titin through the combination of well-characterized protein domains GB1 and resilin. We show that these artificial elastomeric proteins can be photochemically crosslinked and cast into solid biomaterials. These biomaterials behave as rubber-like materials showing high resilience at low strain and as shock-absorber-like materials at high strain by effectively dissipating energy. These properties are comparable to the passive elastic properties of muscles within the physiological range of sarcomere length and so these materials represent a new muscle-mimetic biomaterial. The mechanical properties of these biomaterials can be fine-tuned by adjusting the composition of the elastomeric proteins, providing the opportunity to develop biomaterials that are mimetic of different types of muscles. We anticipate that these biomaterials will find applications in tissue engineering as scaffold and matrix for artificial muscles.

The fabrication of the chitosan-based bioink for in vitro tissue repair and regeneration: A review.

The repair and regeneration of the injured tissues or organs is a major challenge for biomedicine, and the emerging 3D bioprinting technology as a class of promising techniques in biomedical research for the development of tissue engineering and regenerative medicine. Chitosan-based bioinks, as the natural biomaterials, are considered as ideal materials for 3D bioprinting to design and fabricate the various scaffold due to their unique dynamic reversibility and fantastic biological properties. Our review aims to provide an overview of chitosan-based bioinks for in vitro tissue repair and regeneration, starting from modification of chitosan that affect these bioprinting processes. In addition, we summarize the advances in chitosan-based bioinks used in the various 3D printing strategies. Moreover, the biomedical applications of chitosan-based bioinks are discussed, primarily centered on regenerative medicine and tissue modeling engineering. Finally, current challenges and future opportunities in this field are discussed. The combination of chitosan-based bioinks and 3D bioprinting will hold promise for developing novel biomedical scaffolds for tissue or organ repair and regeneration.

Biomimetic mineralization of nacre-inspired multiple crosslinked PVA/CaAlg/SiO2 membrane with simultaneously enhanced mechanical and separation properties.

Inspired by the natural nacre structure, we propose a new strategy to fabricate mineralized, multiple crosslinked hydrogel membranes with the "rigid silica in soft polymer" nacre-like structure. In-situ SiO2 nanoparticles (NPs) and polyvinyl alcohol/sodium alginate (PVA/NaAlg) are used to simulate the rigid "bricks" and soft "mortar" compositions of nacre, respectively. The nacre-like mineralized (PVA/CaAlg/SiO2) membrane showed a higher tensile strength of 4.1 ± 0.08 MPa, excellent pure water flux of 170 ± 3 L/m2h, and an oil/water rejection rate of 99 %. The interwoven hierarchal structure, similar to nacre, was determined by SEM analysis. In addition, incorporating SiO2 NPs increases the anti-swelling, roughness, and hydrophilicity of the membranes. PVA/CaAlg/SiO2 membrane exhibited excellent superhydrophilicity (WCA value was 0°) and superoleophobicity underwater (OCA value was 162°). PVA/CaAlg/SiO2 membrane also showed excellent separation performance for water-soluble organic pollutants and can be used for dye separation with rejection efficiencies of 99.5 %, 99.1 %, and 98.3 % for Congo red (CR), Alizarin red (AR), and Sunset yellow (SY), respectively. Moreover, PVA/CaAlg/SiO2 membrane had outstanding long-term filtration and antifouling performance. The biomineralization-inspired structure provides a promising technique that can be used to prepare high-performance organic-inorganic membranes with great promise for wastewater separation application.

A polyphenol and ε-polylysine functionalized bacterial cellulose/PVA multifunctional hydrogel for wound healing.

Hydrogels for wound dressings have recently attracted considerable attention in the field of biomedical materials. Developing hydrogel dressings with multiple functions, including good antibacterial, mechanical and adhesive properties, to enhance wound regeneration is significant for clinical applications. To this end, a novel hydrogel wound dressing (PB-EPL/TA@BC) was developed, which was prepared by incorporating bacterial cellulose (BC) modified with tannic acid and ε-polylysine (EPL) into a PVA and borax matrix through a simple method without introducing any other chemical reagents. The hydrogel exhibited good adhesion (8.8 ± 0.2 kPa) to porcine skin, and the mechanical properties were significantly improved after adding BC. Meanwhile, it showed good inhibition against Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (84.1 ± 2.6 %, 86.0 ± 2.3 % and 80.7 ± 4.5 %) in vitro and Methicillin-resistant Staphylococcus aureus (MRSA) in vivo without the use of antibiotics, ensuring that the process of wound repair with a sterile environment. The hydrogel also presented good cytocompatibility and biocompatibility and could achieve hemostasis within 120 s. The in vivo experiments indicated that hydrogel could not only instantly complete hemostasis of the injured liver models but also obviously promote wound healing in a full-thickness skin. Furthermore, the hydrogel accelerated wound healing process by reducing inflammation promoting collagen deposition compared with commercial Tegaderm™ films. Therefore, the hydrogel is a promising high-end dressing material for wound hemostasis and repair for to enhance the wound healing.

Engineered bi-histidine metal chelation sites map the structure of the mechanical unfolding transition state of an elastomeric protein domain GB1.

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing ß-strands 1 and 4 is largely disrupted, and the first ß-hairpin is partially disordered while the second ß-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.

A facile way to tune mechanical properties of artificial elastomeric proteins-based hydrogels.

Protein-based hydrogels have attracted considerable interests due to their potential applications in biomedical engineering and material sciences. Using a tandem modular protein (GB1)(8) as building blocks, we have engineered chemically cross-linked hydrogels via a photochemical cross-linking strategy, which is based on the cross-linking of two adjacent tyrosine residues into dityrosine adducts. However, because of the relatively low reactivity of tyrosine residues in GB1, (GB1)(8)-based hydrogels exhibit poor mechanical properties. Here, we report a Bolton-Hunter reagent-based, facile method to improve and tune the mechanical properties of such protein-based hydrogels. Using Bolton-Hunter reagent, we can derivatize lysine residues with phenolic functional groups to modulate the phenolic (tyrosine-like) content of (GB1)(8). We show that hydrogels made from derivatized (GB1)(8) with increased phenolic content show significantly improved mechanical properties, including improved Young's modulus, breaking modulus as well as reduced swelling. These results demonstrate the great potential of this derivatization method in constructing protein-based biomaterials with desired macroscopic mechanical properties.

Engineering shape memory and morphing protein hydrogels based on protein unfolding and folding.

Engineering shape memory/morphing materials have achieved considerable progress in polymer-based systems with broad potential applications. However, engineering protein-based shape memory/morphing materials remains challenging and under-explored. Here we report the design of a bilayer protein-based shape memory/morphing hydrogel based on protein folding-unfolding mechanism. We fabricate the protein-bilayer structure using two tandem modular elastomeric proteins (GB1)8 and (FL)8. Both protein layers display distinct denaturant-dependent swelling profiles and Young's moduli. Due to such protein unfolding-folding induced changes in swelling, the bilayer hydrogels display highly tunable and reversible bidirectional bending deformation depending upon the denaturant concentration and layer geometry. Based on these programmable and reversible bending behaviors, we further utilize the protein-bilayer structure as hinge to realize one-dimensional to two-dimensional and two-dimensional to three-dimensional folding transformations of patterned hydrogels. The present work will offer new inspirations for the design and fabrication of novel shape morphing materials.

Generation of cost-effective MXene@polydopamine-decorated chitosan nanofibrous wound dressing for promoting wound healing.

Herein, we designed and fabricated a MXene@polydopamine (MXene@PDA)-decorated chitosan non-woven fabric (M-CNF) hemostatic dressing with super hydrophilic properties for wound repair and regeneration. The M-CNF exhibit excellently wettability characteristics which can rapidly absorb water from blood. Moreover, M-CNF with 15 mg/mL MXene@PDA (M-CNF-15) show better antibacterial performance, excellent blood-clotting performance, better blood cell and platelet adhesion ability than CNF, displaying both active and passive hemostatic mechanisms to accelerate blood clotting in mouse-liver injury model. In addition, the M-CNF-15 also shows better wound healed performance than Tegaderm™ film in a full-thickness skin defect model, and further demonstrating that the MXene@PDA can promote fibrinogen reformation the at the initial phases of the wound healing process. Therefore, this strategy for designing and manufacturing of multi-functional M-CNF wound dressing will have great potential for active local hemostasis and wound repair and regeneration.

Protein mechanics: from single molecules to functional biomaterials.

Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of multidomain elastomeric proteins, and the design of novel elastomeric proteins that exhibit stimuli-responsive mechanical properties. Moving forward, we are now exploring the use of these artificial elastomeric proteins as building blocks of protein-based biomaterials. Ultimately, we would like to rationally tailor mechanical properties of elastomeric protein-based materials by programming the molecular sequence, and thus nanomechanical properties, of elastomeric proteins at the single-molecule level. This step would help bridge the gap between single protein mechanics and material biomechanics, revealing how the mechanical properties of individual elastomeric proteins are translated into the properties of macroscopic materials.

Freeze-Casting with 3D-Printed Templates Creates Anisotropic Microchannels and Patterned Macrochannels within Biomimetic Nanofiber Aerogels for Rapid Cellular Infiltration.

A new approach is described for fabricating 3D poly(ε-caprolactone) (PCL)/gelatin (1:1) nanofiber aerogels with patterned macrochannels and anisotropic microchannels by freeze-casting with 3D-printed sacrificial templates. Single layer or multiple layers of macrochannels are formed through an inverse replica of 3D-printed templates. Aligned microchannels formed by partially anisotropic freezing act as interconnected pores between templated macrochannels. The resulting macro-/microchannels within nanofiber aerogels significantly increase preosteoblast infiltration in vitro. The conjugation of vascular endothelial growth factor (VEGF)-mimicking QK peptide to PCL/gelatin/gelatin methacryloyl (1:0.5:0.5) nanofiber aerogels with patterned macrochannels promotes the formation of a microvascular network of seeded human microvascular endothelial cells. Moreover, nanofiber aerogels with patterned macrochannels and anisotropic microchannels show significantly enhanced cellular infiltration rates and host tissue integration compared to aerogels without macrochannels following subcutaneous implantation in rats. Taken together, this novel class of nanofiber aerogels holds great potential in biomedical applications including tissue repair and regeneration, wound healing, and 3D tissue/disease modeling.

Complexation-induced resolution enhancement of 3D-printed hydrogel constructs.

Three-dimensional (3D) hydrogel printing enables production of volumetric architectures containing desired structures using programmed automation processes. Our study reports a unique method of resolution enhancement purely relying on post-printing treatment of hydrogel constructs. By immersing a 3D-printed patterned hydrogel consisting of a hydrophilic polyionic polymer network in a solution of polyions of the opposite net charge, shrinking can rapidly occur resulting in various degrees of reduced dimensions comparing to the original pattern. This phenomenon, caused by complex coacervation and water expulsion, enables us to reduce linear dimensions of printed constructs while maintaining cytocompatible conditions in a cell type-dependent manner. We anticipate our shrinking printing technology to find widespread applications in promoting the current 3D printing capacities for generating higher-resolution hydrogel-based structures without necessarily having to involve complex hardware upgrades or other printing parameter alterations.