The Horizon of Materiobiology: A Perspective on Material-Guided Cell

Feb 21, 2017 - ECM proteins show good bioactivity in directing cell behaviors and/or tissue regeneration, while they lack sufficient mechanical proper...
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The Horizon of Materiobiology: A Perspective on Material-Guided Cell Behaviors and Tissue Engineering Yulin Li,† Yin Xiao,‡ and Changsheng Liu*,† †

Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Meilong Road 130, Shanghai 200237, People’s Republic of China ‡ Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Brisbane, Queensland 4059, Australia ABSTRACT: Although the biological functions of cell and tissue can be regulated by biochemical factors (e.g., growth factors, hormones), the biophysical effects of materials on the regulation of biological activity are receiving more attention. In this Review, we systematically summarize the recent progress on how biomaterials with controllable properties (e.g., compositional/degradable dynamics, mechanical properties, 2D topography, and 3D geometry) can regulate cell behaviors (e.g., cell adhesion, spreading, proliferation, cell alignment, and the differentiation or self-maintenance of stem cells) and tissue/organ functions. How the biophysical features of materials influence tissue/organ regeneration have been elucidated. Current challenges and a perspective on the development of novel materials that can modulate specific biological functions are discussed. The interdependent relationship between biomaterials and biology leads us to propose the concept of “materiobiology”, which is a scientific discipline that studies the biological effects of the properties of biomaterials on biological functions at cell, tissue, organ, and the whole organism levels. This Review highlights that it is more important to develop ECM-mimicking biomaterials having a self-regenerative capacity to stimulate tissue regeneration, instead of attempting to recreate the complexity of living tissues or tissue constructs ex vivo. The principles of materiobiology may benefit the development of novel biomaterials providing combinative bioactive cues to activate the migration of stem cells from endogenous reservoirs (i.e., cell niches), stimulate robust and scalable self-healing mechanisms, and unlock the body’s innate powers of regeneration.

CONTENTS 1. Introduction 2. Material Guidance of Cell Behaviors (Cell Fate) 2.1. How Do Cells Sense Materials? 2.2. Material-Guided Cell Adhesion 2.2.1. Material Composition-Mediated Cell Adhesion through the Interaction of Materials with Cellular Integrin Receptors 2.2.2. Cells Prefer To Adhere to Substrates with Similar Stiffness 2.2.3. Nanotopography Is Helpful for the Adjustment of Cell Adhesion, and Micropatterns Direct the Alignment of Cell Adhesions 2.2.4. 3D Microenvironments Facilitate Cell Adhesion More Effectively than 2D Topographical Substrates 2.3. Material-Guided Cell Migration 2.3.1. Compositional Gradients Create a Driving Force To Guide Cell Migration 2.3.2. Mechanical Stimuli Can Change the Cell Migration Mode 2.3.3. Patterned Substrates Accelerate Directional Cell Movement

2.3.4. Scaffolds with 3D Porous Geometry and Cell-Responsive Biodegradability Dynamically Regulate Cell Migration 2.4. Material-Guided Cell Spreading 2.4.1. Materials with a Positive Charge and Moderate Hydrophilicity Promote Cell Spreading 2.4.2. Cells Present Less Spreading on Soft Substrates, Probably Due to Reduced Actin Organization and Local Adhesion 2.4.3. Topographical Substrates Modulate Cellular Gene Expression To Regulate Cell Spreading (Cell Shape and Arrangement) 2.4.4. 3D Microenvironments Guide Cell Spreading Behaviors through Geometrically Constraining Cells 2.5. Material-Guided Cell Proliferation 2.5.1. Compositional Motifs Promote Cell Proliferation through the Activation of Cell Receptors

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Chemical Reviews 2.5.2. Mechanical Information Modulates Cell Proliferation by Activating Mitogenic Signaling Pathways 2.5.3. Micro/Nano Hierarchical Topographies Improve Cell Proliferation, Probably through the FAK Activation 2.5.4. Scaffolds with a 3D Interconnected Porous Structure Promote Cell Proliferation 2.6. Material-Guided Cell Differentiation 2.6.1. Special Material Compositions Induce Specific Cell Differentiation 2.6.2. Stem Cells Preferentially Differentiate into Specific Cell Lineages on Substrates with a Tissue-like Stiffness 2.6.3. A Disordered Topography Promotes Osteogenesis, while an Ordered One Benefits Neuronal/Myogenic Differentiation 2.6.4. A 3D Geometry with Ordered Cylindrical Pores Promotes Osteogenesis 2.7. Material-Guided Stemness Maintenance 2.7.1. A Compositional “Constraint Effect” Promotes Stemness Maintenance 2.7.2. Soft Substrates Keep Stem Cells Quiescent via Triggering Their Autocrine Signaling 2.7.3. Micro-/Nanotopographies Promote Stemness Maintenance by Blocking Cell Differentiation 2.7.4. Stemness Can Be Better Maintained in 3D Geometry than on a 2D Surface 2.8. How Do Cell Behaviors Collectively Interact To Determine Cell/Tissue Fate? 3. Material Guidance of Tissue Engineering 3.1. Material-Guided Bone Regeneration 3.1.1. Inorganic Elements and Molecules Guide Cells To Express Bone-Related ECM Proteins for Bone Regeneration 3.1.2. Materials with Relatively High Elastic Moduli Promote Osteogenesis and Bone Integration 3.1.3. A Disordered Surface Topography Benefits Osteogenesis and Osseointegration 3.1.4. 3D Scaffolds with an Appropriate Porous Structure Improve Bone Regeneration 3.1.5. The Combination of Hierarchical Architecture with Growth Factors Affords a Self-Regenerative Capacity for Bone Healing 3.2. Material-Guided Tendon Regeneration 3.2.1. Compositional Gradients Are Helpful To Regenerate Tendon and Maintain Its Multitissue Integrity 3.2.2. Mechanical Gradients and MechanoStimuli Guide the Specific Differentiation of Stem Cells Across the Multitissue Section of Tendon 3.2.3. Aligned Topographical and Geometrical Nanofeatures Promote Tenogenesis and Cell Alignment 3.3. Material-Guided Skeletal Muscle Regeneration

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3.3.1. Special Material Compositions Improve Muscle Protein Production, Muscle Cell Recruitment, and the Structural Integrity of Skeletal Muscle 3.3.2. Substrates with Moderate Stiffness Guide Stem Cell Differentiation into Muscle Cells To Improve Muscle Regeneration Capacity 3.3.3. A Micro-/Nanopatterned Topography Directs Muscle Cell Alignment and Promotes the Formation of Organized Myotubes and Skeletal Muscle 3.3.4. A 3D Micropatterned Geometry Induces the Formation of Well-Aligned Cell Bundles for Muscle Regeneration 3.4. Material-Guided Heart Regeneration 3.4.1. The Composition of Materials Regulates Cardiac Cell Anisotropy, Growth Factor Delivery for Angiogenesis, and/or Electrical Sensitivity for Heart Regeneration 3.4.2. Substrates with an Appropriate Degree of Elasticity Offer Bioinstructive Cues To Correct Abnormal Cell Activities and Remodel the Degenerative Heart 3.4.3. An Aligned Topography Promotes Myogenesis and Cell Alignment, Accelerating Heart Regeneration 3.4.4. Aligned Porous 3D Architectures Promote Cardiomyocyte Differentiation and Increase Cardiac Bioactivity 3.5. Material-Guided Blood Vessel Regeneration 3.5.1. ECM Proteins Promote Capillary Sprouting and Neovascularization 3.5.2. Elasticity Is Important for Maintaining the Elongation and Bioactivity of Vascular Cells 3.5.3. Cellular Responses to Topographical Patterns Drive Re-endothelialization 3.5.4. Geometry with Parallel Pore Alignments Promotes Vascularization and Blood Vessel Ingrowth 3.6. Material-Guided Nerve Regeneration 3.6.1. Material Compositions with the Abilities To Enhance Neural Differentiation, Vascularization, and/or Neural Signal Transmission Promote Nerve Regeneration 3.6.2. Softer Substrates Guide Neurogenesis, while Stiffer Ones Activate Synaptic Connectivity and Transmission 3.6.3. Topography at Nanoscale Promotes Neuronal Differentiation and Hampers Glial Scar Formation, while Microsized Alignments Guide Neurite Growth 3.6.4. Geometries with Pore Alignments Regulate Localized Angiogenesis and Neurite Growth 3.7. Material-Guided Skin Regeneration 3.7.1. ECM Proteins Are Good Enhancers of Wound Healing 3.7.2. Skin Regeneration Can Be Regulated by the Dynamic Sensing of Endogenous and External Mechanical Stimuli

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Chemical Reviews 3.7.3. A Nano-/Microsized Sandwiched Geometry Drives Re-epithelialization for Skin Regeneration 4. Effects of the Development of Materiobiology on the Materials Genome Initiative (MGI) 5. Conclusions and Perspectives Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

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made up of six elements (O, C, H, N, Ca, and P). The arrangement of atoms and molecules results in the formation of cells and their ECM, which together affect how tissues/organs develop. Second, from the perspective of material science, organisms have special architectures that endow them with unique biological functions. Third, the realization of biological functions is highly dependent on the structures of organisms at different biological levels (i.e., atom, molecule, cell, tissue, and organ). These structures inspire researchers to design biomaterials with special microstructures through biomimetic approaches. However, it remains questionable whether the structure(s) of materials alone is sufficient to induce biological functions, especially for certain complicated functions. Our current understanding of the specific roles that materials with special structures play in conferring biological functions remains partial and preliminary. For instance, although it has been reported that the ECM contains useful structural and biochemical information for the regulation of cell functions, the compositional and structural complexities of the ECM render it difficult to elucidate the precise role of each part. In recent decades, increasing attention has been paid to the development of “ECM-mimicking materials”, which are designed to have ECM-like compositions and architectures and constructed using materials with known structures. These materials have distinct compositions and well-defined structures, thus allowing a more detailed exploration of how the properties of materials affect biological functions. Until now, extensive research has indicated that there is a close relationship between material design/properties/applications and biological responses/outcomes,6−8 specifically in three primary ways: (1) Biomaterials have functions that can influence biological responses. (2) The composition, mechanical properties, surface topography, and three-dimensional (3D) geometrical features of biomaterials can provide bioactive cues that act together to synergistically regulate cell behaviors (e.g., cell adhesion, migration, proliferation, differentiation, and stemness maintenance) and guide tissue/organ development. (3) 3D ECM-mimicking scaffolds can stimulate cells to regulate gene expression and synthesize new ECM, which in return adjusts the degradation/adsorption of biomaterials and the growth/remodeling of tissues. Therefore, the features of materials guide cellular biological behaviors for tissue/organ regeneration. The interdependent relationship between biomaterials and biology leads us to propose the concept of “materiobiology”: materiobiology is a scientific discipline that studies the biological effects of the properties of biomaterials on biological functions at different levels (e.g., cells, tissues, organs, and the whole organism). It considers the mechanisms by which materials affect the biological behaviors of living creatures and seeks to disclose the relationships between the characteristics of materials and the features of biology. As an interdisciplinary science, materiobiology draws from material science, biology, chemistry, and physics. It takes advantage of the techniques/ tools for the synthesis, design, and processing of materials to study and modulate biological systems and to elucidate the material-induced underlying principles of regenerative biology. The importance of materiobiology can be described by the following aspects: (1) Considering the extensive amount of current research on biomaterials for tissue engineering, materiobiology provides general guidelines for studying the biological effects of biomaterials and integrating these materialmediated functions at a systematic level. (2) The mechanisms underlying the regulation of biological functions by materials that

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1. INTRODUCTION During the natural tissue regeneration process, the extracellular matrix (ECM, a complex network of proteins and polysaccharides secreted and assembled by cells) provides bioactive cues for the regulation of cell activities and tissue/organ functions.1 Natural tissue regeneration is highly regulated by mechanical signals, which can be sensed by cells and transformed into biochemical information, and vice versa that the biochemical information can be transformed back to mechanical signal. Cell− cell and cell−ECM interactions regulate cell functions and ECM remodeling, which maintain the dynamic self-regeneration/ renewal of tissues and organs.2 Serious injuries, diseases, and changes with aging may damage the self-regenerative capacity of tissues and organs, necessitating their replacement or regeneration with the help of medical devices. Among these devices are biomaterials that can be used to treat, augment, repair, or replace a tissue and its functions, or for diagnostics.3 In the early 20th century, biomaterials were mainly used in prosthetic devices such as vascular stents and artificial hips. In these devices, the inertness of the materials was emphasized to avoid “biological rejection” from the host organism.4 Although biomaterials have been researched for a long time, their clinical application is still hampered, probably because the “inertness” of the materials means that they cannot provide sufficient bioactive cues to trigger cell functions needed for tissue engineering.5 Recently, the availability of advanced analytic equipment has helped in understanding the principles of cellular activities and their interactions with materials at the genetic and molecular levels. This new understanding is useful for designing biomaterials with controllable properties to mediate specific biological responses. In addition, advances in material fabrication technologies allow the development of biomaterials with welldefined architectures that can temporospatially regulate biochemical and biophysical processes. Revolutionary advances in analytic instruments and material processing technologies have greatly enriched our understanding of the relationships between materials and biological functions. This enhanced understanding allows us to ask new questions. Is it possible to finely regulate biological functions by optimizing the properties and structure of materials through biomimetic design, that is, that is inspired by the natural components of the human body?6 In combination with the innate biological activities of cells and tissues, can materials be designed to enable active control of cell behaviors and tissue/organ functions, and thereby synergistically facilitate host-initiated repair/healing/regeneration? It is known that the assembly of atoms into molecules, cells, tissues, and organs endows the body with the features of life. First, humans consist of biological materials, of which ∼99% are C

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Figure 1. ECM proteins show good bioactivity in directing cell behaviors and/or tissue regeneration, while they lack sufficient mechanical properties. However, these properties can be improved through complexation with synthetic polymers or some inorganic elements. The composites can be further endowed with dynamic degradability for the controllable release of specific composition(s) to stimulate/guide cell functions and create space for cell/ tissue growth. The regulation of cell fate can be further adjusted by varying the stiffness/softness of materials at the 2D or 3D level and is mediated via special intracellular signaling and a mechanotransduction pathway arising from interactions between cells and materials. Materials can also be afforded with special 2D topographical (nano-/micropatterned) and/or 3D geometrical (nano-/microhierarchical architecture) cues to direct cell adhesion, spreading, proliferation, migration, and/or differentiation for tissue regeneration. Specific combinations of these bioactive properties are expected to endow materials with the capacity to modulate biological functions (such as self-regenerative ability) for tissue/organ regeneration.

further used for the manipulation of morphogenesis and organogenesis. Typical examples are that the alignment of nanofibers can promote neuronal regeneration28 and blood vessel regeneration.29,30 Materials with micro-/nanohierarchical architectures can exert profound synergistic effects on the regeneration of tissues such as muscle31 and bone.32 Meanwhile, materials with biomimetic features in their compositions and structures may induce some biological functions (e.g., the homing of endogenous cells) that promote tissue regeneration.8 Until now, most reports regarding the effects of materials on biological functions have been case-by-case studies evaluating discrete phenomena. Systematic investigation of the mechanisms by which material properties affect cell functions to decide cell fate and tissue/organ development is still lacking. Thus, our understanding of the impact of materials on biological functions remains preliminary and needs to be improved. Therefore, in this Review, we systematically summarize the recent progress on how biomaterials with controllable properties (e.g., compositional/ degradable dynamics, mechanical properties, 2D topography, and 3D geometry) can regulate cell behaviors (e.g., cell adhesion, spreading, proliferation, cell alignment, and the differentiation or self-maintenance of stem cells) and tissue/organ functions. Furthermore, we will highlight how the biophysical features of materials influence tissue/organ regeneration. Finally, current challenges and a perspective on the development of novel materials that can modulate specific biological functions are prospected. Such materials can be developed using advanced technologies (e.g., high-resolution fabrication technology) to spatially localize bioactive factors (Figure 1). On the basis of our summary, it is clear that the properties of materials significantly influence the biological functions of the human body. This Review discusses a new paradigm of “materiobiology” and highlights that it is more important to develop ECM-mimicking biomaterials having a self-regenerative capacity to stimulate tissue regeneration, instead of attempting to recreate the complexity of living tissues or tissue constructs ex vivo. The principles of materiobiology may benefit the development of novel biomaterials providing combinative bioactive cues to

materiobiology elucidates may indicate new strategies to design bioinstructive materials with an endogenous regenerative capacity and a high bioactivity for tissue engineering.7,8 As stated by Mooney,3 research on biomaterials is moving toward “bioinspired design and incorporation of dynamic behavior” to optimize the self-regenerative potential of materials, which is the essential nature of materiobiology as proposed herein. From the birth of tissue engineering in 1993,9 investigations have increasingly been focused on the effects of materials on cell behaviors and tissue regeneration. Such efforts have indicated that tissue engineering is a complicated process in which tissue/ organ development is highly dependent on material characteristics. During this process, each feature of a material plays a unique role in the regulation of cell behaviors. For instance, material components (e.g., ECM proteins and metal ions) are capable of adjusting cell−material adhesion, driving cell polarization to enhance cell migration,10 affecting the cell spreading rate and area,11 regulating cell proliferation or differentiation,12 and determining stem cell quiescence and stemness maintenance.13 These cell functions can also be affected by biophysical cues from the mechanical, topographical, and geometrical features of biomaterials. That is, each aspect [e.g., the composition, physical properties, two-dimensional (2D) topography, and 3D geometry] of materials contributes to the regulation of cell functions that collectively promote the regeneration of tissues/organs.6,14−20 For instance, the ECM provides biophysical cues for the regulation of stem cell osteogenesis, which can be further improved by the introduction of specific inorganic elements (e.g., ions of Ca, P, Mg, Li, Ir, Sr, Zr, Zn, and F).21 Stiff substrates accelerate osteogenic differentiation, which can be further promoted by the introduction of disordered nanopatterns, but not highly ordered ones.22,23 Soft substrates with an ordered nanotopography increase neuronal differentiation even in the absence of induction factors.24−26 Stem cells within hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favor osteogenesis (adipogenesis).27 Physical parameters can be D

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Figure 2. Schematic illustrations of the processes by which muscle cells interact with ECM substrates. (A) Role of stiffness: The interactions between ECM stiffness (KE) and integrins (KI) can be transferred to cells through the integrin−adaptor (KA) bond (FIA) and the adaptor−actin bond (FAA). The adhesion process begins with nascent adhesions through which adaptor proteins mediate the formation of initial integrin clusters, and might promote actin polymerization from clusters (formins). Myosin II filaments exert local contractile forces between filaments (blue arrows). As adhesions mature and grow, they connect to actin fibers moving rearward toward the cell center due to global myosin contractility (blue arrows). Rearward-moving actin pulls on bound adaptor proteins, which in turn pull on integrins. Because of transient stick−slip bonds, rearward speeds are reduced with respect to actin to a certain extent in adaptor proteins, and even further in integrins (black arrows). (B) Molecular mechanosensing mechanisms: force could be detected by (i) exposing cryptic domains in ECM molecules, (ii) increasing the lifetime of fibronectin-α5β1−integrin bonds (catch bonds) and activating integrins, (iii) increasing the phosphorylation of the p130Cas substrate domain, (iv) releasing focal adhesion kinase (FAK) autoinhibition and increasing its tyrosine kinase activity, (v) exposing previously buried vinculin-binding sites in the talin rod domain, (vi) exposing previously buried binding sites to integrin-β tails or filamin-interacting protein in filamin immunoglobulin-like domains, (vii) releasing myosin light-chain kinase autoinhibition and activating myosin II, and (viii) increasing the lifetime of myosin-IIA−actin bonds (catch bonds). (C) Signaling processes downstream of force detection: The upper panel shows receptor-type tyrosine-protein phosphatase (RPTP)-α-, Fyn-, and talin-dependent signaling events. (i) Cell attachment, integrin clustering, and deformation of the ECM result in RPTP-α activation. This allows the Src-family kinase Fyn to access the catalytic site. Fyn is dephosphorylated at tyrosine 531 and switches to the open conformation. (ii) Fyn subsequently phosphorylates Rac1 guanine nucleotide exchange factors (GEFs), such as Tiam1, that in turn activate Rac1. (iii) Rac1 activity induces actin polymerization from integrin adhesion sites by formin family proteins and/or the ARP2/3 complex. (iv) Rearward actin flow driven by myosin II contractility stretches talin to expose cryptic vinculin-binding sites, leading to adhesion strengthening and cluster growth. (v) When high forces are generated by actin rearward flow and contractility, the bond between actin and talin slips and talin relaxes. The lower panel depicts FAK- and p130Cas-dependent downstream signaling. (vi) Downstream of integrin clustering, FAK is autophosphorylated at tyrosine 397. Phosphorylated FAK binds to talin and phosphorylates the Rac1 GEF b-PIX (encoded by ARHGEF7). (vii) FAK also forms a complex with Src to activate p130Cas, which is hyperphosphorylated and binds to Crk and DOCK180 to activate small Rho GTPases, such as Rap. (viii) Rac1 induces actin polymerization and cell protrusion, whereas Rap enhances adhesion maturation (ix). (x) Additionally, FAK can activate p190GEF to enhance RhoA activity, stress fiber formation (xi), and adhesion turnover. (xii) Cross regulation can alter the region of activity of Rho GTPases. Reprinted with permission from ref 34. Copyright 2012 The Company of Biologists Ltd.

the systematic database of the Materials Genome Initiative

activate the migration of stem cells from endogenous reservoirs (i.e., cell niches), stimulate robust and scalable self-healing mechanisms, and unlock the body’s innate powers of regeneration.33 Furthermore, with our deeper and more systemic understanding of “materiobiology”, various experimental and theoretical materiobiological data are expected to greatly enrich

(MGI) strategic plan launched in 2014,423 in which materiobiology is the core content in the aspect of biomaterials. E

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Figure 3. Cell−cell and cell−ECM interactions for the regulation of cell sorting and tissue assembly. (A) Integrins mainly mediate cell−ECM assembly, and cadherins mainly regulate cell−cell adhesions, both of which contribute to orchestrating vertebrate morphogenesis. (a) Integrins canonically mediate ECM assembly, but also promote cellular cohesion as a secondary effect. (b) Cadherins canonically mediate cell−cell cohesion, but stimulate ECM assembly as a secondary effect. (c) The convergence of these secondary effects suggests that integrins and cadherins, via very different mechanisms, act semiredundantly to effect the same outcome. (B) Inside tissues, cadherin 2 can maintain cell−cell adhesion, stabilize intercellular α5-integrin association, and repress integrin activity, while on the tissue surface, integrins are activated to bind with ligands, such as the ECM component fibronectin. (a) A schematic transverse cross-section of paraxial mesoderm with adherent mesenchymal cells (red hexagons) and fibronectin matrix (yellow) on the tissue surface. (b) Within the mesenchyme, integrins and cadherins on adjacent cell membranes associate and repress integrin activity. (c) On the tissue surface, there is no cadherin 2 and integrin is activated, resulting in fibronectin fibril formation. Adapted with permission from ref 41. Copyright 2015 Elsevier. (C) Cell−ECM adhesions can be mediated by focal adhesions that have a nanoscale architecture. Adapted with permission from ref 46. Copyright 2010 Springer.

regeneration of tissues.36 Previous studies have indicated that

2. MATERIAL GUIDANCE OF CELL BEHAVIORS (CELL FATE) It is recognized that the ability of cells to undergo fundamental cellular processes (e.g., cell adhesion, migration, proliferation, and differentiation) on the surface or interface of biomaterials is crucial for the successful regeneration of tissues.35 If biomaterials can be endowed with bioactivity for the regulation of cell functions, they are expected to have the potential to direct the

certain compositions, physical properties, and surface and structural characteristics of biomaterials can regulate cellular behaviors and direct the process of tissue regeneration, mainly through direct interactions between cells and biomaterials.34,37 F

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Figure 4. (A) The incorporation of magnesium phosphate cement (MPC) into calcium phosphate cement (MCPC) can regulate cell adhesion affinity and the alkaline phosphatase (ALP) expression of stem cells (an indicator for osteogenic differentiation). (B) A moderate Mg2+ content can optimize the conformation of fibronectin (Fn) to activate its interaction with integrins, improving cell adhesion, osteogenic differentiation, and bone regeneration (C). Reprinted with permission from ref 37. Copyright 2015 Elsevier.

2.1. How Do Cells Sense Materials?

activation, the activities of Rac1 and Rap are enhanced, resulting in the improvement of actin polymerization and cell protrusion as well as adhesion maturation. Furthermore, FAK can also increase Rho activity to induce stress fiber formation and adhesion turnover (Figure 2C).34

Traction forces exerted by cells through their interactions with the ECM influence cytoskeletal tension and induce changes in cell morphology and associated signaling cascades that ultimately alter gene expression to regulate cell functions (e.g., cell migration, differentiation, proliferation, and apoptosis) and the development/regeneration of tissues, organs, or diseases.34,38 As an example, muscle cells interact with the ECM through the binding of cell surface integrins with ECM adhesion ligands via a process of mechanotransduction, which is dependent on the mechanics, composition, and structure of the microenvironment.39 In the initial stage, integrin clusters induce contractile forces that stimulate actin filament growth and actin−myosin assembly via the formin-mediated polymerization of single actin filaments. Upon the maturation of focal adhesions, myosin II endogenous contractility becomes the main force driving actin filament polymerization to control cell movement (Figure 2A). Integrins are indirectly linked with the actin cytoskeleton via protein adaptor(s), such as talin, filamin, tensin, parvin, and/or myosin X, forming different kinds of cell−ECM interactions, such as nascent adhesions, focal complexes, focal adhesions, and podosomes (Figure 2B). Mechanical signals can be sensed by cells through mechanotransduction, mainly via cellular pathways including phosphorylation/dephosphorylation alongside small GTPase and phospholipid signaling.34 For example, cell−ECM mechanical interactions can activate RPTP-α to increase Rac1 activity through Fyn phosphorylation/dephosphorylation. Activated Rac1 induces actin polymerization from integrin adhesion sites and actin-myosin assembly to increase adhesion interactions and cluster growth. Integrin clustering activates focal adhesion kinase (FAK) through autophosphorylation, which reactivates integrins to increase their binding strength. Through FAK

2.2. Material-Guided Cell Adhesion

Cell adhesion is involved in various biological processes, including embryogenesis, tissue regeneration, and wound healing.40 Cell adhesive interactions with neighbors and/or the ECM regulate different cell activities (cell spreading, cell migration, cell sorting, and cell assembly), as well as the assembly, structure, and mechanical integrity of tissues.41 Cell adhesions are mediated by transmembrane cell−cell and cell− matrix adhesion molecules, which provide a direct connection between the cell cytoskeleton and neighboring cells and/or ECM proteins. Such molecular networks are fundamental to the transmission of the bioactive signals originating from microenvironments to cells and tissues to regulate the morphology of cells (the orientation and localization of subcellular organelles and cell polarity), and the structure and mechanical properties of tissues.41 Generally, there are two kinds of cell adhesions (i.e., cell−cell and cell−ECM adhesions). Through these interactions, cells can sense external forces and geometrical constraints. Cell−cell adhesion is dominated by transmembrane adhesion receptors such as cadherins, while cell−ECM adhesion is mainly mediated by integrins.41 Cells expressing cadherins of similar types tend to adhere together, which is important for cell sorting.42 Morphogenesis during embryonic development occurs through cell−cell adhesion and coordinated interactions/communications between cells both nearby and more distant. Cadherins can G

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Figure 5. Micro-/nanopatterned substrates with specific cell-adhesive and cell-resistant arrangements can regulate the size and distribution of integrinmediated adhesions, cell shape, and cytoskeletal architecture (A) and guide the formation of multicellular organization (B), which can be further controlled by adjusting the stiffness of the substrate material (C, D). Substrates can be endowed with nanotopological features to regulate cell−matrix adhesions for the manipulation of the size and geometry of cells cultured on them (E, F). Reprinted with permission from ref 67. Copyright 2012 The Company of Biologists Ltd.

integrin binding.48 Cell adhesion can also be regulated by the direct decoration of substrates with cell adhesion ligands such as proteins, peptides, and oligosaccharides49,50 or other organic compositions (e.g., CH3, OH, COOH, and NH2).51 As a metalloprotein, integrin has a high affinity with certain metal ions (e.g., Mg2+, Mn2+, and Ca2+), which can be introduced into biomaterials to regulate cell adhesion activity.52 For instance, doping calcium phosphate cement with Mg2+, which has a high binding affinity with integrin receptors, can effectively upregulate the expression of α5β1-integrin in bone marrow stromal cells (BMSCs), improving cell−material adhesion, actin filaments assembly, osteogenic differentiation, and bone regeneration efficacy (Figure 4).37 2.2.2. Cells Prefer To Adhere to Substrates with Similar Stiffness. The stiffness of materials is often measured in terms of Young’s modulus (E) and expressed in units of pascals (Pa). The human body consists of soft and hard tissues with elastic moduli ranging from hundreds of Pa to GPa, which can be sensed by cells mainly via mechanotransduction for the regulation of cell behaviors, disease development, and embryonic morphogenesis.53,54 It was commented in an impressive review by Discher et al. that “tissue cells feel and respond to the stiffness of their substrates” in an active way.53 Cells tend to grow on material surfaces that are similar in stiffness.55 In normal muscle tissues, cells probe elasticity as they anchor and pull on their surroundings, where myosin-based contractility and transcellular adhesions transmit cellular forces to the substrates.53 Most cells tend to anchor more strongly to stiffer 2D substrates instead of soft ones.53 Interestingly, in contrast to cell adhesion to 2D

mediate the assembly of the intracellular actin cytoskeletal network (ACN) at conjunctions, while the ACN maintains cadherin integrity at cell−cell contacts.43 Both of these functions play important roles in maintaining a morphologically stable structure during the dynamic changes in tissue architecture that occur via remodeling.44 Cell−ECM adhesion occurs through the formation of supramolecular protein complexes called focal adhesions, which are nanoscale (1600 nm had adhesive behaviors similar to those of cells cultured on flat surfaces.64 Cell adhesion on substrates is more effective when the spacing width of their adhesive ligands is below 60 nm (instead of being either 74 or 120 nm), probably because the clustering of integrins only occurs at such a scale (4−5 integrins).65,66 However, micropatterns are helpful for tuning the arrangement of integrin-mediated adhesions, thus guiding the shape/orientation of adhesive cells. In this sense, nano-/microstructured biomaterials can synergistically regulate cell adhesion behaviors (Figure 5).67 2.2.4. 3D Microenvironments Facilitate Cell Adhesion More Effectively than 2D Topographical Substrates. In the past few decades, the topographical characteristics of 2D surfaces and their effects on cellular biological responses have been widely investigated due to the relatively high accessibility and reproducibility of such experiments. However, it is more important to elucidate how cells behave in 3D scaffolds instead of on 2D substrates, because cells in vivo mostly live in complex 3D microenvironments. Notably, “topography” refers to 2D structural factors, while “geometry” is defined as 3D structural microenvironments, determined by the surrounding fiber matrix and the arrangement of neighboring cells.2 Although 2D and 3D systems have similarities in cellular adhesion and contraction, they present distinct differences in several aspects (e.g., the composition of focal adhesions, cell morphology, and contractility).47,68−72 As compared to 2D substrates, 3D scaffolds have a lower effective stiffness, different geometric parameters (e.g., porous structure and surface/interface state), and special dynamic degradability.73−76 Therefore, cells may present different adhesive behaviors upon switching from 2D substrates to 3D microenvironments, which is largely dependent on whether cells adhere to the ECM via partial cell surface contact or via most/all of the cell surface through an integrin-mediated mode (Figure 6).67 Distinct from 2D substrates where focal and fibrillar adhesions occur in different areas, 3D microenvironments allow for the simultaneous formation of focal and fibrillar adhesions at the same location in vivo (Figure 7).47 3D microenvironments may provide combinative temporospatial biochemical/physical cues to regulate cell functions more easily, as compared to 2D substrates.77 For instance, fibroblasts can form 3D-matrix adhesions within 5 min, while it takes several days to culture cells at a high density on 2D substrates to evolve 3D-matrix adhesions, which may be associated with the cooperative effects in 3D systems.47 Unlike a stiff 2D substrate, a pliable 3D matrix allows the free assembly of 3D-matrix adhesions.47 Therefore,

Figure 6. Schematic representation of adhesive, topographical, mechanical, and/or geometrical factors on 2D substrates or in 3D microenvironments. The bioactive cues encountered by a cell are strikingly different between 2D ECM-coated glass and 3D ECM (e.g., collagen) scaffolds, thus affecting cell behaviors in different modes. Reprinted with permission from ref 67. Copyright 2012 The Company of Biologists Ltd.

cell adhesion can be effectively regulated by adjusting the 3D geometrical parameters (e.g., pore size and pore shape). For example, a small pore size may limit the extension of stem cells and result in a round shape with smaller cell adhesions, while a large pore size with curved turns may favor the formation of larger cell adhesions.78,79 Combining appropriate topographic features (surface/interface structure) with geometric ones (pore size, porous interconnectivity, and architecture) in the design of scaffolds can exert synergistic effects on cell functions (e.g., osteogenesis).80 It should be noted that the formation of a moderate number of cell adhesions, but not an excessive number, is desirable to improve cell functions in 3D microenvironments.81,82 2.3. Material-Guided Cell Migration

Besides cell adhesion, another important cell activity is cell migration. Cell migration is involved in many complicated processes, including pseudopodia protrusion, mechanical force transfer, the formation of new adhesions, and the release of old ones.83,84 Cells can migrate individually (single migration) or move together (collective migration). Different cells have different types of movement. For instance, fibroblasts move slowly and neutrophils move quickly in single migration mode. Proper cell−cell adhesion is required to induce cell polarity, and cell−cell assembly is required for collective migration.85 For collective migration, cell−cell junctions (e.g., cadherins) are required to maintain the integrity of cell groups.86,87 Cells can I

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Figure 7. In vivo 3D-matrix adhesions differ from focal or fibrillar adhesions on 2D substrates. (A−E) Confocal images of indirect immunofluorescence staining of an NIH-3T3 mouse fibroblast in vitro on a 2D fibronectin-coated coverslip; (F−J) transverse cryostat craniofacial mesenchyme sections of an E13.5 mouse embryo. α5-Integrin [(A, F), green] and paxillin [(B, G), red] colocalize with a fibrillar assembly in mesenchymal tissue [(H), yellow in merged image indicates overlap of red and green labels], but not on a 2D substrate in vitro (C). Fibronectin [(D, I), blue] localizes to fibrillar structures in vivo, and merged images indicate a substantial overlap of all three molecules [(J), white as compared to (E)]. Note that focal adhesions (filled arrowheads) and fibrillar adhesions (open arrowheads) show differential localizations of the α5 integrin and paxillin markers only on traditional flat 2D substrates in vitro. The 3D-matrix adhesions (arrows) identified by triple localization are present in 3D environments in vivo. Scale bar, 5 μm. Reprinted with permission from ref 47. Copyright 2001 American Association for the Advancement of Science.

Figure 8. Cells migrate in different modes, including lamellipodia (LM), lobopodia (LB), and amoeboid. 2D lamellipodia protrude in a wide flattened shape, while 3D lamellipodia assume a small and sharper fan-like structure at the migration tip, where myosin II and ROCK are needed to drive 3D cell migration. Cells exhibiting LB migration present blunt cylindrical protrusions, and this migration mode tends to occur in linearly elastic 3D scaffolds. Both LM and LB migration require the formation of robust adhesive interactions with the surrounding matrix via integrin function. In contrast to normal cells, many cancer cells may switch from LM-based mesenchymal migration to amoeboid migration upon the inhibition of extracellular proteolysis in 3D systems. Reprinted with permission from ref 95. Copyright 2012 The Company of Biologists Ltd.

move as sheets, chains, or clusters in collective migration mode.88,89 Cell migration is involved in various physiological and pathological processes, such as morphogenesis,90 immune responses,91 the renewal of skin and intestinal cells,92 and tumor metastasis.88,89 Cell migration can be classified into lamellipodia, lobopodia, amoeboid, pseudopodia (including rhizopodia and axopodia), and mesenchymal types according to the shapes adopted by cells during their movement.93 Lamellipodia are thin fan-shaped protrusions enriched in F-actin and actin-binding proteins such as cortactin.94 Lobopodia are blunt cylindrical protrusions that might be driven by intracellular pressure rather than actin polymerization. Cancer cells in 3D collagen scaffolds adopt round amoeboid migration.95 Amoeboid migration is proteinaseindependent and contractility-dependent, while mesenchymal migration is proteinase-dependent and contractility-independent (Figure 8).10

To drive cell migration, an asymmetric force should be generated through a cell polarity process that is often involved in the activation of actin polymerization at the leading edge of migration, localized focal adhesion, actomyosin contractility, or osmotic pressure.96−99 Similar to cell adhesions, cell migration can also be regulated by the specific temporospatial variation of compositional, mechanical, and/or structural factors in the microenvironments of cells.97,100 2.3.1. Compositional Gradients Create a Driving Force To Guide Cell Migration. The human body has compositional gradients (e.g., of proteins and proteoglycans) in the ECM, which create a natural driving force to direct cell migration (chemotaxis). For instance, gradients in the abundance of endothelial surface adhesion proteins are required for the recruitment of T lymphocytes onto endothelial cells at sites of infection in vivo. Gradients in the abundances of collagen and laminin are associated with angiogenesis and cancer invasion.10 J

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can be classified into four types: durotaxis (stiffness-mediated migration), plithotaxis (tensile-induced directional collective migration), ECM deformation-induced single migration, and cohesotaxis (migration guided by gradients of intracellular tension) (Figure 10A−D).95 In contrast to chemotaxis, mechanotaxis regulates cell migration through direct mechanical transmission and/or indirect mechano-biochemical transduction.109,110 Mechanotaxis is involved in various biological processes, including epithelial-to-mesenchymal transition,111,112 nervous system development,113,114 innate immunity,115 and cancer metastasis.116,117 In a thoughtful commentary, Petrie et al. reviewed the effects of the mechanical properties of 2D or 3D microenvironments on cell migration and disease development.95 It is indicated that cells may migrate by different modes in scaffolds with different mechanical properties. The actin-binding motor protein myosin II maintains a low level of tension on actin fibers that are coupled to the ECM through cell−ECM adhesions. The RhoA−ROCK− myosin-II signaling axis is capable of sensing changes in ECM structure and responding by increasing actomyosin contractility.95 Crosstalk between Rac1 and RhoA signaling potentially regulates the mechanosensing of matrix rigidity and the mode of 3D cell migration.95 For instance, nonlinear elastic materials trigger lamellipodia-based motility, while elastic materials promote lobopodia migration.95 Applying a cross-linking treatment to 3D collagen hydrogels can transform their nonlinear elasticity to linear elasticity, which changes cell migration behavior from a mesenchymal mode to an amoeboid one.118 Cancer cell migration is linked to increased force generation in both 2D and 3D microenvironments.119−122 Cancer cells that use lamellipodia might have a low membrane tension, while increased actomyosin contractility could elevate membrane tension to promote lobopodia migration.95 Migrating cancer cells alter the geometry of the ECM and define tracks for following cells, causing cancer cells to migrate in an inherently collective process that accelerates cancer metastasis123 (Figure 10E). These results provide indirect evidence for how the correct “material properties” are needed to provide proper “biological information” to instruct biological functions at both the cell and the tissue levels. 2.3.3. Patterned Substrates Accelerate Directional Cell Movement. Directional cell migration can be achieved by culturing cells on surfaces with special patterns.124 For instance, oriented nanofibers cause higher cell mobility than randomly oriented ones.125 In addition, cells tend to move along the grooves of substrates, while random migration occurs on flat substrates.126 NIH 3T3 fibroblasts cultured on grooved substrates with 550−1100 nm spacing presented quicker migration than those cultured on one with 2750 nm spacing.127 A luminal stent with 15 μm-wide surface microgrooves created in the direction of coronary flow accelerated endothelial cell migration, resulting in lower levels of neointimal formation.128 It is believed that pattern-guided cell migration may be attributed to preferential actin polymerization along the alignment groove.129−131 A patterned topography enables the directional movement of specific cells for wound healing (corneal regeneration) or treatment of infectious diseases.132 2.3.4. Scaffolds with 3D Porous Geometry and CellResponsive Biodegradability Dynamically Regulate Cell Migration. As compared to 2D systems, 3D models more closely mimic physiological conditions. It is indicated that cells in 3D microenvironments migrate in a distinct mode from that of cells on 2D substrates. Cells undergoing 2D migration often

Figure 9. Specific chemokine receptors (e.g., G-protein-coupled receptors) can drive the directional movement of a chemotaxing cell along chemogradients. At the migration front, a chemokine receptor coupled with a heterotrimeric G-protein controls an actin network that includes Arp2/3, WASp/Scars, CARMIL, myosin I, and ADF/cofilin. At the back and sides, the receptor and G-protein regulate actin/myosin II complexes. Reprinted with permission from ref 92. Copyright 2008 Elsevier.

Chemotaxis can be also used for the mediation of cell movement in biological processes such as the regeneration of nerves92 or skin,101 immune responses,102,103 and cancer metastasis.92 Migration can be driven by the formation of dynamic assemblies of the actin cytoskeleton in cells (Figure 9). In chemotaxis, chemokine gradients strongly direct actin assembly to the cell’s leading edge and, hence, determine the direction of cell movement.92 Fast-moving cells such as neutrophils are directed by gradients of fMLP peptide released by bacteria during immune responses.10,100 On the basis of their physiological roles, chemokines can be classified as “inflammatory” factors for the recruitment of immune cells to inflamed regions or “homeostatic” ones for the trafficking and positioning of cells belonging to the adaptive immune system.102,103 The overexpression of platelet-derived growth factor at a site of injury can recruit dermal fibroblasts to the provisional clot for wound healing.101 In a newly forming blood vessel, a tip cell is formed by sensing the chemotactic gradient.104 It has to be noted that the excessive recruitment of these cells results in tissue damage and chronic inflammatory diseases.92 In addition, cellular migratory behavior is closely regulated by two interdependent cellular functions: binding of the cell surface adhesion receptors and ECM proteolytic degradation.83,105 In this sense, the introduction of proteolytic components (e.g., collagen, laminin, fibronectin, and synthetic blocks containing matrix metalloproteinases) may allow the regulation of cell-induced degradation in specific areas of scaffolds to create dynamic pathways for cells to move along.106−108 2.3.2. Mechanical Stimuli Can Change the Cell Migration Mode. The mechanical properties of materials are capable not only of regulating cell adhesion, but also of mediating cell migration.11 The process of using mechanical gradients to regulate cell migration is called “mechanotaxis”. Mechanotaxis K

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Figure 10. Schematic representation of how the mechanical properties of materials mediate cell migration behaviors (mechanotaxis). There are four types of mechanotaxis (A−D): (A) Durotaxis, cell migration directed by stiffness gradients of the ECM. (B) Plithotaxis, the tendency of cells within a monolayer to migrate along the local orientation of the maximal principal stress. (C) Isolated cells can attract each other across long distances by deforming their common ECM. (D) Cohesotaxis, the tendency of cells within a monolayer to follow gradients of intracellular tension. Reprinted with permission from ref 109. Copyright 2013 Elsevier. (E) Mechanical properties can regulate the mode of 3D cell migration. The 3D migration of metazoan cells using lobopodia occurs under conditions of high cell−matrix interaction and RhoA activity in scaffolds with linear moduli. In contrast, conditions of low cell−matrix adhesion and low RhoA activity in scaffolds with nonlinear moduli benefit 3D migration using lamellipodia. Cancer cells switch to a rounded amoeboid mode that requires low adhesion and high contractility upon inhibition of protease activity or modulation of Rho GTPase crosstalk. It remains to be determined how cells can transfer from lobopodia-based migration to round bleb-based motility (indicated by the question mark). Reprinted with permission from ref 95. Copyright 2012 The Company of Biologists Ltd.

move in lamellipodia mode, maintaining actin polymerization against the plasma membrane over a broad area to push their leading edge forward, followed by integrin-dependent adhesion to the underlying substrate.133−135 However, 3D cell migration occurs in loose or denser connective tissues. Single migration in 3D microenvironments can be seen in primordial germ cells, leukocytes, hematopoietic stem cells, and cancer cells during metastasis.136 Collective cell migration is often constrained, generally depending on the proteolytic activity of the migrating cells,137 although there are some examples of proteolyticindependent 3D migration (e.g., amoeboid movement) where the cytosol is pushed into the surrounding matrix.138,139 To migrate in 3D environments, all leukocytes use amoeboid movement, while macrophages use both amoeboid and mesenchymal migration modes. Mesenchymal migration takes

place in dense matrixes and involves podosomes and proteolysis of the ECM to create paths. Podosome disruption has been correlated with reduced mesenchymal migration of macrophages and unaffected amoeboid migration.140 The introduction of a porous structure or dynamic degradation links in 3D scaffolds may be useful for the regulation of cell migration behaviors.73,74 For instance, the presence of matrix metalloproteinase-sensitive degradable elements in 3D scaffolds can increase cell migration. Faster migration occurs in scaffolds with a pore size of 12 μm (equivalent to cell size) than in those with pore sizes of 7 μm (smaller than cell size) and 17 μm (larger than cell size). Therefore, the optimization of cell migration behaviors can be achieved through the synergistic optimization of the composition, mechanical properties, topography, and geometry of 3D scaffolds.141 L

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which cells interact with their substrates.144 Materials with specific compositions, mechanical properties, and structures can provide bioactive cues for the regulation of cell spreading, probably through the adjustment of extensional and contractile forces and dynamic ECM remodeling (Figure 11).144,145 2.4.1. Materials with a Positive Charge and Moderate Hydrophilicity Promote Cell Spreading. Normally, sufficient adhesive interactions are required for cells to spread on their substrates. A lack of cell adhesion may result in apoptosis. Because ECM proteins have cell−ECM adhesive sites through which they stimulate cells to express specific types of integrins and synthesize new adhesive proteins, ECM proteins such as fibronectin and laminin can be introduced onto substrates to mediate cell adhesion and spreading. For instance, both the degree and the speed of fibroblast spreading are accelerated when the fibroblasts are cultured on substrates that are decorated with appropriate concentrations of fibronectin.11 In addition to proteins and peptides, cell spreading is also sensitive to certain other organic molecules on substrates.148 It is indicated that the decoration of substrates with functional groups of −CF3H, −COOH, −SO3H, epoxy, −OH, and NH2 results in increased cell spreading. The highest improvement in cell spreading may be associated with the positive surface potential and moderate hydrophilicity (contact angle 40−60°) of amino-modified substrates,149 which promote cell adhesion and cell polarity, thus accelerating the spreading process of cells.146 2.4.2. Cells Present Less Spreading on Soft Substrates, Probably Due to Reduced Actin Organization and Local Adhesion. Cell spreading involves the dynamic adjustment of cell morphology via balancing the mechanical states of extension and contraction, which are mediated by actin traction and integrin-mediated adhesion.11 Cells and tissues often undergo dynamic mechanical interactions with their neighbors, and these mechanical signals are necessary for biological development,

2.4. Material-Guided Cell Spreading

Cell spreading is a key factor in the regulation of cell functions (e.g., cell proliferation, differentiation, and apoptosis) during tissue/organ development.142 Cell spreading is a determinant of the final morphology of cells and is driven through their interactions with the ECM.83,143 Therefore, the investigation of cell spreading may inform us about the basic mechanisms by

Figure 11. Scheme representing how cell spreading can be regulated by adjusting the composition, mechanical properties, topography, and geometry of biomaterials. Substrates with a positive charge (e.g., decorated with NH2) and moderate hydrophilicity (contact angle, 40− 60°) are beneficial to cell spreading. Stiffer substrates make cells present a more elongated shape than soft ones.146 2D nano-/micropatterns and 3D porous architectures with a high orientation degree induce cells to spread into extended morphologies.147

Figure 12. Effect of material stiffness on stem cell spreading and proliferation was examined using hydrogels (top row, soft 290 Pa, stiff 19.1 kPa) and fiber networks (middle row, soft 140 MPa fiber, 2.8 kPa network; stiff 3.1 GPa fiber, 55 kPa network). Low (0.5 mg mL−1, 10 μm patterns and those cultured on a flat surface,384 probably because the large micropatterns behave more like a flat surface and lose their guiding effects on cell morphology and differentiation.391−393 However, micropatterns can regulate other nerve-related biological activities. For example, microscaled structures allow the infiltration of hippocampal astrocytes and endothelial cells, whereas submicrometer-scale features lack this ability.394 Microgrooves (10−20 μm width) were demonstrated to create a pathway that directed cell migration, proliferation, and neurite growth.57,234,395 Therefore, nano-/micro topographies may

provide multiple bioactive cues for the spatial control of cell behaviors (adhesion, migration, proliferation, alignment, and differentiation) and neuronal assembly (neurite growth and neurite pattern), resulting in a synergistic effect on nerve regeneration.233,235,323,396 3.6.4. Geometries with Pore Alignments Regulate Localized Angiogenesis and Neurite Growth. Developing a method for the specific control of nerve growth is important for improving nerve regeneration. Different from 2D substrates, 3D porous scaffolds can regulate cell functions (e.g., neurogenesis) and the tissue regeneration process more effectively.323 Scaffolds with >100 μm pore size allow for tissue ingrowth from the host. Hyaluronic acid-poly-D-lysine hydrogels with pore sizes of 90− 230 μm can successfully integrate with host tissue, promoting endothelialization and new ECM formation.397 Nanoporous structures can also be introduced to endow scaffolds with drug administration functions (e.g., dexamethasone administration to stabilize the neural interface and prevent astrocyte adhesion).398 Besides pore size, the porous arrangement is another factor affecting nerve regeneration. Interconnected porous hydrogels with aligned pores (40 μm) and orthogonal pores (80 μm) promoted localized angiogenesis and neurite growth.399 AD

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Figure 29. Intracellular mechanisms of mechanotransduction. Mechanical force is sensed by the integumentary system and activates multiple intracellular signaling pathways. Several membrane-bound mechanosensory complexes have been described and include stretch-activated ion channels, growth factor receptors, integrins, and G-protein-coupled receptors. Of primary significance in fibroblasts and keratinocytes is the matrix−integrin activation of focal adhesion complexes that contain FAK. Mechanical force is transmitted across the cell membrane to activate downstream biochemical pathways including but not limited to calcium-dependent targets, nitric oxide (NO) signaling, mitogen-associated protein kinases (MAPKs), Rho GTPases, and phosphoinositol-3-kinase (PI3K). The convergence of these signals results in the activation of transcription factors that translocate to the nucleus and activate mechanoresponsive genes. Reprinted with permission from ref 411. Copyright 2011 Elsevier.

3.7.1. ECM Proteins Are Good Enhancers of Wound Healing. As the major ECM component, collagen, which is able to regulate the proliferation and migration of fibroblasts, keratinocytes, and endothelial cells, has been incorporated into porous grafts for skin regeneration to treat severe skin injuries, including acute skin burns and chronic skin diseases.406 Laminin can be incorporated into scaffolds to improve the proliferation and migration of dermal fibroblasts. Heparin with heparinbinding domains is able to spatially administer growth factors (e.g., bFGF) to accelerate the wound healing process.407 The combination of gelatin, hyaluronan, and chondroitin sulfate can improve both the proliferation of human foreskin fibroblasts and the epithelial differentiation of human keratinocytes and MSCs.408 3.7.2. Skin Regeneration Can Be Regulated by the Dynamic Sensing of Endogenous and External Mechanical Stimuli. Mechanical cues play a significant role in maintaining the functions of cells and tissues/organs.409 As the outer layer of the human body, skin dynamically senses endogenous and external mechanical signals. Mechanical properties exert effects on cell functions (e.g., cell adhesion, spreading, proliferation, differentiation, cytoskeletal contraction, and reorientation), as well as on ECM synthesis.410,411 For instance, substrates with an elastic modulus of 10 kPa promote the spreading of fibroblasts154 and their differentiation into myotubes.319 Smooth muscle cells on substrates with an intermediate stiffness (21−52 kPa) exhibit fast motility.412 Fibroblasts can adjust their internal mechanical properties to match the substrate stiffness. Therefore, mechanical gradients may be used to drive them to migrate toward skin defects to accelerate wound healing.413 Mistaken communications between skin cells and their physical environment may lead to skin disorders, because mechanical signals can be transformed into biochemical responses via a mechanotransduction pathway (Figure 29).411 Skin scar hardness may be associated with the

Furthermore, porous scaffolds may be selectively decorated with bioactive motifs (e.g., RGD peptides) to give ECM-like cues for nerve regeneration.400 BMSCs can be recruited into the RGDpeptide-decorated aligned microchannels (100 μm diameter) of alginate hydrogels, which promote the differentiation of BMSCs into neuron and glial cells exclusively in the microchannels. The microchannels can further provide space to encapsulate VEGFloaded microparticles to induce vascular growth solely along the channel direction in vivo, posing the future promise of synergistically regulating the spatial organization of nerve-like tissue or blood vessels (Figure 27).50 In this sense, the rational design of scaffolds with a well-defined architecture could provide ideal microenvironments for the temporospatial administration of bioactive factors (e.g., interleukin 9 for T-cell activation and differentiation,401 and CCL3, CCL4, CCL5, and CCL2 for guiding the directional migration of NSCs).402 This avenue of drug-imbued scaffold design presents a high potential for increasing the self-repair capacity of materials for nerve regeneration. 3.7. Material-Guided Skin Regeneration

Skin, the largest organ of the human body, is a hierarchical network of barriers to protect the internal tissues and organs from the environment, allowing them to maintain their normal biological activities.403 Skin mainly consists of three major layers (e.g., the epidermis, dermis, and hypodermis) with nerves and blood vessel between the layers (Figure 28).404 Skin healing is a complicated process mainly accomplished through the coordination of three stages, that is, inflammation, cell migration/ proliferation, and new skin formation and remodeling.405 The limited origins of natural skin autografts pose the necessity of developing artificial scaffolds for wound healing to face the increasing requirements of healing skin following trauma/ disease. The bioactivity of artificial skin grafts can be regulated via optimizing their composition, structure, and physical properties. AE

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overexpression of collagen under rigid microenvironments. Skin scars during wound healing may be eliminated via the application of cyclic mechanical stimuli.411 Generally, mechanical forces, which highly depend on material properties (composition, stiffness, topography, and geometry), are sensed by cell membrane receptors and/or through ion channels, and can be transformed into ion/chemical/enzyme-related biochemical signals. These signals then switch on transcription factors to activate their target genes in the nucleus. These may be the main mechanisms through which the physical properties of materials can regulate biological functions (Figure 29).411 3.7.3. A Nano-/Microsized Sandwiched Geometry Drives Re-epithelialization for Skin Regeneration. Reepithelialization is a significant process for the regeneration of skin with its multilayer architecture and depends not only on rapid cell migration and proliferation but also on effective cell infiltration. 3D scaffolds with an appropriate porous structure may offer sufficient space to promote these functions. For instance, collagen−glycosaminoglycan scaffolds with 20−120 μm pore size and >95% porosity allow cell infiltration in vitro. When transplanted onto the dermal bed of injured skin, such scaffolds can promote the rapid migration and proliferation of epidermal cells around the wound, resulting in effective skin regeneration in vivo.414−420 Collagen/chitosan scaffolds with pore size >200 μm support and accelerate the infiltration of fibroblasts from the surrounding tissue.421 Aligned nanofibers, as compared to random ones, significantly increased epidermal skin cell migration and infiltration into porous scaffolds in vitro.422 Recently, a nanofibrous architecture has been introduced onto the surface of microporous mesh skin grafts to obtain nano-/ microsized sandwiched scaffolds. The sandwiched transplants significantly improved re-epithelialization for wound healing in vivo, probably because the nanostructured surface can rapidly switch on cell regeneration signals, and the inner microporous geometry allows for cell migration and proliferation, as well as new tissue growth.423

Figure 30. Schematic representation of how the development of materiobiology can enrich and deepen the contents of the Materials Genome Initiative (MGI) database.

and theory and equipping the materials community with advanced tools and techniques”, which may provide more effective approaches for solving these problems.423 Recently, the development of materiobiology has been advanced under the guidance of experimental design using advanced technologies (e.g., computational simulation and theoretical analysis). The principal materiobiological interactions have been abstracted by taking advantage of various resources (e.g., international research teams and shared material genome databases). Such efforts may facilitate the prediction of future biomaterials through computational optimization and the design of personalized medical devices. Pursuing these areas of research would provide new insights to uncover more facets of materiobiology. It is expected that the study of materiobiological principles can deepen our understanding of the regulation of biological processes by materials and facilitate the rational design of tissue-specific biomaterials to harness their endogenous regenerative capacity.7 These studies will accelerate the development of “real” ECM-like scaffolds, possibly in a predictable and scalable way. Different patients may respond differently to specific biomaterials. However, by taking advantage of the MGI database and patients’ endogenous materiobiological functions, personalized transplantable ECM-like scaffolds with a self-regenerative capacity could one day be developed to improve regeneration in vivo.

4. EFFECTS OF THE DEVELOPMENT OF MATERIOBIOLOGY ON THE MATERIALS GENOME INITIATIVE (MGI) From the perspective of materials, their composition and structure decide their physical or chemical properties and their functions (e.g., electronic, photonic, and/or thermal features). To explore the special properties and functions of materials in a more effective way, the Materials Genome Initiative (MGI) strategic plan was launched in the year 2014.423 This plan aims to develop a systematic materials database to facilitate the rational design of functional materials, including biomaterials and catalytic, electronic, photonic, lightweight, and structural materials. Among these, biomaterials are the core part of the MGI program; such materials can be used for regeneration of diseased/damaged tissues, which is a key factor for the improvement of human welfare. Recently, researchers have been providing extensive experimental and theoretical data in the “material-biology” field, which can help to guide the biomimetic design of various advanced materials, and are expected to greatly enrich the MGI database (Figure 30). Conventionally, it has been extremely difficult to predict the biological features of biomaterials because their physical properties (e.g., composition, mechanical properties, 2D topography, and 3D geometry) affect the biological responses of cells and/or tissues in a very complicated way. The MGI mission statement includes making available “experiment, computation,

5. CONCLUSIONS AND PERSPECTIVES The powerful self-regenerative capacity of natural tissues/organs has been driving researchers to explore novel biomaterials using a biomimetic approach. Artificial ECM-mimicking scaffolds that are designed according to the special features of a tissue (e.g., its composition, mechanical properties, topography, and 3D geometry) have been indicated to provide bioactive cues to regulate cell functions for effective tissue/organ regeneration (Figure 31). For instance, an artificial construct with an oriented AF

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Figure 31. Materials with tissue-mimetic physical properties (e.g., compositional, mechanical, and structural features) offer combinative biological cues to accelerate tissue regeneration. (A) Microtubular constructs guide the alignment of neuronal cells along their long axis to promote the regeneration of nerve (1D-like tissue). Reprinted with permission from ref 424. Copyright 2009 American Chemical Society. (B) The decoration of microporous mesh with an oriented nanotopological surface significantly improved skin (2D-like tissue) regeneration in vivo, because nanofeatures accelerate epidermal skin cell responses (e.g., cell migration) to switch on the skin regeneration process at an early stage, and the microporous architecture provides space for cell invasion and proliferation, as well as new tissue growth. Reprinted with permission from ref 423. Copyright 2014 Elsevier. (C) Artificial tubular constructs with a 3D-patterned geometry benefit the formation of a spatial alignment of endothelial cells for the regeneration of blood vessels (3D-like tubular construct).348 (D) Elastic scaffolds with a tubular porous structure and heart-matched elasticity promote cell recruitment, mobility, differentiation, and myocardial ischemic elasticity for the regeneration of diseased heart (elastic 3D tissue).340,344 (E) Hierarchical macro-/micro-/ nanoporous scaffolds with bioactive compositions and the capacity for the spatially controlled administration of growth factors show a self-regenerative ability and can accelerate cell recruitment and differentiation for the rapid regeneration of injured bone (rigid 3D tissue). Reprinted with permission from ref 290. Copyright 2016 Elsevier.

differentiation, and the recovery of myocardial elasticity.340,344 Bioglass-based scaffolds with a macro-/micro-/nanoporous structure can successfully regenerate critical-size bone defects, where their (a) highly interconnected macropores (pore size 200−500 μm) benefit cell migration, vascularization, and bone growth, (b) micropores (pore size