Table Of ContentBio-Instructive Scaffolds for
Musculoskeletal Tissue Engineering
and Regenerative Medicine
Bio-Instructive
Scaffolds for
Musculoskeletal
Tissue Engineering and
Regenerative Medicine
Edited by
Justin L. Brown
The Pennsylvania State University,
University Park, PA, United States
Sangamesh G. Kumbar
UConn Health, Farmington, CT, United States
University of Connecticut, Storrs, CT, United States
Brittany L. Banik
The Pennsylvania State University, University Park,
PA, United States
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Contributors
L. Altomare Politecnico di Milano; INSTM, Local Unit Politecnico di Milano, Milan,
Italy
B.L. Banik The Pennsylvania State University, University Park, PA, United States
W.S.V. Berg-Foels UConn Health, Farmington, CT, United States
D.T. Bowers The Pennsylvania State University, University Park, PA, United States
B. Brazile Mississippi State University, Mississippi State, MS, United States
E. Brinkman-Ferguson Mississippi State University, Mississippi State, MS, United
States
D.P. Browe Rutgers, The State University of New Jersey, Piscataway, NJ, United States
J.L. Brown The Pennsylvania State University, University Park, PA, United States
J.R. Butler Mississippi State University, Mississippi State, MS, United States
L. Chen Orthopaedic Institute, Soochow University, Suzhou, China
K.L. Collins Duke University, Durham, NC, United States
J. Cooley Mississippi State University, Mississippi State, MS, United States
K.M. Copeland Mississippi State University, Mississippi State, MS, United States
M. Cristina Tanzi Politecnico di Milano; INSTM, Local Unit Politecnico di Milano,
Milan, Italy
M.S. Detamore University of Oklahoma, Norman, OK, United States
S. Farè Politecnico di Milano; INSTM, Local Unit Politecnico di Milano, Milan, Italy
P. Fattahi The Pennsylvania State University, University Park, PA, United States
J.W. Freeman Rutgers, The State University of New Jersey, Piscataway, NJ, United
States
E.M. Gates Duke University, Durham, NC, United States
C.L. Gilchrist Duke University, Durham, NC, United States
A.S. Goldstein Virginia Tech/Wake Forest University School of Biomedical Engineering
and Sciences; Virginia Tech, Blacksburg, VA, United States
J. Guan The Ohio State University, Columbus, OH, United States
F. Han Orthopaedic Institute, Soochow University, Suzhou, China
B.D. Hoffman Duke University, Durham, NC, United States
xi
xii Contributors
S.G. Kumbar UConn Health, Farmington; University of Connecticut, Storrs, CT, United
States
B. Li Orthopaedic Institute, Soochow University, Suzhou, China
J. Liao Mississippi State University, Mississippi State, MS, United States
S. Lin Mississippi State University, Mississippi State, MS, United States
S. Mahzoon University of Oklahoma, Norman, OK, United States
N. Mistry UConn Health, Farmington; University of Connecticut, Storrs, CT,
United States
J. Moskow UConn Health, Farmington; University of Connecticut, Storrs, CT,
United States
N.B. Shelke UConn Health, Farmington, CT, United States
T.J. Siahaan University of Kansas, Lawrence, KS, United States
P.S. Thayer Virginia Tech/Wake Forest University School of Biomedical Engineering
and Sciences; Virginia Tech, Blacksburg, VA, United States
J. Wicks Orthopaedic Institute, Soochow University, Suzhou, China
S. Yadav UConn Health, Farmington, CT, United States
C. Zhu Orthopaedic Institute, Soochow University, Suzhou, China
Chapter 1
Bio-Instructive Cues in Scaffolds
for Musculoskeletal Tissue
Engineering and Regenerative
Medicine
K.L. Collinsa, E.M. Gatesa, C.L. Gilchrist, B.D. Hoffman
Duke University, Durham, NC, United States
1.1 INTRODUCTION
The primary goal of the field of tissue engineering is the generation of biological
substitutes to repair tissues damaged by injury, disease, or aging. When tissue en-
gineering emerged in the mid-20th century, initial efforts focused on treatments
for skin wounds of burn victims [1]. These treatments involved the cultivation,
preservation, and transplantation of skin from various sources, including speci-
mens from the same species, termed allografts, as well as those from different
species, termed xenografts [2–4]. Despite some success, both allografts and xe-
nografts had many drawbacks: there was a high degree of variability between do-
nors, tissue preservation was challenging, and disease transmission from donor
to patient was possible [5–7]. To overcome these obstacles, researchers began
to investigate ways to create artificial tissues by cultivating cells in vitro and
placing them on or within biomaterial scaffolds that provided adhesive and me-
chanical support for neotissue growth, giving rise to the primary components of
modern day tissue engineering [8,9]. A major advance in tissue engineering oc-
curred with the discovery that a patient's own stem cells could be harvested, cul-
tivated, and re-implanted for therapeutic effect, with precautions taken to control
pluripotency and differentiation [10]. Over the past several decades since these
key developments, researchers have investigated an array of new bio-scaffold
materials (both synthetic and naturally derived), fabrication techniques, stem cell
sources, and differentiation protocols—all with the goal of creating new functional
tissues for repair or replacement. At the heart of this field lies the central chal-
lenge of controlling cell behavior: determining which signals or input instructions
a. These authors contributed equally.
Bio-Instructive Scaffolds for Musculoskeletal Tissue Engineering and Regenerative Medicine.
http://dx.doi.org/10.1016/B978-0-12-803394-4.00001-X 3
Copyright © 2017 Elsevier Inc. All rights reserved.
4 PART | I Introduction
are necessary to direct cells to assemble and maintain a new functional tissue.
Towards this end, much effort has focused on engineering the environment im-
mediately surrounding the cells, termed the cellular microenvironment, to affect
key cell behaviors, including growth, migration, and differentiation.
1.1.1 Role of the Cellular Microenvironment
In vivo, cells reside within a complex and dynamic microenvironment that pro-
vides a variety of biochemical and biophysical cues to the cell. In recent years
there has been an increasing recognition of both the complexity of the cellular
microenvironment and its importance in directing cell behaviors. This is of par-
ticular relevance to the field of tissue engineering, where microenvironmental
cues have been shown to direct an array of cell behaviors critical to tissue de-
velopment and regeneration, including differentiation, growth, migration, and
extracellular matrix (ECM) production and assembly [11–13]. The primary
components of the cellular microenvironment include soluble biochemicals, the
insoluble ECM, and other nearby cells. It has been well established that soluble
biochemical molecules such as growth factors, morphogens, and hormones can
strongly influence cell behavior and stem cell differentiation [14]; however,
only more recently have studies revealed that the native ECM and other adja-
cent cells, in addition to providing structural and adhesive support, also regulate
key aspects of cell function by presenting a variety of biochemical and biophys-
ical cues [15]. Thus, all aspects of the microenvironment should be considered
potent regulators of cell behavior. In order to faithfully recapitulate complex tis-
sue structure and function, it is necessary to determine not only which of these
microenvironmental signals are critical in directing specific cell behaviors, but
also the manner in which they are presented to cells.
1.1.2 Current Challenges
A major emphasis in the field of tissue engineering is the development of bio-
materials, most commonly bioinstructive scaffolds, which incorporate a variety
of microenvironmental cues to direct cell behaviors. Specifically, studies have
examined the effects of specific ECM ligands, scaffold topographies or archi-
tecture, and mechanical properties such as stiffness on cell behavior and tis-
sue formation [12,16–19]. While these studies have significantly improved our
understanding of the roles of particular cues, they have also demonstrated that
presenting a single or a few select cues may not be sufficient to generate a fully
functional tissue, with neotissues often falling short of native tissue structure
and function. These findings suggest that the successful recapitulation of com-
plex combinations of cues is likely necessary to achieve fully functional tis-
sues. As the cellular microenvironment contains a wealth of biochemical and
biophysical cues that are often interconnected, a key challenge in tissue engineer-
ing is to understand which of these cues are necessary in directing formation of
Bio-Instructive Cues in Scaffolds Chapter | 1 5
a specific functional tissue. Therefore, a more complete understanding of how
cells sense, interpret, and convert these microenvironmental cues into down-
stream biochemical responses is needed to accelerate the advancement of tissue
engineering approaches. Ideally, this mechanistic understanding will eventually
enable the “rational” design of new biomaterials that specifically control these
cellular signaling systems to affect tissue-level outcomes.
In this chapter, we will examine the roles of both chemical and physical cell
regulators within the cellular microenvironment. We will begin by defining key
aspects of the ECM and describing methods for their recapitulation. We will
then discuss several key cell signaling pathways important in the detection of
the cellular microenvironment, with particular emphasis on those involved in
the sensing of biophysical microenvironmental cues. An emerging theme in the
field is that an understanding of the specific cell signaling pathways that a given
environmental cue regulates will be necessary for the development of rationally
designed bioinstructive scaffolds.
1.2 THE CELLULAR MICROENVIRONMENT: KEY ASPECTS
1.2.1 What is the Microenvironment?
The cellular microenvironment is defined as the local, microscale surroundings
with which cells interact. In general, the cellular microenvironment is com-
prised of three main components: biochemical signaling molecules, the ECM,
and other nearby cells [20] (Fig. 1.1). These local surroundings contribute both
biophysical and biochemical regulatory cues that influence cell growth and di-
rect cell behaviors [20]. Initial efforts in determining key regulators of cell be-
havior focused on biochemical cues, particularly the activity of small proteins,
FIG. 1.1 Constituents of the cellular microenvironment which play various roles in influencing
cell function. These include neighboring cells, soluble signals, and the extracellular matrix. External
forces are also present in various forms such as fluid shear stress. Understanding the regulatory
roles of these aspects may be important in recapitulating the microenvironment with biomimetic
scaffolds.
6 PART | I Introduction
like cytokines and growth factors, or chemical messengers, such as hormones.
Recently, biophysical cues have become increasingly recognized as an integral
means for controlling cell behavior [21]. In general, biophysical cues within the
microenvironment consist of any physical property of the environment [22].
Pertinent examples include the mechanics, shape, and orientation of key struc-
tures. This is evident in the context of the diverse fibrillar ECM proteins, which
have varying dimensions, mechanical properties, and local alignment. Notably,
these molecular-scale properties are not always apparent in the bulk mechanical
measurements typical of standard material science approaches such as rheom-
etry [12,23,24]. When designing scaffolds for manipulating cell behavior, it is
imperative to remember that cells exist and function on the micron length-scale
and generally probe environments using protein receptors that function on the
nanometer length-scale [25]. The cell will respond to the characteristics of the
local microenvironment it perceives, which are not necessarily the characteris-
tics determined through traditional assays that typically work on millimeter and
longer length-scales.
To detect both biochemical and biophysical cues from the microenviron-
ment, cells express specialized receptors on their surface that can bind to a
diversity of ligands. In general, these receptors can be divided into two main
classes: receptors for soluble ligands and receptors for insoluble ligands. In
this chapter, soluble will be used to refer to protein or small molecule that
freely diffuses, while insoluble will be used to refer to an immobilized protein.
Cytokines, growth factors, and hormones typify soluble ligands, while insolu-
ble ligands are commonly a portion of an ECM protein or a protein expressed
on the surface of a neighboring cell. However, we note that inclusion into each
of these groupings is not mutually exclusive, as certain growth factors have
important biological functions in both their soluble and insoluble forms. A key
example of this behavior is transforming growth factor β (TGFβ), which read-
ily binds to the ECM proteins fibronectin (FN) and fibrillin [26,27]. Notably,
TGFβ is a potent regulator of many aspects of cell behavior, including cell
growth, ECM production, and inflammation. This growth factor is secreted
as a two-part complex and held in an inactive, latent form when bound to the
ECM [26]. TGFβ is then activated upon release from the ECM and its subse-
quent disassociation with its binding partner, the latency-associated peptide
(LAP) [28].
Receptor ligation, involving the binding of a specific receptor–ligand pair,
often results in biochemical responses in the cell interior, including activation
of downstream signaling pathways. For instance, biochemical cues are potent
regulators of cell adhesion, migration, and differentiation [14]. Additionally, the
cell surface receptors that facilitate adhesion to the ECM and other cells, partic-
ularly those belonging to the integrin and cadherin protein families, often medi-
ate detection of biophysical microenvironmental cues. To better understand the
composition of the microenvironment and its role in influencing cell behavior,
each of the main components will be briefly discussed.
Bio-Instructive Cues in Scaffolds Chapter | 1 7
1.2.2 Components of the Microenvironment
1.2.2.1 Cells
While most studies of cell signaling and tissue-engineered scaffold design have
focused on the ability of isolated biochemical and biophysical cues in the mi-
croenvironment to affect cell behavior, ultimately it is the cells within these
tissues that create and maintain their local microenvironment. Therefore, it is
important to understand the bi-directional feedback between the cell and its
microenvironment. At the most basic and general level, the ability of cells to
affect their environment can be categorized into three main processes: the pro-
duction of constituent elements including both soluble and insoluble factors, the
assembly or re-arrangement of the insoluble ECM scaffold, and homotypic and
heterotypic interactions between neighboring cells.
An emerging idea is that the ability of cells to adhere and generate force is
critical to its ability to alter the microenvironment. Within each cell, a biological
polymer-based scaffold called the cytoskeleton provides structural support, while
motor proteins and adenosine triphosphate (ATP)-driven polymerization mediate
the generation of internal forces which drive motion and shape change [29]. Indeed,
cell force generation locally facilitates ECM assembly, particularly of FN [30],
and can lead to large-scale reorganization of ECM matrices [31]. Furthermore,
by adhering to the ECM and to neighboring cells through cytoskeleton-a ssociated
linkages (i.e., focal adhesions and cell–cell contacts, respectively), cells can send
and receive mechanical signals to their environment and other cells [32]. Many
cellular processes, including migration and division, rely on the coordinated activ-
ity between cell-generated forces and cell adhesion [33].
1.2.2.2 Soluble Signaling Molecules
The most established and well-studied role of cells in affecting their microen-
vironment is their secretion of soluble biochemical compounds such as growth
factors, hormones, cytokines, and chemokines. These molecules readily diffuse
throughout the microenvironment and are recognized by specialized cell surface
receptors that can induce the cell to modulate its signaling pathways [14]. Using
this mechanism, cells can affect both their own function (autocrine signaling) as
well as the function of other cells (paracrine signaling) [14]. In tissue engineer-
ing approaches, this property can be used to augment cell culture systems with
bioactive factors [34]. The details of biochemically mediated cell regulation
through soluble signaling molecules have been well established and thoroughly
presented elsewhere [14,35], and will not be a focus of this chapter.
1.2.2.3 Extracellular Matrix
In the most general terms, the ECM is a complex milieu of biopolymers that
surround cells. In many tissues, such as cartilage and tendon, the ECM is the
dominant component, as exhibited by the fact that the ECM dictates the bulk
mechanical response of these tissues [36,37]. Although the ECM was initially
