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.     

Highly Tunable Elastomeric Silk Biomaterials

Elastomeric, fully degradable, and biocompatible biomaterials are rare, with current options presenting significant limitations in terms of ease of functionalization and tunable mechanical and degradation properties. A new method for covalently crosslinking tyrosine residues in silk proteins, via horseradish peroxidase and hydrogen peroxide, to generate highly elastic hydrogels with tunable properties, is reported. These materials offer tunable mechanical properties, gelation kinetics, and swelling properties. In addition, these new polymers withstand shear strains on the order of 100%, compressive strains greater than 70% and display stiffness between 200–10 000 Pa, covering a significant portion of the properties of native soft tissues. Molecular weight and solvent composition allow control of material mechanical properties over several orders of magnitude while maintaining high resilience and resistance to fatigue. Encapsulation of human bone marrow derived mesenchymal stem cells (hMSC) shows long term survival and exhibits cell‐matrix interactions reflective of both silk concentration and gelation conditions. Further biocompatibility of these materials is demonstrated with in vivo evaluation. These new protein‐based elastomeric and degradable hydrogels represent an exciting new biomaterials option, with a unique combination of properties, for tissue engineering and regenerative medicine.     

Highly Aligned Nanofibrous Scaffold Derived from Decellularized Human Fibroblasts

Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nano­fibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) are used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm‐thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter is generated after removing the cellular components from the fibroblast sheet. The elastic modulus of the scaffold is well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) shows the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold's biocompatibility is further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induces a significantly lower immune response compared to its unaligned counterpart, as detected by the pro‐inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.     

Sulfated Hydrogel Matrices Direct Mitogenicity and Maintenance of Chondrocyte Phenotype through Activation of FGF Signaling

Deciphering the roles of chemical and physical features of the extracellular matrix (ECM) is vital for developing biomimetic materials with desired cellular responses in regenerative medicine. Here, it is demonstrated that sulfation of biopolymers, mimicking the proteoglycans in native tissues, induces mitogenicity, chondrogenic phenotype, and suppresses catabolic activity of chondrocytes, a cell type that resides in a highly sulfated tissue. Through tunable modification of alginate it is shown that increased sulfation of the microenvironment promotes fibroblast growth factor (FGF) signaling‐mediated proliferation of chondrocytes in a 3D matrix independent of stiffness, swelling, and porosity. Furthermore, for the first time it is shown that a biomimetic hydrogel acts as a 3D signaling matrix to mediate a heparan sulfate/heparin‐like interaction between FGF and its receptor leading to signaling cascades inducing cell proliferation, cartilage matrix production, and suppression of dedifferentiation markers. Collectively, this study reveals important insights on mimicking the ECM to guide self‐renewal of cells via manipulation of distinct signaling mechanisms.     

Remote Manipulation of Slidable Nano‐Ligand Switch Regulates the Adhesion and Regenerative Polarization of Macrophages

The development of materials capable of varying macroscale ligand distributions can emulate an extracellular matrix (ECM) remodeling and regulate the adhesion and polarization of macrophages. In this report, negatively charged slidable nano‐ligands are assembled and then conjugated to a positively charged substrate via electrostatic interaction. The negatively charged slidable nano‐ligands are prepared by coating magnetic nanoparticles with a polymer linker and negatively charged RGD ligand. The nano‐ligand sliding is characterized under an external magnetic field, which spatiotemporally alters macroscale ligand density. To the best of knowledge, this is the first demonstration that magnetic maipulation of the macroscale ligand density inhibits inflammatory M1 phenotype but stimulates the adhesion and regenerative M2 phenotype of host macrophages. Furthermore, it is elucidated that the magnetic attraction of the slidable nano‐ligand facilitates the assembly of adhesion structures in macrophages, thereby stimulating their regenerative M2 phenotype. The design of ECM‐emulating materials that allow remote, spatiotemporal, and reversible controllability of macroscale ligand density provides an appealing strategy in the spatiotemporal regulation of immunomodulatory tissue‐regenerative responses to implants in vivo.     

Electrospun Extracellular Matrix: Paving the Way to Tailor‐Made Natural Scaffolds for Cardiac Tissue Regeneration

Biomimetic scaffolds generally aim at structurally and compositionally imitating native tissue, thus providing a supportive microenvironment to the transplanted or recruited cells in the tissue. Native decellularized porcine extracellular matrix (ECM) is becoming the ultimate bioactive material for the regeneration of different organs. Particularly for cardiac regeneration, ECM is studied as a patch and injectable scaffolds, which improve cardiac function, yet lack reproducibility and are difficult to control or fine‐tune for the desired properties, like most natural materials. Seeking to harness the natural advantages of ECM in a reproducible, scalable, and controllable scaffold, for the first time, a matrix that is produced from whole decellularized porcine cardiac ECM using electrospinning technology, is developed. This unique electrospun cardiac ECM mat preserves the composition of ECM, self‐assembles into the same microstructure of cardiac ECM ,and ,above all, preserves key cardiac mechanical properties. It supports cell growth and function, and demonstrates biocompatibility in vitro and in vivo. Importantly, this work reveals the great potential of electrospun ECM‐based platforms for a wide span of biomedical applications, thus offering the possibility to produce complex natural materials as tailor‐made, well‐defined structures.     

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.     

3D Printed Cartilage‐Like Tissue Constructs with Spatially Controlled Mechanical Properties

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage‐like tissue is fabricated using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a bath composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel (MPa order compressive modulus) is developed as an extracellular matrix (ECM) with self‐healing properties. Within this bath supplemented with thrombin, human mesenchymal stem cell (hMSC) spheroids embedded in fibrinogen are 3D bioprinted, creating a soft microenvironment composed of fibrin (kPa order compressive modulus) that simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids present high viability and chondrogenic‐like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to locally bioprint a soft and cell stimulating biomaterial inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro‐ and macromechanical properties of the 3D printed tissues such as cartilage.     

Manipulation of Stem Cells Fates: The Master and Multifaceted Roles of Biophysical Cues of Biomaterials

Owing to their self-renewal and differentiation ability, stem cells are conducive for repairing injured tissues, making them a promising source of seed cells for tissue engineering. The extracellular microenvironment (ECM) is under dynamic mechanical control, which is closely related to stem cell behaviors. During the design and fabrication of biomaterials for regenerative medicine, the physiochemical properties of the natural ECM should be closely mimicked, which can reinforce stem cell lineage choice and tissue engineering. By reproducing the biophysical stimulations that stem cells may experience in vivo, many studies have highlighted the key role of biophysical cues in regulation of cell fate. Optimization of biophysical factors leads to desirable stem cell functions, which can maximize the effectiveness of regenerative treatment. In this review, the main biophysical cues of biomaterials, including stiffness, topography, mechanical force, and external physical fields are summarized, and their individual and synergistic influence on stem cell behavior is discussed. Subsequently, the current progress in tissue regeneration using biomaterials is presented, which directs the design and fabrication of functional biomaterial. The mechanisms via which biophysical cues activate cellular responses are also analyzed. Finally, the challenges in basic research as well as for clinical translation in this field are discussed.     

Local Immune‐Triggered Surface‐Modified Stem Cells for Solid Tumor Immunotherapy

Here, described are additional treatment strategies that make use of human mesenchymal stem cell (hMSC)‐based local immunotherapeutic agents for the treatment of solid tumors. Dibenzocyclooctyne‐poly(ethylene glycol)‐pheophorbide A conjugates are engineered for cell surface conjugation by copper‐free click chemistry and are subsequently conjugated to hMSC (hMSC‐DPP). hMSC‐DPP can recognize and migrate toward cancer lesions, where they secrete pro‐inflammatory cytokines such as interleukin (IL)‐6, IL‐8, and heat shock protein 70 in pursuance of photodynamic therapy‐mediated cell death. The secreted immune factors trigger interferon gamma, IL‐2, IL‐4, IL‐12, and granulocyte‐macrophage colony‐stimulating factor, resulting in the local accumulation of T cells, B cells, natural killer cells, and antigen presenting cells at the tumor site. Treatment with hMSC‐DPP induces the accumulation of cytokines at the cancer site and minimizes systemic immune‐based side effects. This strategy is expected to increase the vulnerability of cancer cells to immune cells and cytokines, thus aiding in the development of a robust treatment platform for cancer immunotherapy.     

Polyelectrolyte‐Clay‐Protein Layer Films on Microfluidic PDMS Bioreactor Surfaces for Primary Murine Bone Marrow Culture

Poly(dimethylsiloxane) (PDMS) microbioreactors with computerized perfusion controls would be useful for engineering the bone marrow microenvironment. However, previous efforts to grow primary bone marrow cells on PDMS substrates have not been successful due to the weak attachment of cells to the PDMS surface even with adsorption of cell adhesive proteins such as collagen or fibronectin. In this work, modification of the surface of PDMS with biofunctional multilayer coatings is shown to promote marrow cell attachment and spreading. An automated microfluidic perfusion system is used to create multiple types of polyelectrolyte nanoscale coatings simultaneously in multiple channels based on layer‐by‐layer deposition of PDDA (poly(diallyldimethyl ammonium chloride)), clay, type IV collagen and fibronectin. Adherent primary bone marrow cells attached and spread best on a surface with composition of (PDDA/clay)₅ (Collagen/Fibronectin)₂ with negatively charged fibronectin exposed on the top, remaining well spread and proliferating for at least two weeks. Compared to traditional more macroscopic layer‐by‐layer methods, this microfluidic nanocomposite process has advantages of greater flow control, automatic processing, multiplexed fabrication, and use of lesser amounts of polymers and protein solutions.     

Nanostructured Biomaterials for Regeneration

Biomaterials play a pivotal role in regenerative medicine, which aims to regenerate and replace lost/dysfunctional tissues or organs. Biomaterials (scaffolds) serve as temporary 3D substrates to guide neo tissue formation and organization. It is often beneficial for a scaffolding material to mimic the characteristics of extracellular matrix (ECM) at the nanometer scale and to induce certain natural developmental or/and wound healing processes for tissue regeneration applications. This article reviews the fabrication and modification technologies for nanofibrous, nanocomposite, and nanostructured drug‐delivering scaffolds. ECM‐mimicking nanostructured biomaterials have been shown to actively regulate cellular responses including attachment, proliferation, differentiation, and matrix deposition. Nanoscaled drug delivery systems can be successfully incorporated into a porous 3D scaffold to enhance the tissue regeneration capacity. In conclusion, nanostructured biomateials are a very exciting and rapidly expanding research area, and are providing new enabling technologies for regenerative medicine.     

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.     

Processed Tissue–Derived Extracellular Matrices: Tailored Platforms Empowering Diverse Therapeutic Applications

Tissue‐derived decellularized extracellular matrices (dECM) have gradually become the gold standard of scaffolds for tissue engineering, owing to their close mirroring of the intricate composition, architecture, and topology of the native extracellular matrix (ECM). Intriguingly, further manipulation of these acellular tissues through various processing techniques has been demonstrated to be an effective strategy to control their characteristics and impart them with ample valuable new traits, thereby expanding their applicability to a significantly wider spectrum of research and translational applications. Herein, state‐of‐the‐art processed dECM platforms and their potential applications are focused on. The ECM characteristics that make it so appealing for tissue engineering are presented, followed by a concise discussion on the main considerations for choosing a dECM source for such applications. The key methodologies for dECM processing, including hydrogel production, bioprinting, electrospinning, and production of porous scaffolds, microcarriers, and microcapsules, as well as their inherent advantages and challenges, are introduced. To demonstrate the use of processed dECM platforms for tissue engineering, selected in vivo and in vitro applications recently developed utilizing these platforms are highlighted. Finally, concluding remarks and a prospective outlook for future developments and improvements in the field of processed dECM‐based devices are given.     

Using Mean Field Theory to Guide Biofunctional Materials Design

Cell‐instructive characteristics of extracellular matrices (ECM) resulting from a subtle balance of biomolecular and biophysical signals must be recapitulated in engineered biomaterials to facilitate regenerative therapies. However, no material explored so far allows the independent tuning of the involved molecular and physical cues due to the inherent correlation between biopolymer concentration and material properties. Addressing the resulting challenge, a rational design strategy for ECM‐inspired biohybrid hydrogels based on multi‐armed poly(ethylene glycol) and heparin, adapting a mean field approach to identify conditions at which the balance of elastic, electrostatic, and excluded volume forces results in constant heparin concentrations within swollen polymer networks with gradually varied physical properties is introduced. Applying heparin‐based biofunctionalization schemes, multiple distinct combinations of matrix parameters could be identified to effectively stimulate the pro‐angiogenic state of human endothelial cells and the differentiation of human mesenchymal stem cells. The study demonstrates the power of joint theoretical and experimental efforts in creating bioactive materials with specifically and independently controllable characteristics.     

Development of back‐junction back‐contact silicon solar cells based on industrial processes

We have presented simplified industrial processes to fabricate high performance back‐junction back‐contact (BJBC) silicon solar cells. Good optical surface structures (solar averaged reflectance 2.5%) and high implied open‐circuit voltage (0.695 V) have been realized in the BJBC cell precursors through wet chemical processing, co‐diffusion, P ion implantation and annealing oxidation, as well as laser patterning and plasma enhanced chemical vapour deposition passivation processes. We have achieved a certified high efficiency of close to 22% on BJBC silicon solar cells with the size of 4.04 cm² by using screen printing and co‐firing technologies. The manufacturing process flow further successfully yields efficiency of around 21% BJBC silicon solar cells with enlarged sizes of 6 × 6 cm². The present work has demonstrated that the commercialization of low‐cost and high‐efficiency BJBC solar cells is possible because we have used processes compatible with existing production lines. Copyright © 2017 John Wiley & Sons, Ltd.     

A Hydrogel Platform that Incorporates Laminin Isoforms for Efficient Presentation of Growth Factors – Neural Growth and Osteogenesis

Laminins (LMs) are important structural proteins of the extracellular matrix (ECM). The abundance of every LM isoform is tissue-dependent, suggesting that LM has tissue-specific roles. LM binds growth factors (GFs), which are powerful cytokines widely used in tissue engineering due to their ability to control stem cell differentiation. Currently, the most commonly used ECM mimetic material in vitro is Matrigel, a matrix of undefined composition containing LM and various GFs, but subjected to batch variability and lacking control of physicochemical properties. Inspired by Matrigel, a new and completely defined hydrogel platform based on hybrid LM-poly(ethylene glycol) (PEG) hydrogels with controllable stiffness (1–25 kPa) and degradability is proposed. Different LM isoforms are used to bind and efficiently display GFs (here, bone morphogenetic protein (BMP-2) and beta-nerve growth factor (β-NGF)), enabling their solid-phase presentation at ultralow doses to specifically target a range of tissues. The potential of this platform to trigger stem cell differentiation toward osteogenic lineages and stimulate neural cells growth in 3D, is demonstrated. These hydrogels enable 3D, synthetic, defined composition, and reproducible cell culture microenvironments reflecting the complexity of the native ECM, where GFs in combination with LM isoforms yield the full diversity of cellular processes.     

Quantitative Recovery of Magnetic Nanoparticles from Flowing Blood: Trace Analysis and the Role of Magnetization

Magnetic nanomaterials find increasing application as separation agents to rapidly isolate target compounds from complex biological media (i.e., blood purification). The responsiveness of the used materials to external magnetic fields (i.e., their saturation magnetization) is one of the most critical parameters for a fast and thorough separation. In the present study, magnetite (Fe₃O₄) and non‐oxidic cementite (Fe₃C) based carbon‐coated nanomagnets are characterized in detail and compared regarding their separation behavior from human whole blood. A quantification approach for iron‐based nanomaterials in biological samples with strong matrix effects (here, salts in blood) based on platinum spiking is shown. Both materials are functionalized with polyethyleneglycol (PEG) to improve cytocompatibility (confirmed by cell toxicity tests) and dispersability. The separation performance is tested in two setups, namely under stationary and different flow‐conditions using fresh human blood. The results reveal a superior separation behavior of the cementite based nanomagnets and strongly suggest the use of nanomaterials with high saturation magnetizations for magnetic retention under common blood flow conditions such as in veins.     

Light-Activated Decellularized Extracellular Matrix-Based Bioinks for Volumetric Tissue Analogs at the Centimeter Scale

Tissue engineering requires not only tissue-specific functionality but also a realistic scale. Decellularized extracellular matrix (dECM) is presently applied to the extrusion-based 3D printing technology. It has demonstrated excellent efficiency as bioscaffolds that allow engineering of living constructs with elaborate microarchitectures as well as the tissue-specific biochemical milieu of target tissues and organs. However, dECM bioinks have poor printability and physical properties, resulting in limited shape fidelity and scalability. In this study, new light-activated dECM bioinks with ruthenium/sodium persulfate (dERS) are introduced. The materials can be polymerized via a dityrosine-based cross-linking system with rapid reaction kinetics and improved mechanical properties. Complicated constructs with high aspect ratios can be fabricated similar to the geometry of the desired constructs with increased shape fidelity and excellent printing versatility using dERS. Furthermore, living tissue constructs can be safely fabricated with excellent tissue regenerative capacity identical to that of pure dECM. dERS may serve as a platform for a wider biofabrication window through building complex and centimeter-scale living constructs as well as supporting tissue-specific performances to encapsulated cells. This capability of dERS opens new avenues for upscaling the production of hydrogel-based constructs without additional materials and processes, applicable in tissue engineering and regenerative medicine.     

Bioorthogonal Strategies for Engineering Extracellular Matrices

Hydrogels are commonly used as engineered extracellular matrix (ECM) mimics in applications ranging from tissue engineering to in vitro disease models. Ideal mechanisms used to crosslink ECM‐mimicking hydrogels do not interfere with the biology of the system. However, most common hydrogel crosslinking chemistries exhibit some form of crossreactivity. The field of bioorthogonal chemistry has arisen to address the need for highly specific and robust reactions in biological contexts. Accordingly, bioorthogonal crosslinking strategies are incorporated into hydrogel design, allowing for gentle and efficient encapsulation of cells in various hydrogel materials. Furthermore, the selective nature of bioorthogonal chemistries can permit dynamic modification of hydrogel materials in the presence of live cells and other biomolecules to alter matrix mechanical properties and biochemistry on demand. This review provides an overview of bioorthogonal strategies used to prepare cell‐encapsulating hydrogels and highlights the potential applications of bioorthogonal chemistries in the design of dynamic engineered ECMs.