Advanced Surfaces for Stem Cell Research E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Serie: Advance Materials Series
• Sprache: Englisch

Seitenzahl: 831

Veröffentlichungsjahr: 2016

Beschreibung

The book outlines first the importance of Extra Cellular Matrix (ECM), which is a natural surface for most of cells. In the following chapters the influence of biological, chemical, mechanical, and physical properties of surfaces in micro and nano-scale on stem cell behavior are discussed including the mechanotransduction. Biomimetic and bioinspired approaches are highlighted for developing microenvironment of several tissues, and surface engineering applications are discussed in tissue engineering, regenerative medicine and different type of biomaterials in various chapters of the book. This book brings together innovative methodologies and strategies adopted in the research and development of Advanced Surfaces in Stem Cell Research. Well-known worldwide researchers deliberate subjects including: * Extracellular matrix proteins for stem cell fate * The superficial mechanical and physical properties of matrix microenvironment as stem cell fate regulator * Effects of mechanotransduction on stem cell behavior * Modulation of stem cells behavior through bioactive surfaces * Influence of controlled micro and nanoengineered surfaces on stem cell fate * Nanostructured polymeric surfaces for stem cells * Laser surface modification techniques and stem cells applications * Plasma polymer deposition: a versatile tool for stem cell research * Application of bioreactor concept and modeling techniques in bone regeneration and augmentation treatments * Substrates and surfaces for control of pluripotent stem cell fate and function * Application of biopolymer-based, surface modified devices in transplant medicine and tissue engineering * Silk as a natural biopolymer for tissue engineering

Leseprobe

Contents

Cover

Title page

Copyright page

Preface

Chapter 1: Extracellular Matrix Proteins for Stem Cell Fate

1.1 Human Stem Cells, Sources, and Niches

1.2 Role of Extrinsic and Intrinsic Factors

1.3 Extracellular Matrix of the Mesenchyme: Human Bone Marrow

1.4 Biomimetic Peptides as Extracellular Matrix Proteins

References

Chapter 2: The Superficial Mechanical and Physical Properties of Matrix Microenvironment as Stem Cell Fate Regulator

2.1 Introduction

2.2 Fabrication of the Microenvironments with Different Properties in Surfaces

2.3 Effects of Surface Topography on Stem Cell Behaviors

2.4 Role of Substrate Stiffness and Elasticity of Matrix on Cell Culture

2.5 Stem Cell Fate Induced by Matrix Stiffness and Its Mechanism

2.6 Competition/Compliance between Matrix Stiffness and Other Signals and Their Effect on Stem Cells Fate

2.7 Effects of Matrix Stiffness on Stem Cells in Two Dimensions versus Three Dimensions

2.8 Effects of External Mechanical Cues on Stem Cell Fate from Surface Interactions Perspective

2.9 Conclusions

Acknowledgments

References

Chapter 3: Effects of Mechanotransduction on Stem Cell Behavior

3.1 Introduction

3.2 The Concept of Mechanotransduction

3.3 The Mechanical Cues of Cell Differentiation and Tissue Formation on the Basis of Mechanotransduction

3.4 Mechanotransduction via External Forces

3.5 Mechanotransduction via Bioinspired Materials

3.6 Future Remarks and Conclusion

Declaration of Interest

References

Chapter 4: Modulation of Stem Cells Behavior Through Bioactive Surfaces

4.1 Lithography

4.2 Micro and Nanopatterning

4.3 Microfluidics

4.4 Electrospinning

4.5 Bottom-up/Top-down Approaches

4.6 Substrates Chemical Modifications

4.7 Conclusion

Acknowledgements

References

Chapter 5: Influence of Controlled Micro- and Nanoengineered Environments on Stem Cell Fate

5.1 Introduction to Engineered Environments for the Control of Stem Cell Differentiation

5.2 Mechanoregulation of Stem Cell Fate

5.3 Controlled Surface Immobilization of Biochemical Stimuli for Stem Cell Differentiation

5.4 Three-dimensional Micro- and Nanoengineered Environments for Stem Cell Differentiation

5.5 Conclusions and Future Perspectives

References

Chapter 6: Recent Advances in Nanostructured Polymeric Surface: Challenges and Frontiers in Stem Cells

6.1 Introduction

6.2 Nanostructured Surface

6.3 Stem Cell

6.4 Stem Cell/Surface Interaction

6.5 Microscopic Techniques Used in Estimating Stem Cell/Surface

6.6 Conclusions and Future Perspectives

References

Chapter 7: Laser Surface Modification Techniques and Stem Cells Applications

7.1 Introduction

7.2 Fundamental Laser Optics for Surface Structuring

7.3 Methods for Laser Surface Structuring

7.4 Stem Cells and Laser-modified Surfaces

7.5 Conclusions

References

Chapter 8: Plasma Polymer Deposition: A Versatile Tool for Stem Cell Research

8.1 Introduction

8.2 The Principle and Physics of Plasma Methods for Surface Modification

8.3 Surface Properties Influencing Stem Cell Fate

8.4 New Trends and Outlook

8.5 Conclusions

References

Chapter 9: Three-dimensional Printing Approaches for the Treatment of Critical-sized Bone Defects

9.1 Background

9.2 Overview of 3D Printing Technologies

9.3 Surgical Guides and Models for Bone Reconstruction

9.4 Three-dimensionally Printed Implants for Bone Substitution

9.5 Scaffolds for Bone Regeneration

9.6 Bioprinting

9.7 Conclusion

List of Abbreviation

References

Chapter 10: Application of Bioreactor Concept and Modeling Techniques to Bone Regeneration and Augmentation Treatments

10.1 Bone Tissue Regeneration

10.2 Actual Therapeutic Strategies and Concepts to Obtain an Optimal Bone Quality and Quantity

10.3 Bioreactors Employed for Tissue Engineering in Guided Bone Regeneration

10.4 Bioreactor Concept in Guided Bone Regeneration and Tissue Engineering:

In Vivo

Application

10.5 New Multidisciplinary Approaches Intended to Improve and Accelerate the Treatment of Injured and/or Diseased Bone

10.6 Computational Modeling: An Effective Tool to Predict Bone Ingrowth

References

Chapter 11: Stem Cell-based Medicinal Products: Regulatory Perspectives

11.1 Introduction

11.2 Defining Stem Cell-based Medicinal Products

11.3 Regional Regulatory Issues for Stem Cell Products

11.4 Regulatory Systems for Stem Cell-based Technologies

11.5 Stem Cell Technologies: The European Regulatory System

References

Chapter 12: Substrates and Surfaces for Control of Pluripotent Stem Cell Fate and Function

12.1 Introduction

12.2 Pluripotent Stem Cells

12.3 Substrates for Maintenance of Self-renewal and Pluripotency of PSCs

12.4 Substrates for Promoting Differentiation of PSCs

12.5 Conclusions

Acknowledgments

References

Chapter 13: Silk as a Natural Biopolymer for Tissue Engineering

13.1 Introduction

13.2 SF as a Biomaterial

13.3 Biomedical Applications of Silk-based Biomaterials

13.4 Conclusion and Future Directions

References

Chapter 14: Applications of Biopolymer-based, Surface-modified Devices in Transplant Medicine and Tissue Engineering

14.1 Introduction to Cardiovascular Disease

14.2 Need Assessment for Biopolymer-based Devices in Cardiovascular Therapeutics

14.3 Emergence of Surface Modification Applications in Cardiovascular Sciences: A Historical Perspective

14.4 Nitric Oxide Producing Biosurface Modification

14.5 Surface Modification by Extracellular Matrix Protein Adherence

14.6 The Role of Surface Modification in the Construction of Cardiac Prostheses

14.7 Biopolymer-based Surface Modification of Materials Used in Bone Reconstruction

14.8 The Use of Biopolymers in Nanotechnology

References

Chapter 15: Stem Cell Behavior on Microenvironment Mimicked Surfaces

15.1 Introduction

15.2 Stem Cells

15.3 Stem Cells: Microenvironment Interactions

15.4 Biomaterials as Stem Cell Microenvironments

15.5 Biomimicked and Bioinspired Approaches

15.6 Conclusion

References

Index

End User License Agreement

Guide

Cover

Copyright

Contents

Begin Reading

List of Tables

Chapter 1

Table 1.1

Adhesive sequences in matrix proteins and their integrin receptor.

Table 1.2

Activation of several signaling pathways by integrins with growth factor receptors.

Table 1.3

ECM proteins and their localization within the BM.

Chapter 4

Table 4.1

Advanced technologies for the fabrication of bioactive surfaces for stem cell research.

Chapter 5

Table 5.1

Stem cells response on micro- and nanostructured 2D surfaces.

Table 5.2

Examples of stem cell response on physical gradients.

Table 5.3

Examples of stem cells response on micropatterned 2D surfaces.

Table 5.4

Examples of stem cells response to nanopatterned RGD on 2D surfaces.

Table 5.5

Examples of stem cells response to 2D biochemical gradients.

Table 5.6

Stem cell response on 3D scaffolds.

Chapter 7

Table 7.1

Real and imaginary parts of titanium and its reflectance for selected lasers and wavelengths.

Table 7.2

List of applied surface modifications techniques on various materials with chosen stem cell type and their cellular results.

Chapter 8

Table 8.1

List of the organic precursors used to prepare plasma-deposited film, summarizing their chemical formula, the nature of the surface functionality formed, nature of the chemical bond formed with biomolecules, class of biomolecules and cell type tested, and corresponding literature references.

Chapter 9

Table 9.1

Selection of bone implant fabricated by AM techniques.

Table 9.2

Selection of bone scaffold fabricated by AM techniques.

Table 9.3

Selection of bone scaffold fabricated by bioprinting.

Chapter 11

Table 11.1

Types of cell-based therapy products according to the US Pharmacopeia.

Table 11.2

Regulatory requirements for HCT/Ps.

Table 11.3

The responsibilities of NAC and EMA/CAT.

Chapter 12

Table 12.1

Substrate–medium combinations for maintenance of PSCs.

Table 12.2

Substrates used for the differentiation of PSCs.

Chapter 13

Table 13.1

Biocompatible forms and properties of SF.

Table 13.2

Current applications of SF as biomaterials.

Table 13.3

Studies on the properties and compatibility of stem cells with silk-based matrices.

Chapter 15

Table 15.1

Effects of surface functional groups on cellular behavior of SCs.

Table 15.2

Different biomimicked and bioinspired approaches in biomaterial design and their effects on stem cell behavior.

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Advanced Materials Series The Advanced Materials Series provides recent advancements of the fascinating field of advanced materials science and technology, particularly in the area of structure, synthesis and processing, characterization, advanced-state properties, and applications. The volumes will cover theoretical and experimental approaches of molecular device materials, biomimetic materials, hybrid-type composite materials, functionalized polymers, supramolecular systems, information- and energy-transfer materials, biobased and biodegradable or environmental friendly materials. Each volume will be devoted to one broad subject and the multidisciplinary aspects will be drawn out in full.

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Managing Editors: Sachin Mishra and Sophie Thompson

Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Advanced Surfaces for Stem Cell Research

Edited by

Ashutosh Tiwari

Bora Garipcan

Lokman Uzun

Copyright © 2017 by Scrivener Publishing LLC. All rights reserved.

Co-published by John Wiley & Sons, Inc. Hoboken, New Jersey, and Scrivener Publishing LLC, Beverly, Massachusetts. Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

ISBN 978-1-119-24250-5

Preface

Stem cells have attracted much attention in the fields of regenerative medicine and tissue engineering for their important role in the treatment of several diseases. This is due to their unique properties such as their self-renewal capability and ability to differentiate into specific cell types. New research and therapies in these fields are mainly focused on a better understanding of the natural mechanisms of stem cells and the control and regulation of their behavior under in-vivo or in-vitro conditions. Since a natural and/or synthetic surface is an important physical structure for most of the cells, the effect of surface properties, such as chemistry, charge, energy, hydropathy, pattern, topography, and stiffness, with or without differentiation media, influences stem cell behavior as well as controls and directs stem cell differentiation. Biomaterials that are developed by altering surface properties are a promising challenge for regenerative medicine and tissue engineering fields, drug investigation/toxicity studies and stem cell-based therapies.

This book, Advanced Surfaces in Stem Cell Research, part of the Advanced Materials Series, first outlines the importance of extra cellular matrix (ECM), which is a natural surface for most cells, and is discussed in the first chapter entitled “Extracellular Matrix Proteins for Stem Cell Fate.” Chapters 2 through 6 discuss the influence of biological, chemical, mechanical, and physical properties on stem cell behavior and fate. The mechanical and physical properties of matrix microenvironment as stem cell fate regulator are reviewed in Chapter 2, followed by a discussion on the effect of mechanotransduction on stem cell behavior in chapter 3. In chapter 4, stem cell modulation on bioactive surfaces is disputed. Since micro- and nanoscale structure and surfaces have an influence on stem cell behavior and fate, these properties are discussed in chapters 5 and 6, respectively entitled “Influence of Controlled Micro- and Nano-Engineered Surfaces on Stem Cell Fate” and “Recent Advances in Nanostructured Polymeric Surface: Challenges and Frontiers in Stem Cells.” Chapters 7 through 10 deliberate 2D and 3D surface fabrication and modification using different techniques on stem cell fate. Laser surface modification techniques and stem cell applications and plasma polymer deposition as a versatile tool for stem cell research are discussed in chapters 7 and 8, respectively. The effect of 3D structures and dynamic cell environment, such as bioreactors, on stem cell fate are presented in detail in chapters 9 and 10, respectively entitled “3D Printing Approaches for the Treatment of Critical-Sized Bone Defect” and “Application of Bioreactor Concept and Modeling Techniques in Bone Regeneration and Augmentation Treatments.” Chapter 11 is an important and interesting chapter which will inform readers from a different point of view, with regulatory perspectives on medical products as stem cell-based medicinal products. One of the recent stem cell sources, pluripotent stem cells, are discussed in chapter 12, “Substrates and Surfaces for Control of Pluripotent Stem Cell Fate and Function.” Surface engineering applications are discussed in tissue engineering, regenerative medicine and different types of biomaterials in chapters 13 and 14, respectively entitled “Silk as a Natural Biopolymer for Tissue Engineering” and “Application of Biopolymer-Based, Surface Modified Devices in Transplant Medicine and Tissue Engineering.” Biomimetic and bioinspired approaches are also indicated for developing microenvironment of several tissues in chapter 15, “Stem Cell Behavior on Microenvironment Mimicked Surfaces.”

We would like to thank the authors that have contributed to the chapters of this book, including all scientists who have contributed to this topic. We hope and believe that this book will be very useful to those in the biomaterials, tissue engineering, regenerative medicine, stem cell research and material science communities.

Editors Ashutosh Tiwari, PhD, DSc Bora Garipcan, PhD Lokman Uzun, PhD September 2016

Chapter 1Extracellular Matrix Proteins for Stem Cell Fate

Betül Çelebi-Saltik

Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey

Center for Stem Cell Research and Development, Hacettepe University, Ankara, Turkey

Corresponding author: [email protected]

Abstract

Stem cell-based regenerative medicine aims to repair and regenerate injured and/or diseased tissues by implanting a combination of cells, biomaterials, and soluble factors. Unfortunately, due to an incomplete understanding and knowledge of the interactions between biomaterials and specific stem cell types, and the inability to control the complex signaling pathways ensured by these interactions, the ability to design functional tissue and organ substitutes has been limited. The greatest challenge remains the ability to control stem cells’ fate outside of the cell’s natural microenvironment or “niche”. Stem cell fate is known to be regulated by signals from the microenvironment, such as extracellular matrix (ECM) including glycosaminoglycans and proteoglycans to which stem cells adhere. They represent an essential player in stem cell microenvironment because they can directly or indirectly modulate the maintenance, proliferation, self-renewal, and differentiation of stem cells. The interactions between stem cells and the ECM play a critical role in living tissue development, repair, and regeneration as well. The design of artificial ECM and/or binding site is important in tissue engineering because artificial ECM and/or binding regulates cellular behaviors. Identification of binding sites and key motifs in ECM proteins that interact with cellular receptors can allow researchers to generate small peptides that can mimic the function of large ECM proteins.

Keywords: Extracellular matrix proteins, stem cells, niche, integrin, signaling

1.1 Human Stem Cells, Sources, and Niches

Stem cells have two distinct abilities: self-renewal of themselves and differentiation into tissue/organ-specific cells. Based on their differentiation potential, they can be classified as totipotent, pluripotent, multipotent, or unipotent cells. The totipotent fertilized egg exhibits the stem cell that gives rise to all embryonic and extra-embryonic structures of the developing embryo [1]. Human embryonic stem cells (hESCs) derived from the inner cell mass of the blastocyst have the ability to self-renew over a long period without undergoing senescence [1]. Consequently, cells with higher regeneration capacity and plasticity are needed which lead to use of pluripotent stem cells. Identifying suitable source of stem cells is elemental for regeneration of any tissue. Mature and differentiated multipotent stem cells are easily available but least preferred due to their limited cell division and differentiation capacity. Indeed, the number of stem cells in adults is very low, and it depletes with age. Bone marrow (BM)-derived stem cells first described by Friedenstein et al. are still the most frequently investigated cell type [2]. These cells are lineage-restricted, and in contrast to hESCs, multipotent adult stem cells undergo replicative senescence and their lifespan in culture is limited. The existence of multipotent postnatal stem cells has been reported in BM, peripheral blood, umbilical cord, umbilical cord blood (UCB), Wharton’s jelly, placenta, neuronal, and adipose tissues [3–7]. Takahashi and Yamanaka developed a new technique by describing the reprogramming of human somatic fibroblasts into primitive pluripotent stem cells by over-expressing OCT4, SOX2, KLF4, and MYC [8]. These human induced pluripotent stem cells are similar to hESCs in the sense that they also express pluripotency genes, have telomerase activity, and are able to differentiate into all cell types of the three embryonic germ layers (endoderm, ectoderm, and mesoderm) (Figure 1.1).

Figure 1.1 Embryonic stem cells, adult stem cells, and induced pluripotent stem cells.

Stem cell niche consists of stem cells, supporting cells, extracellular matrix (ECM), soluble factors, and nervous systems. All these factors have an important role in stem cell niche; however, ECM that holds stem cells in a niche and controls their cellular processes plays a critical role in the control of stem cell fate [9]. Since its first definition originally proposed in 1978 by Schofield for the hematopoietic stem cell (HSC) microenvironment, the concept of the niche has increased in complexity [10]. Niches are highly specialized for each type of stem cell, with a defined anatomical localization, and they are composed by stem cells and by supportive stromal cells (which interact each other through cell surface receptors, gap junctions and soluble factors), together with the ECM in which they are located (Figure 1.2). In addition, secreted or cell surface factors, signaling cascades and gradients, as well as physical factors such as shear stress, oxygen tension, and temperature, promote to control stem cell behavior [11].

Figure 1.2 Stem cell niche. Adapted from Jhala et al. [9].

Stem cell niche cells mesenchymal and HSCs play an important role in many regeneration processes in human body. HSCs are blood forming cells which were recognized more than 50 years ago [12]. They are produced during embryogenesis in a complex developmental process in several anatomical sites (the yolk sac, the aorta-gonad-mesonephros region, the placenta, and the fetal liver), after which HSCs colonize the BM at birth [13]. These cells are divided into two major progenitor lineages; common myeloid (CMP) and common lymphoid progenitors (CLP). CMPs give rise to megakaryocyte (MK)/erythroid and granulocyte/macrophage progenitors developing into platelet producing MKs, erythrocytes, mast cells, neutrophils, eosinophils, monocytes, and macrophages. CLPs will mature into B-lymphocytes, T-lymphocytes, and natural killer cells. They are positive to cell surface markers such as CD34, CD45, and CD117 [14]. HSCs have the ability to leave their tissue of origin, enter circulation, identify, and relocate to an available niche elsewhere during early development [15]. In the adult, they can leave the BM and return back to it through homing mechanisms [16].

Since the identification of mesenchymal stem cells (MSCs) as colony-forming unit fibroblasts (CFU-Fs) by Friedenstein et al. in 1970 and the first detailed description of the tri-lineage capacity by Pittenger et al., our understanding of these cells has taken great strides forward [17]. These plastic adherent multipotent cells are able to differentiate into bone, cartilage, and fat cells and can be isolated from many adult tissue types. They have been obtained from a wide variety of fetal and adult tissues: adipose tissue, placenta, umbilical cord, Wharton’s Jelly, synovial membrane, and dental pulp [18]. Specifically, a recent study reported plastic-adherent MSC-like colonies derived from the non-mesodermal tissues such as brain, spleen, liver, kidney, lung, BM, muscle, thymus, and pancreas [19]. They are positive to cell surface markers such as CD29, CD44, CD105, CD90, and CD73 [20]. MSCs secrete cytokines, chemokines, growth factors, and ECM either spontaneously or after induction by other cytokines [21]. The mesenchymal ECM consists of various proteins such as collagens types I, III, IV, V, and VI, laminin, fibronectin, proteoglycans, such as syndecan, Pln, decorin, and the glycosaminoglycan (GAG) hyaluronan (Figure 1.3) [22]. Proteoglycans are macromolecules consisting of a core protein attached to several polysaccharides (GAGs). They are able to bind cells to the matrix and bind growth factors to GAGs, thus preventing extracellular protease degradation of growth factors [23].

Figure 1.3 The mesenchymal ECM proteins network.

The ECM is a highly dynamic structure that is constantly being remodeled, either enzymatically or non-enzymatically, and its molecular components are subjected to many post-translational modifications [24]. ECM proteins vary not only in composition but also in physical parameters including elasticity and topography. Such physical properties are known to affect the micro- to nanotopography of integrin receptors (alpha and beta subunits) and to influence a range of cellular processes through changes in cell shape and the actin cytoskeleton [25–27]. Cell adhesion to the ECM is mediated by ECM receptors, such as integrins, discoidin domain receptors, and syndecans [24]. ECM properties including stiffness, fiber orientation, and ligand presentation dimensionality provoke specific cellular behaviors [24]. It is clear that ECM-based control of the cell may also occur through multiple physical mechanisms, such as ECM geometry at the micro- and nanoscale (shape, porosity, and topography), ECM elasticity or mechanical signals (stiffness and stress) transmitted from the ECM to the stem cells that turn into biochemical response (Figure 1.4). An understanding of the interaction of these mediators with signaling pathways may provide new insights into the regulation of self-renewal and differentiation of stem cells [28].

Figure 1.4 ECM-based control of the cell.

1.2 Role of Extrinsic and Intrinsic Factors

1.2.1 Shape

The interactions between many extrinsic and intrinsic factors that govern cell shape are varied and may involve relatively long-term interactions with the cellular niche, as well as more acute changes due to physical factors such as mechanical or osmotic stress. In 1911, Carrel and Burrows showed that cells were responsive to shape cues [29] and over the last decade, the effects of surface shape have been well documented. Chen et al. performed a study to clarify how cells can differentiate on different surface shape via molecular signaling pathways. Based on their research, human MSCs were induced to take on either round or spread out shapes by fibronectin-patterned surfaces. The cells on the round islands underwent adipogenesis but those on spread out islands underwent osteogenesis. This phenomenon was reported due to the activation of RhoA which is induced by surface shape [30]. In other research, MSCs that cultured on small 30 nm nanotubes showed no differentiation whereas MSCs on 70–100 nm nanotubes showed cytoskeletal stress and osteoblastic lineage differentiation. This change was also reported due to the activation of RhoA and its effector ROCK kinase [31].

1.2.2 Topography Regulates Cell Fate

Cells are encountering different topographies sized clues in vivo from macro- (bone and ligament) to micro- (other cells) and nanotopography (proteins and ligands), all of which influence cell behavior and cell fate. It is known clear that topographical clues alone can produce the same effect as chemical induction including growth factors, chemokines and cytokines [32]. Topographical (physical) cues in the sub-micrometer range such as increasing the roughness or displaying specific geometrical shapes elicit specific cell responses. Pattering techniques such as soft lithography, photolithography, electrospinning, layer-by-layer microfluidic patterning, three-dimensional (3D) printing, ion milling, and reactive ion etching create scaffolds with precise controlled geometry, texture, porosity, and rigidity [32]. Micro-topographies that include micropits, microgrooves, and micropillars induce the cell body by physical confinement or alignment [33]. Focal adhesions (FAs), the sites of cell attachment to the underlying surfaces, play a pivotal role in all cell actions in response to nano- and microtopography. These dynamic adhesions are subject to complex regulation involving integrin binding to ECM components and the reinforcement of the adhesion plaque by recruitment of additional proteins [33].

1.2.3 Stiffness and Stress

The mechanical properties of the ECM not only allow such tissues to cope with stresses but also regulate various cellular functions such as spreading, migration, proliferation, stem cell differentiation, and apoptosis [28, 34]. Gels based on natural ECM, such as collagen type I, Matrigel, and fibrin, were the first materials used to suggest an impact of stiffness on cell fate [34]. Increasing the cross-linking of these matrixes that modulates matrix stiffness impacts integrin signaling and acto-myosin-mediated cellular tension [35]. For example, when a low concentration of cross-linker was used to attach collagen to stiff hydrogels, epidermal stem cells were stimulated to terminally differentiate, and MSCs differentiated into adipocytes rather than osteoblasts [36]. In the case of MSCs, osteogenic differentiation is favored by stiff substrates, whereas adipogenic differentiation is promoted by soft substrates [25]. Increases in microenvironment stiffness led to greater ECM and adhesion protein expression in cardiac stem cells [37]. Substrate stiffness also directs skeletal muscle stem cells and neuronal stem cells to either self-renew or differentiate. It has been also reported that cell spreading, self-renewal, and differentiation were inhibited on soft substrates (10 Pa), whereas with moduli of 100 Pa or greater, cells exhibited peak levels of a neuronal marker, beta-tubulin III, on substrates that had the approximate stiffness of brain tissue. Softer substrates (100–500 Pa) promoted neuronal differentiation (neural stem cells cultured on fibronectin peptide containing hydrogels in serum-free neuronal differentiation medium exhibit maximal differentiation at 500 Pa), whereas stiffer substrates (1,000–10,000 Pa) led to glial differentiation [38].

1.2.4 Integrins

Stem cells interact with ECM proteins via a family of surface receptors called integrins. Their types and quantities on each stem cell are specific to the cell and tissue type. Integrins facilitate the interactions between stem cells and ECM proteins and transduce chemical and physical signals from the matrix to the cells. They are involved in many cellular functions, including quiescence, cell cycle progression, cell adhesion, migration, and survival. Integrins are heterodimeric transmembrane receptors. An integrin molecule is mainly composed of two glycoprotein subunits, alpha (α) and beta (β). In vertebrates, 18 α subunits and 8 β subunits have been found, and they can form 24 different heterodimeric structures [39]. α Subunit has four Ca2+-binding domain on its extracellular part of polypeptide chain, and β subunit bears a number of cysteine-rich domains on its extracellular part of polypeptide chain. The extracellular domain of integrins binds to ECM ligands, such as laminin, fibronectin, collagen, and vitronectin, while the intracellular domain connects with cytoskeletal proteins, such as α-actinin and talin, as well as regulatory proteins, such as calreticulin and cytohesin. Integrins interact with ligands through weak interactions, but the ligand-binding affinity may be modulated by intracellular signals. After the ligand binds between the two subunits, the induced conformational change physically pushes the two subunits apart and initiates downstream signaling [e.g. activation of cytoplasmic TPK (FAK) and serine/threonine kinases, activation of small GTPases, induction of calcium transport, or changes of phospholipid synthesis]. This transmembrane linker makes integrins important in cell–cell and cell–ECM adhesion, signal transduction, and growth factor receptor responses [40]. Basement membranes are typically rich in laminins and non-fibrillar collagen type IV, whereas soft connective tissue is dominated by the presence of fibrillar collagens types I and III [41]. Cells respond to the mechanical and biochemical changes in ECM through the cross-talk between integrins and the actin cytoskeleton [42].

Integrins have been recognized as a key regulator in embryogenesis. Endoderm is one of the three primary germ layers. The expression of integrin subunits α3, α6, and β4 decreased in definitive endoderm (DE) compared to undifferentiated cells, but αV and β5 are highly expressed in undifferentiated hESCs, and this expression is increased after DE formation. When ESCs are directed to differentiate toward endoderm lineages, laminin substrates are useful during the differentiation toward DE. This response is mediated by integrins αV and β5. Besides, when cells are differentiated toward pancreatic or hepatic lineages, integrin β1 becomes important [40]. Mesoderm derived from ESC has potential to regenerate multiple tissues and organs. Integrins such as α5β1 and α6β1 may be useful in mesoderm induction. The activation of α5β1 modulated bone morphogenetic protein (BMP)-4 expression and BMP-4 induction during differentiation activates BMP, Wnt, fibroblast growth factor (FGF), and transforming growth factor (TGF)-β/nodal/activin signaling [43]. The generation of neuroectoderm lineages from ESC becomes the main focus of present ectoderm differentiation study. Deficiency of the α3 integrin subunit has been observed in mice with defective neuron migration. On the other hand, the interaction of laminin and fibronectin with β1 integrins promotes the maintenance and migration of neural precursor cells [44]. It has been reported that α1β1, α6β1, and α5β1 are crucial in the differentiation of specific neural crest lineages [40]. Skin substitutes derived from ESC differentiation may serve as a continual source for the treatment of wound healing. β1 Integrin is responsible for melanocyte proliferation, α2β1-collagen IV, α3β1-laminin, and α6β4-laminin are responsible for keratinocytes differentiation [45], α6β1-laminin and α2, α5, αvβ3-collagen type IV are responsible for melanocytes differentiation (Table 1.1) [46].

Table 1.1 Adhesive sequences in matrix proteins and their integrin receptor.

ECM proteins

Sequence

Integrins

Collagens

GFOGER

α

1

β

1,

α

2

β

1,

α

10

β

1,

α

11

β

1

Fibronectin

RGD

α

5

β

1,

α

V

β

3,

α

8

β

1,

α

V

β

1,

α

V

β

6,

α

IIΕ

β

3

LDV

α

4

β

1,

α

4

β

7

REDV

α

4

β

1

Laminins

E1 fragment

α

1

β

1,

α

2

β

1,

α

10

β

1

E8 fragment

α

3

β

1,

α

6

β

1,

α

7

β

1,

α

6

β

4

Vitronectin

RGD

α

V

β

3,

α

IIb

β

3,

α

V

β

5,

α

V

β

1,

α

V

β

8

Fibrinogen

RGD

α

V

β

3

KQAGV

α

IIb

β

3

Von Willebrand factor

RGD

α

V

β

3,

α

IIb

β

3

1.2.5 Signaling via Integrins

Expression of integrins is regulated by ECM to cell signaling. It has been reported that TGF-β1 induced elevated expression of α2β1 integrin in fibroblasts. Therefore, cytokines regulate the expression of integrins [47]. FGFs and vascular endothelial growth factors (VEGFs) have been shown to bind to heap and can be cleaved off from the GAG components of heparan sulfate proteoglycans (HSPG) by the enzyme heparanase and released as soluble ligand [48]. Fibronectin and tenascin-C bind to VEGF, which potentiates the VEGF-mediated signaling through its receptor VEGFR2 (Table 1.2) [49].

Table 1.2 Activation of several signaling pathways by integrins with growth factor receptors.

Growth factor receptors

Ref.

Insulin receptor

[56]

Type 1 insulin-like growth factor (IGF1) receptor

[57]

VEGF receptor

[58]

TGF-

β

receptor

[59]

Hepatocyte growth factor (HGF) receptor

[60]

Platelet-derived growth factor-

β

(PDGF-

β

) receptor

[61]

Epidermal growth factor (EGF) receptor

[62]

Several tyrosine kinases and phosphatases are necessary for the regulation of mechano-sensory activity of FAs; such as FA kinase (FAK), Src, receptor type tyrosine protein phosphatase α (RPTP α) and SH2 domain containing protein tyrosine phosphatase 2 (SHP2) [50]. FAK localizes at FAs on integrin clustering to regulate cell adhesion, migration and mechanotransduction [51]. FAK is a non-receptor tyrosine kinase which is activated upon integrin binding to autophosphorylate Y397—this induces subsequent binding of Src by the SH2 domain, leading to stable and increased activation of Src–FAK complex [42]. Src is a non-receptor tyrosine kinase associated with the cytoplasmic tail of β3 integrins via its SH3 domains. Overexpression and phosphorylation of FAK correlate with the increase in cell motility and invasion. Adhesion and spreading of cells on a variety of ECM proteins, including collagen type IV, lead to an increase in tyrosine phosphorylation and activation of FAK. Furthermore, suppression of adhesion-induced tyrosine phosphorylation of FAK may interrupt cancer cell–ECM interactions and affect the invasive and metastatic potential of cancer cells [52]. Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) has also been shown to bind FAK in a cell adhesion-dependent manner at the major autophosphorylation site Y397 leading to activation of AKT, a ubiquitously expressed serine/threonine kinase that regulates integrin-mediated cell survival [53]. The AKT pathway is essential for proliferation because a dominant negative mutant of PI3K prevents cyclin D1 expression. However, proliferation also requires ERK signaling. Both AKT and ERK phosphorylations are required to induce cell growth when stimulated with mitogens [54]. Members of the GTPase family, Rac1, RhoA and Cdc42, are associated with adhesion-dependent cell cycle regulation. Rac and Rho are activated by integrin and they control cell cycle. Besides, Rac1 has a central role in linking cell adhesion to G1/S transition by activating mitogenic pathways via a range of effectors. In contrast, RhoA controls cell cycle in part via the organization of actin stress fibres and cell shape [54]. To promote quiescence, stem cells express high levels of cyclin-dependent kinase inhibitors (CDKIs) such as p21, p27, p57, and p15. Following antimitogenic signals, p21 and p27 bind to cyclin–CDK complexes to inhibit their catalytic activity and induce cell cycle arrest [55].

The alignment and orientation of ECM molecules contributes to mechanosensing. Progenitor stem cells enhanced cell cycle on aligned ECM proteins, and this effect is mediated by β1 integrin and ERK activation [63]. It has been reported by Roncoroni et al. that BM MSC express the α1, α2, α3, α6, α7,α9, α11, and β1 subunits of integrins, and TGF-β1 regulates the expression of α2, α6, and β1 integrins, thus helps forward the attachment of MSC to ECM proteins [64]. Therefore, cytokines regulate the expression of integrins too. ECM remodeling takes place by means of proteases like matrix metalloproteases, serine and cysteine proteases. These interactions stimulate or inhibit various signaling pathways in the stem cell niche.

1.3 Extracellular Matrix of the Mesenchyme: Human Bone Marrow

In adult, hematopoiesis is restricted to the extravascular compartment where HSCs are in contact or close proximity with a heterogeneous population of stromal cells in the niche. Cellular interactions between HSCs and stromal cells involve various cell surface molecules, including integrins, selectins, sialomucins, and the immunoglobulin gene superfamily, that are subsequently translated into cell signaling regulating the localization and function of the cells within the niche [65].

In BM, integrin receptors interact with ligands that include ECM proteins, immunoglobulin superfamily members, and vascular cell adhesion protein 1 (VCAM-1) and, therefore, they play an important role in cell adhesion and signaling. Several integrins have been identified in the BM microenvironment and more specifically on HSCs [66, 67]. Functional integrins depend on the cytokine medium in which they reside, making adhesive events regulatable by cytokines. Among the different integrin subtypes, the β1 integrins have shown to play an important role in HSC migration and homing to the BM [67]. Although β1 integrin is generally known to promote proliferation, it can also be inhibitory, constituting a dominant negative signal over the stimulatory effects [54]. In the HSCs, the α4, α5, α6, α7, and α9 integrin subunits are expressed [68]. A study on the spatial location of ECM proteins including fibronectin, collagen types; I, III, and IV, and laminin in murine femoral BM by immunofluorescence has revealed distinct locations for each protein, supporting the notion that they have an important role in the homing and lodgment of transplanted cells (Table 1.3, [69]). Collagen type IV, laminin, nidogen/entactin, and perlecan are the major components of this network [70]. Minor components bind to the major components in a tissue-specific manner via their chains, and they contribute to BM’s heterogeneity [70]. Many reports have documented the importance of α4β1 and α5β1, in modulating adhesive interactions between HPCs and ECM components that comprise the stem cell niche [71, 72]. A study conducted by Van Der Loo et al. demonstrated that α5β1 is expressed on mouse and human long-term repopulating hematopoietic cells and binds to fibronectin in the ECM and that disruption of this binding can lead to decreased engraftment in the BM [73]. Also, expression of the laminin receptor has been found on erythroid progenitors and its inhibitions blocks BM homing of Burst forming unit-erythroid (BFU-E) [74]. MK maturation and platelet generation are consequent to MK migration from the osteoblastic to the vascular niche, where MKs extend proplatelets, and newly generated platelets are released into the blood [75].

Table 1.3 ECM proteins and their localization within the BM.

Bone compact and/or trabeculae

Endosteal surface/marrow

Periosteal surface

Central marrow

Marrow vessel: sinuses and arteries

Fibronectin

+++

+++

+++

++

–

Collagen type I

+++

+++

–

–

–

Collagen type II

–

–

++

–

–

Collagen type IV

+

++

++

–

++

Laminin

+

–

+++

+

+++

+++, bright expression; ++, moderate expression; +, faint expression; –, absent. Adapted from [69].

It has been demonstrated that interactions of MKs with fibrinogen or von Willebrand factor in vascular space, are able to sustain MK maturation and proplatelets, whereas collagen type I suppresses these events and prevents premature platelet release in the osteoblastic niche [76]. The negative regulation of proplatelets by collagen type I is mediated by the interaction with the integrin α2β1. Hence, recent studies have demonstrated that fibronectin may represent a new regulator of MK maturation and platelet release [77]. Collagen fibers stimulate platelet activation, leading to inside out regulation of the integrin GP IIb–IIIa, secretion from dense and alpha granules, generation of thromboxanes, and expression of procoagulant activity, all of which support the hemostatic process. ECM proteins such as laminin, collagen type IV self-assemble in the BM into a honeycomb-like polymer. The role of collagen in supporting platelet adhesion to the endothelium is mediated through indirect and direct interactions [78]. Another ECM protein, osteopontin, is a phosphorylated glycoprotein that can be produced by a variety of BM cells, especially bone surface. Osteopontin has the ability to bind multiple integrins as well as CD44. It has some roles affecting many physiological and pathological processes including chemotaxis, adhesion, apoptosis, inflammatory responses, and tumor metastasis. Several integrins with potential binding to osteopontin are expressed by HSCs, making them molecular candidates in the cross-talk between osteoblasts and HSCs at the level of BM stem cell niches [79]. We previously investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of UCB CD34+ hematopoietic progenitor cells (HPCs). Toward this, collagen types I, IV, laminin, and fibronectin were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favored the differentiation of some lineages over that of others (e.g. fibronectin for erythroids), some the expansion of HPCs (e.g. laminin and MK progenitor), while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second ‘basement membrane-like structure’ was without collagen type I (3ECMp). Removal of collagen type I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ and CD41+ (MK) cell expansions and induced greater myeloid progenitor expansion than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of hematopoietic progenitors than individual ECM protein [80].

1.4 Biomimetic Peptides as Extracellular Matrix Proteins

Physicochemical and biological functions of the cell membrane create new opportunities for developing bioactive peptides for biomedical applications. Membrane proteins are asymmetrically distributed in the lipid bilayer of all biological membranes. Transmembrane proteins have some specific orientations; one side of the protein might be shaped such that it can act as a receptor for a signaling molecule, while the other side changes shape in response to the binding signal [81]. The communication of cells with surfaces is mediated by pre-adsorbed protein layers. Understanding the interactions of the ECM on stem cell fate will provide the framework for designing and engineering ECMs to control cell behavior that can be used to direct stem cell expansion and differentiation.

The bioactive peptides can perform the desired function of ECM proteins and have additional advantages such as they are smaller than parent proteins, more stable, precisely synthesized, and have tunable properties such as selectivity and solubility [82]. Peptide sequences with different properties such as cell adhesion, growth factor binding, and enzymatically degradable sequences are most commonly used [9]. The commonly used arginine–glycine–aspartic acid (RGD) peptides for cell adhesion have shown to affect differentiation of various stem cells but inhibit adhesion when in solution by blocking integrin binding sites. RGD peptides do not support the growth of hESCs as these cells utilize non-RGD-binding integrins for attachment to adherent substrates. On the other hand, laminin, fibrinogen, vitronectin, tenascin, thrombospondin, entactin, bone sialoprotein, osteopontin, and certain collagens contain RGD sites [83]. It has been reported that RGD plays an important role in initiating chondrogenic differentiation [84]. RGD peptides can be used to provide cell attachment to non-adhesive surfaces and can be easily combined with scaffolds or biomaterials [85, 86]. Naghdi et al. demonstrated that polyethylene glycol hydrogel conjugated with RGD improved neurite outgrowth [87]. Another amino acid sequence, YIGSR, resides on the β1-chain of the laminin. The YIGSR peptide can be used to mimic the properties of soluble laminin, such as inhibiting neurite growth, while the IKVAV peptide antagonizes the activity of soluble laminin and activates cell spreading, neurite outgrowth, and angiogenesis [88]. The peptide sequence GFOGER in collagen type I specifically binds the α2β1 receptor and promotes osteoblastic differentiation [89]. It has been previously shown that GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes [90]. Connelly et al. explained that RGD peptides but not GFOGER increased collagen type I expression of hMSCs. Moreover, the GFOGER and RGD peptides enhanced osteocalcin at similar levels [91]. It has been previously reported that MSCs cultured in DGEA-collagen type I mimetic peptide exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs [92]. Hyaluronan, also known as hyaluronic acid (HA), is a large GAG that typically surrounds non-polarized and migratory cells. The various activities of HA are a consequence of its binding to hyaluronan binding proteins. It contains the 9–11-residue long B-X7