Arrayed Hollow Channels in Silk‐Based Scaffolds Provide Functional Outcomes for Engineering Critically Sized Tissue Constructs

In the field of regenerative medicine there is a need for scaffolds that support large, critically‐sized tissue formation. Major limitations in reaching this goal are the delivery of oxygen and nutrients throughout the bulk of the engineered tissue as well as host tissue integration and vascularization upon implantation. To address these limitations, the development of a porous scaffold platform made from biodegradable silk protein that contains an array of vascular‐like structures that extend through the bulk of the scaffold was previously reported. Here, the hollow channels play a pivotal role in enhancing cell infiltration, delivering oxygen and nutrients to the scaffold bulk, and promoting in vivo host tissue integration and vascularization. The unique features of this protein biomaterial system, including the vascular structures and tunable material properties, render this scaffold a robust and versatile tool for implementation in a variety of tissue engineering, regenerative medicine, and disease modeling applications.     

Matrix‐Bound VEGF Mimetic Peptides: Design and Endothelial‐Cell Activation in Collagen Scaffolds

Long‐term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial‐cell (EC) differentiation and vasculature formation are dependent on spatiotemporal cues in the extracellular matrix that dynamically interact with cells; a process that is difficult to reproduce in artificial systems. Here, a novel bifunctional peptide is presented that mimics matrix‐bound vascular endothelial growth factor (VEGF) which can be used to encode spatially controlled angiogenic signals in collagen scaffolds. The peptide is comprised of a collagen mimetic domain that was previously reported to bind to type I collagen by a unique hybridization mechanism, and a VEGF‐mimetic domain with pro‐angiogenic activity. Circular dichroism and collagen‐binding studies confirm the triple‐helical structure and the collagen binding affinity of the collagen‐mimetic domain, and EC‐culture studies demonstrate the peptide's ability to induce endothelial cell morphogenesis and network formation as a matrix‐bound factor in 2D and 3D collagen scaffolds. Spatial modification of collagen substrates is also shown with this peptide, which allows localized EC activation and network formation. These results demonstrate that the peptide can be used to present spatially directed angiogenic cues in collagen scaffolds, which may be useful for engineering organized microvasculature.     

Direct‐Write Assembly of Microperiodic Silk Fibroin Scaffolds for Tissue Engineering Applications

Three–dimensional, microperiodic scaffolds of regenerated silk fibroin have been fabricated for tissue engineering by direct ink writing. The ink, which consisted of silk fibroin solution from the Bombyx mori silkworm, was deposited in a layer‐by‐layer fashion through a fine nozzle to produce a 3D array of silk fibers of diameter 5 µm. The extruded fibers crystallized when deposited into a methanol‐rich reservoir, retaining a pore structure necessary for media transport. The rheological properties of the silk fibroin solutions were investigated and the crystallized silk fibers were characterized for structure and mechanical properties by infrared spectroscopy and nanoindentation, respectively. The scaffolds supported human bone marrow‐derived mesenchymal stem cell (hMSC) adhesion, and growth. Cells cultured under chondrogenic conditions on these scaffolds supported enhanced chondrogenic differentiation based on increased glucosaminoglycan production compared to standard pellet culture. Our results suggest that 3D silk fibroin scaffolds may find potential application as tissue engineering constructs due to the precise control of their scaffold architecture and their biocompatibility.     

Direct Writing of Three‐Dimensional Polymer Scaffolds Using Colloidal Gels

Polymer scaffolds intended to provide a substrate for cell attachment and proliferation benefit if the geometric architecture, mechanical properties, and surface chemistry are controllable within the range applicable for the target tissue. Such scaffolds may be made bioinductive through the inclusion of surface proteins and release of growth factors. Furthermore, the polymer support may be formed of biodegradable polymers for use as tissue‐engineering scaffolds. In this study, a new scaffold‐fabrication technique based on the direct writing of polymer colloidal‐gel‐based inks is described. The colloidal approach allows for the modular design of inks where the structure and composition of the colloidal particles, surface adsorbed molecules, and dissolved species may be easily controlled. Polyacrylate latex particles are formulated into colloidal gels by using a thermoreversible gel‐forming poly(ethylene oxide)–poly(propylene oxide) block‐copolymer adsorbed layer. The resulting colloidal gels are laced with the model protein bovine serum albumin (BSA) either dissolved in the solvent phase of the ink or dispersed in chitosan nanoparticles as a second colloid. Ink development and rheological characterization are presented along with demonstration of assembly of mesoporous scaffolds. After assembly and drying of the scaffold structure, the drug‐release kinetics are measured upon re‐exposure to an aqueous environment. Protein activity appears to be unaffected by the processing route of these scaffolds. Finally, the assembly of heterogeneous scaffolds is demonstrated to illustrate the possibilities for staged or heterogeneous drug release. This approach to scaffold fabrication offers a new route for scaffold assembly from water‐insoluble polymers while allowing the inclusion of sensitive biomolecules without risk of denaturation.     

Water‐Stable Silk Films with Reduced β‐Sheet Content

Silk fibers have outstanding mechanical properties. These fibers are insoluble in organic solvents and water, are biocompatible, and exhibit slow biodegradation in vitro and in vivo due to the hydrophobic nature of the protein and the presence of a high content of β‐sheet structure. Regenerated silk fibroin can be processed into a variety of materials normally stabilized by the induction of β‐sheet formation through the use of solvents or by physical stretching. To extend the biomaterial utility of silk proteins, options to form water‐stable silk‐based materials with reduced β‐sheet formation would be desirable. To address this need for more rapidly degradable silk biomaterials, we report the preparation of water‐stable films from regenerated silk fibroin solutions, with reduced β‐sheet content. The keys to this process are the preparation of concentrated (8 % by weight) aqueous solutions of fibroin and a subsequent water‐based annealing procedure. These new materials degrade more rapidly due to the reduced β‐sheet content, as determined in vitro via enzymatic hydrolysis, yet support human adult stem‐cell expansion in vitro in a similar or improved fashion to the crystallized proteins in film form. These new silk‐based materials extend the range of biomaterial properties that can be generated from this unique family of proteins.     

Capillary Network‐Like Organization of Endothelial Cells in PEGDA Scaffolds Encoded with Angiogenic Signals via Triple Helical Hybridization

Survival of tissue engineered constructs after implantation depends on proper vascularization. The differentiation of endothelial cells into mature microvasculature requires dynamic interactions between cells, scaffold, and growth factors, which are difficult to recapitulate in artificial systems. Previously, photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs), dubbed PEGDA‐CMP, that can be further conjugated with bioactive molecules via CMP‐CMP triple helix hybridization were reported. Here, it is shown that a bifunctional peptide featuring pro‐angiogenic domain mimicking vascular endothelial growth factor (VEGF) and a collagen mimetic domain that can fold into a triple helix conformation can hybridize with CMP side chains of the PEGDA‐CMP hydrogel, which results in presentation of insoluble VEGF‐like signals to endothelial cells. Presentation of VEGF‐like signals on the surface of micropatterned scaffolds in this way transforms cells from a quiescent state to elongated and aligned phenotype suggesting that this system could be used to engineer organized microvasculature. It is also shown that the pro‐angiogenic peptide, when applied topically in combination with modified dextran/PEGDA hydrogels, can enhance neovascularization of burn wounds in mice demonstrating the potential clinical use of CMP‐mediated matrix‐bound bioactive molecules for dermal injuries.     

Macroscopic Supramolecular Assembly to Fabricate 3D Ordered Structures: Towards Potential Tissue Scaffolds with Targeted Modification

3D ordered structures beyond microscale with targeted modification are catching increasing attention due to its application as tissue scaffolds. Especially scaffolds with necessary growth factors at designated locations are meaningful for induced cell differentiation and tissue formation. However, few fabrication methods can address the challenge of introducing bioactive species to the interior targeted places during the preparation process. Herein, for the first time macroscopic supramolecular assembly is applied to obtain such 3D ordered structures and established a proof‐of‐concept idea of complex scaffold with targeted modification. Taking strip‐like polydimethylsilicon building block as a model system, microscaled multilayered structures have been fabricated with parallel aligned building blocks in each layer. The morphology can be adjusted in a flexible way by tuning the number of layer, the space between two adjacent building blocks, and the position and orientation of each PDMS. The as‐prepared 3D structures are demonstrated biocompatible and potential as scaffolds for 3D cell culture. Moreover, bioactive species can be in situ incorporated into designated locations within the 3D structure precisely. In this way, a novel strategy is provided to address the current challenges in fabricating complex 3D tissue scaffolds with localized protein for future induced cell differentiation.     

Biomimetic Elastomeric Bioactive Siloxane‐Based Hybrid Nanofibrous Scaffolds with miRNA Activation: A Joint Physico‐Chemical‐Biological Strategy for Promoting Bone Regeneration

Rapid and efficient disease‐induced or critical‐size bone regeneration remains a challenge in tissue engineering due to the lack of highly bioactive biomaterial scaffolds. Physical structures such as nanostructures, chemical components such as silicon elements, and biological factors such as genes have shown positive effects on bone regeneration. Herein, a bioactive photoluminescent elastomeric silicate‐based nanofibrous scaffold with sustained miRNA release is reported for promoting bone regeneration based on a joint physico‐chemical‐biological strategy. Bioactive nanofibrous scaffolds are fabricated by cospinning poly (ε‐caprolactone) (PCL), elastomeric poly (citrates‐siloxane) (PCS), and bioactive osteogenic miRNA nanocomplexes (denoted PPM nanofibrous scaffolds). The PPM scaffolds possess uniform nanostructures, significantly enhanced tensile stress (≈15 MPa) and modulus (≈32 MPa), improved hydrophilicity (30–60°), controlled biodegradation, and strong blue fluorescence. Bioactive miRNA complexes are efficiently loaded into the nanofibrous matrix and exhibit long‐term release for up to 70 h. The PPM scaffolds significantly promote the adhesion, proliferation, and osteoblast differentiation of bone marrow stem cells in vitro and enhanced rat cranial defect restoration (12 weeks) in vivo. This work reports an attractive joint physico‐chemical‐biological strategy for the design of novel cell/protein‐free bioactive scaffolds for synergistic tissue regeneration.     

Biomimetic Scaffolds for Tissue Engineering

Biomimetic scaffolds mimic important features of the extracellular matrix (ECM) architecture and can be finely controlled at the nano‐ or microscale for tissue engineering. Rational design of biomimetic scaffolds is based on consideration of the ECM as a natural scaffold; the ECM provides cells with a variety of physical, chemical, and biological cues that affect cell growth and function. There are a number of approaches available to create 3D biomimetic scaffolds with control over their physical and mechanical properties, cell adhesion, and the temporal and spatial release of growth factors. Here, an overview of some biological features of the natural ECM is presented and a variety of original engineering methods that are currently used to produce synthetic polymer‐based scaffolds in pre‐fabricated form before implantation, to modify their surfaces with biochemical ligands, to incorporate growth factors, and to control their nano‐ and microscale geometry to create biomimetic scaffolds are discussed. Finally, in contrast to pre‐fabricated scaffolds composed of synthetic polymers, injectable biomimetic scaffolds based on either genetically engineered‐ or chemically synthesized‐peptides of which sequences are derived from the natural ECM are discussed. The presence of defined peptide sequences can trigger in situ hydrogelation via molecular self‐assembly and chemical crosslinking. A basic understanding of the entire spectrum of biomimetic scaffolds provides insight into how they can potentially be used in diverse tissue engineering, regenerative medicine, and drug delivery applications.     

Microfluidic 3D Printing Responsive Scaffolds with Biomimetic Enrichment Channels for Bone Regeneration

Tissue-engineered scaffolds have been extensively explored for treating bone defects; however, slow and insufficient vascularization throughout the scaffolds remains a key challenge for further application. Herein, a versatile microfluidic 3D printing strategy to fabricate black phosphorus (BP) incorporated fibrous scaffolds with photothermal responsive channels for improving vascularization and bone regeneration is proposed. The thermal channeled scaffolds display reversible shrinkage and swelling behavior controlled by near-infrared irradiation, which facilitates the penetration of suspended cells into the scaffold channels and promotes the prevascularization. Furthermore, the embedded BP nanosheets exhibit intrinsic properties for in situ biomineralization and improve in vitro cell proliferation and osteogenic differentiation. Following transplantation in vivo, these channels also promote host vessel infiltration deep into the scaffolds and effectively accelerate the healing process of bone defects. Thus, it is believed that these near-infrared responsive channeled scaffolds are promising candidates for tissue/vascular ingrowth in diverse tissue engineering applications.     

Polymer Scaffolds for Small‐Diameter Vascular Tissue Engineering

To better engineer small‐diameter blood vessels, a few types of novel scaffolds are fabricated from biodegradable poly(L ‐lactic acid) (PLLA) by means of thermally induced phase‐separation (TIPS) techniques. By utilizing the differences in thermal conductivities of the mold materials and using benzene as the solvent scaffolds with oriented gradient microtubular structures in the axial or radial direction can be created. The porosity, tubular size, and the orientational direction of the microtubules can be controlled by the polymer concentration, the TIPS temperature, and by utilizing materials of different thermal conductivities. These gradient microtubular structures facilitate cell seeding and mass transfer for cell growth and function. Nanofibrous scaffolds with an oriented and interconnected microtubular pore network are also developed by a one‐step TIPS method using a benzene/tetrahydrofuran mixture as the solvent without the need for porogen materials. The structural features of such scaffolds can be conveniently adjusted by varying the solvent ratio, phase‐separation temperature, and polymer concentration to mimic the nanofibrous features of an extracellular matrix. These scaffolds were fabricated for the tissue engineering of small‐diameter blood vessels by utilizing their advantageous structural features to facilitate blood‐vessel regeneration.     

Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium‐on‐a‐Chip Application

Bioprinting holds great promise toward engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, a novel and mechanically robust biomaterial‐ink based on nonmulberry silk fibroin protein is developed. The silk‐based ink demonstrates suitable mechanical properties required in terms of elasticity and stiffness (≈40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allows the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which are able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin‐43, and maintain synchronously beating of cardiomyocytes. The printed constructs are found to be nonimmunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue‐on‐a‐chip, the silk‐based ink is used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC‐CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. The ability is confirmed of silk‐based supporting hydrogel toward maturation and viability of hiPSC‐CSs and endothelial cells, and for applications in evaluating drug toxicity.     

Scalable High‐Throughput Production of Modular Microgels for In Situ Assembly of Microporous Tissue Scaffolds

Hydrogel scaffolds that template the regeneration of tissue structures are widely explored; however, there is often a trade‐off between material properties, such as stiffness and interconnected pore size, that may be equally important in supporting tissue growth. Microporous annealed particle scaffolds are introduced to address this trade‐off while maintaining a flowable precursor; however, manufacturing throughput, reproducibility, and flexibility of hydrogel microparticle building blocks are limited, hindering widespread adoption. The scalable high‐throughput production of bioactive microgels for the formation of microporous tissue scaffolds in situ is presented. Using a parallelized step emulsification device, scalable high‐throughput generation of monodisperse microgels is achieved. Crosslinking is initiated downstream of droplet generation using pH modulation via proton acceptors dissolved in the oil phase. This approach enables continuous production of microgels for over 12 h while ensuring highly uniform physicochemical properties. Using this platform, the effects of local matrix stiffness on cell growth orthogonal to scaffold porosity are studied. Formation of injectable cell‐laden mechanically heterogeneous microporous scaffolds is also demonstrated. This approach is particularly suited for the formation of modular, multimaterial scaffolds in situ, which could be applied to 3D bioprinting or to form more complex scaffolds to enhance regeneration of irregular wounds.     

Genetically Engineered Chimeric Silk–Silver Binding Proteins

Composite or hybrid materials are commonly found in Nature, formed through the concentration and subsequent nucleation of ions upon organic templates that are most often protein based. Examples include the deposition of calcium containing salts in bone, teeth and the inner ear and iron oxide structures in magnetotactic bacteria. Biological organisms use a limited number of metal ions, the principal ones being calcium and iron, with lesser amounts of strontium, and barium. The ability to utilize other ions to generate composites offers the possibility of new material properties. New materials incorporating silver would be useful in the context of antimicrobial functions. Therefore, in the present study, a new route to such functionalized biomaterials is reported. Genetically engineered fusion proteins are created by the incorporation of nucleotides corresponding to short silver binding peptides identified by a combinatorial biopanning process into the consensus sequence of silk from the spider, Nephila clavipes. The resulting chimeric silk–silver binding proteins nucleated Ag ions from a solution of silver nitrate while the silk protein provided a stable template material which could be processed into films, fibers, and three‐dimensional scaffolds. The silk films inhibited microbial growth of both Gram‐positive and Gram‐negative microrganisms on agar plates and in liquid culture, thus highlighting the potential of these chimeric material systems as antimicrobial biomedical coatings.     

Self‐Assembling Peptides as Cell‐Interactive Scaffolds

Cell and tissue engineering therapies for regenerative medicine as well as cell‐based assays require an understanding of the interactions between cells with the surrounding microenvironment at the nanoscale. Engineering a cell‐interactive scaffold therefore entails control over the nanostructure of the biomaterial. Peptides that are able to self‐assemble into 3D scaffolds have emerged as interesting biomaterials for directing cell behavior, with desirable properties such as the capability of tuning the nanostructure by modulating the amino acid composition. Here, an overview of the development of self‐assembling peptide hydrogels as functional cell scaffolds is presented, highlighting recent work on incorporating features such as bioactive ligands, growth factor delivery, controlled degradation, and formulation into microgels for defined cell microenvironments.     

Hydrogel−Solid Hybrid Materials for Biomedical Applications Enabled by Surface‐Embedded Radicals

Combinations of hydrogels and solids provide high level functionality for devices such as tissue engineering scaffolds and soft machines. However, the weak bonding between hydrogels and solids hampers functionality. Here, a versatile strategy to develop mechanically robust solid?hydrogel hybrid materials using surface embedded radicals generated through plasma immersion ion implantation (PIII) of polymeric surfaces is reported. Evidence is provided that the reactive radicals play a dual role: inducing surface‐initiated, spontaneous polymerization of hydrogels; and binding the hydrogels to the surfaces. Acrylamide and silk hydrogels are formed and covalently attached through spontaneous reactions with the radicals on PIII activated polymer surfaces without cross‐linking agents or initiators. The hydrogel amount increases with incubation time, monomer concentration, and temperature. Stability tests indicate that 95% of the hydrogel is retained even after 4 months in PBS solution. T‐peel tests show that failure occurs at the tape?hydrogel interface and the hydrogel‐PIII‐treated PTFE interfacial adhesion strength is over 300 N m^?1. Cell assays show no adhesion to the as‐synthesized hydrogels; however, hydrogels synthesized with fibronectin enable cell adhesion and spreading. These results show that polymers functionalized with surface‐embedded radicals provide excellent solid platforms for the generation of robust solid?hydrogel hybrid structures for biomedical applications.     

A Spider‐Capture‐Silk‐Like Fiber with Extremely High‐Volume Directional Water Collection

A spider web collects water by its capture silk for recovering the daytime‐distorted shape during night through water‐sensitive shape memory effect. This unique smart function and geometrical structure of spider‐capture‐silk inspires the development of artificial fibers with periodic knots for directional water collection with vast potential applications in water scarce regions. Existing such fibers are mainly based on nylon filaments coated with petroleum‐originated synthetic polymer solutions. Distinct from using synthetic materials, an all silk‐protein fiber (ASPF) with periodic knots endows extremely high volume‐to‐mass water collection capability. This fiber has a main body consisting of B. mori degummed silk coated with recombinant engineered major ampullate spidroin 2 of spider dragline silk. It is 252 times lighter than synthetic polymer coated nylon fibers that once was reported to have the highest water collection performance. The ASPF collects a maximum water volume of 6.6 µL and has a 100 times higher water collection efficiency compared to existing best water collection artificial fibers in terms of volume‐to‐mass index at the shortest length (0.8 mm) of three‐phase contact line. Since silkworm silks are available abundantly, effective use of recombinant spidroins tandemly shows great potential for scalability.     

Conductive Scaffolds for Cardiac and Neuronal Tissue Engineering: Governing Factors and Mechanisms

Tissue engineering is a promising therapeutic approach in medicine, targeting the replacement of a diseased tissue with a healthy one grown within an artificial scaffold. Due to the high prevalence of cardiac and brain‐related ailments that involve some necrosis of tissue, cardiac and neuronal tissue engineering are intensely studied fields in regenerative medicine. A growing trend in the use of conductive scaffolds for the growth of these tissues has been witnessed recently. While the results are irrefutable, the mechanism of how an electrically conducting scaffold interacts with an electroactive tissue remains has remained elusive. An up‐to‐date summary of all work done in the field is reported, with a special focus on the specific contribution of the conductive scaffold on the performance of the formed tissue. The cell–scaffold electronic interface is then explored from an electrical engineering perspective. The electronic configuration of the system and the mechanisms and governing factors controlling the ability of a conductive scaffold to support cardiac and neuronal tissues are discussed. Using several simulations, the required conductivity of the scaffold in order for it to be suitable for tissue engineering—which also depends on the nature of the charge carriers—is also discussed.     

Biomimetic Ultralight,Highly Porous,Shape‐Adjustable,and Biocompatible 3D Graphene Minerals via Incorporation of Self‐Assembled Peptide Nanosheets

Hybrid nanomaterials with tailored functions, consisting of self‐assembled peptides, are intensively applied in nanotechnology, tissue engineering, and biomedical applications due to their unique structures and properties. Herein, a peptide‐mediated biomimetic strategy is adopted to create the multifunctional 3D graphene foam (GF)‐based hybrid minerals. First, 2D peptide nanosheets (PNSs), obtained by self‐assembling a motif‐specific peptide molecule (LLVFGAKMLPHHGA), are expected to exhibit biofunctionality, such as the biomimetic mineralization of hydroxyapatite (HA) minerals. Subsequently, the noncovalent conjugation of PNSs onto GF support is utilized to form 3D GF‐PNSs hybrid scaffolds, which are suitable for the growth of HA minerals. The fabricated biomimetic 3D GF‐PNSs‐HA minerals exhibit adjustable shape, superlow weight (0.017 g cm^?3), high porosity (5.17 m² g^?1), and excellent biocompatibility, proving potential applications in both bone tissue engineering and biomedical engineering. To the best of the authors' knowledge, it is the first time to combine 2D PNSs and GF to fabricate 3D organic–inorganic hybrid scaffold. Further development of these hybrid GF‐PNSs scaffolds can potentially lead to materials used as matrices for drug delivery or bone tissue engineering as proven via successful 3D scaffold formation exhibiting interconnected pore‐size structures suitable for vascularization and medium transport.     

Channeled β‐TCP Scaffolds Promoted Vascularization and Bone Augmentation in Mandible of Beagle Dogs

A major hindrance to successful alveolar bone augmentation and ridge preservation using synthetic scaffolds is insufficient vascularization in the implanted bone grafts. The slow ingrowth of host vasculature from the bone bed of alveolar bone to the top of the implanted bone grafts leads to limited bone formation in the upper layers of the implanted grafts, which hinders the subsequent implantation of titanium dental implants. In this study, macroporous beta‐tricalcium phosphate (β‐TCP) scaffolds with multiple vertical hollow channels are fabricated that play a similar role as blood vessels for nutrient diffusion and cell migration. The results show that the hollow channels accelerate the degradation rate of the β‐TCP scaffolds and the in vitro release of a bone forming peptide‐1, which significantly promote proliferation and osteogenesis of human bone mesenchymal stem cells on the channeled scaffolds, compared to nonchanneled scaffolds in vitro. More volume of newly formed bone tissues with more blood vessels are augmented in the channeled scaffolds when implanted in mandibular bone defects of beagle dogs. Channeled scaffolds significantly promote new bone formation and augment the height of the mandible. These findings indicate channeled scaffolds facilitate vascularization and bone formation and have great potential for vascularized bone augmentation.