2.6.

3
Tissue Engineering Scaffolds
HANNAH A. PEARCE, YU SEON KIM, LUIS DIAZ-GOMEZ, ANTONIOS G. MIKOS

Department of Bioengineering, Rice University, Houston, TX, United States

Introduction original material should also be taken into consideration

when choosing a material for scaffold design. Implanted scaf-
Over the past 30 years, tissue engineering has experienced an folds undergo degradation most often via hydrolysis and/or
intense evolution facing the need to restore, replace, or regener- enzymatic activity, depending on the chemical structure of
ate tissues that have suffered structural or functional damage the scaffold material. Polymers with hydrolysable bonds such
following injury or disease. To induce and guide tissue growth, as esters and amides are often chosen as biodegradable mate-
tissue engineering has aimed to combine scaffolds that provide rials. Notable examples include poly(lactic-co-glycolic acid)
a mechanical support structure, cells that populate the scaffolds (PLGA), poly(ε-caprolactone) (PCL), and polyurethanes.
and drive the regeneration process, and bioactive molecules Scaffolds fabricated from natural polymers such as collagen,
that regulate cell differentiation and functionality (Langer and gelatin, and polysaccharides will be enzymatically degraded by
Vacanti, 1993). Scaffolds are highly interconnected porous proteases that are present in the extracellular matrix (ECM).
structures that aim to recreate the extracellular environment The rate of scaffold degradation must also be considered
while providing appropriate mechanical support and stabil- for tissue regeneration. Ideally, the degradation rate of an
ity until the new tissue is regenerated (O’Brien, 2011). For implanted scaffold should be similar to the regeneration rate
these reasons, scaffolds should be prepared with biodegrad- of the surrounding tissue. The degradation rate of hydropho-
able materials and the degradation rate should ideally match bic polymers can be enhanced by increasing the content of
the regeneration rate of the tissue where it will be implanted hydrophilic groups. For instance, PLGA foams that contain
while maintaining mechanical support (Thomson et al., 1995). higher glycolic acid content (50:50 lactic acid:glycolic acid)
This chapter describes the functional requirements, processing were shown to degrade more quickly than those that have
techniques, applications, and characterization methods of bio- higher lactic acid content (85:15) (Lu et al., 2000a). Similar
materials to provide a state-of-the-art overview of scaffolds for results can be obtained by incorporating hydrophilic segments
tissue engineering developments in the last decade. within the polymer chain; block copolymers of poly(lactic
acid) (PLA) and poly(ethylene glycol) (PEG) have shown an
Scaffold Design Criteria increased degradation rate when compared to PLA homo-
polymers due to the increased hydrophilicity the PEG blocks
Three criteria that need to be considered when designing a bring to the polymer. The polymer’s molecular weight will
polymeric scaffold for tissue engineering applications are (1) also affect the degradation time, where scaffolds fabricated
biocompatibility, (2) physicochemical properties, and (3) bio- with higher molecular weight polymers will degrade slower
activity. Biocompatibility of a material will dictate whether it than those fabricated with lower molecular weight polymers.
is suitable to be used as a scaffold in vitro and in vivo, whereas In addition to its chemical properties, the scaffold’s physical
its physicochemical properties and bioactivity are factors that properties will also affect the scaffold’s degradation rate. It has
can be tuned to be specific for a certain tissue/cell type, and to been demonstrated that pore size and pore wall thickness will
optimize the scaffold’s ability to regenerate the tissue. affect the rate of degradation of poly(l-lactic acid) (PLLA)
Biocompatibility, when used to describe a material, refers foams in vitro, where thicker pore walls and larger pore size
to its ability to perform with an appropriate host response in contributed to faster degradation via an autocatalytic effect
a specific application (Temenoff and Mikos, 2008). Unlike (Lu et al., 2000a). The effect was also enhanced in vivo, where
implants that are not meant to undergo degradation such acidic degradation products remained within the pores and
as poly(methyl methacrylate) (PMMA) bone cements and further accelerated the degradation reaction (Lu et al., 2000b).
metallic hip joints, tissue engineering scaffolds will gradu- Porosity and pore size/distribution will not only affect
ally degrade over time and be replaced by the native tissue. the scaffold’s mechanical properties and rate of degrada-
As such, the cytotoxicity of degraded components of the tion, but also how seeded cells respond to the environment

1317
1318 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

(Petite et al., 2000). High porosity must also be accompa- Yoo et al., 2009). Short peptide sequences that mimic the
nied with pore interconnectivity, as pores that are trapped active domain of a specific protein, such as the popular argi-
within the scaffold are not accessible to cells and will reduce nine-glycine-aspartate (RGD) sequence, can be used instead
the scaffold’s mechanical properties. Interconnected pores of native ECM proteins which can have issues with stability
will also act as conduits for blood vessels to infiltrate and during and after surface adsorption (Shin et al., 2003).
form a well-vascularized network within the scaffold, which Cell function can also be affected by substrate stiffness.
is a necessary step toward the regeneration of tissues such It has been shown that the microenvironment stiffness sig-
as bone (Petite et al., 2000). A highly porous scaffold has nificantly affects the commitment and differentiation of
a high surface area to volume ratio, which provides suffi- mesenchymal stem cells (MSCs); softer matrices encourage
cient space for cells to attach onto the scaffold’s surfaces, the neurogenic or myogenic differentiation of MSCs, while
enhances the rate of mass transport of oxygen and nutrients stiffer substrates are known to exert a strong influence in
both to seeded cells, and aids in the removal of cell waste supporting osteogenic differentiation (Engler et al., 2006).
(Mikos and Temenoff, 2000; Yang et al., 2001). Different Other cell functions such as migration and proliferation are
pore sizes have been shown to trigger diverse responses from also affected by substrate stiffness (Breuls et al., 2008). Cell
the tissue (Rouwkema et al., 2008; Yang et al., 2001); for lineages have unique optimum conditions for proliferation
instance, pores larger than 300 μm were shown to be ideal and respond differently to microenvironmental stiffnesses;
for bone in-growth, whereas fibrovascular tissues required for instance, human dermal fibroblasts seeded on stiff sub-
500 μm or greater (Kuboki et al., 2001; Wake et al., 1994). strates show higher degrees of organization in their actin
These results indicate that a modular approach is required cytoskeleton and slower migration speed (Dado and Leven-
to regenerate different types of tissues. Nevertheless, while berg, 2009; Ghosh et al., 2007).
high porosity is an important factor for tissue regeneration,
the mechanical integrity of the scaffolds can be negatively
influenced (Karageorgiou and Kaplan, 2005).
In addition to soluble factors, surface characteristics such Scaffold Applications
as hydrophobicity, charge, roughness, and stiffness will also Cell Delivery
impact the behavior of cells seeded on the scaffold (Chang
and Wang, 2011). Without the presence of proteins, cells A widespread application of scaffolds is as a carrier for
tend to adhere and proliferate better on moderately hydro- cell delivery. Although delivering cells in suspension has
philic surfaces (i.e., high wettability) than on hydropho- shown positive results in improving disease conditions such
bic surfaces. One benefit of hydrophobic surfaces though as osteoarthritis and myocardial infarction (Amado et al.,
is the ability for proteins to adsorb through hydrophobic 2005; Berry, 2006; Black et al., 2008; Koh et al., 2013), the
interactions and thus serve as a surface for cells to bind and technique suffers from low cell retention and poor integra-
proliferate. Synthetic polymers that are widely used for scaf- tion of cells at the site of delivery (Chen et al., 2012). By
fold fabrication such as PLGA and PCL are hydrophobic providing an environment where cells can be seeded, tissue-
and thus are not ideal substrates for cell adhesion on their engineering scaffolds provide a platform that can deliver
own, but with proteins adsorbed on the surface they make such cell populations directly into the defect site. Surgical
promising tissue-engineering scaffolding systems. Sur- techniques that incorporate scaffold carriers such as matrix-
face modification strategies such as plasma treatment and assisted chondrocyte implantation (MACI) have proven to
hydrophilic polymer coating have therefore been utilized to be a more effective alternative to direct delivery of cell sus-
enhance cell–surface interaction (Arima and Iwata, 2007; pension (Kon et al., 2009; Steinwachs, 2009).
Cui et al., 2009; Place et al., 2009). An increase in rough- Cells, regardless of their origin, may be seeded onto a
ness has been shown to not only enhance cell adhesion by scaffold via a static or dynamic method (Marler et al., 1998).
improving adsorption of cell adhesion-mediating proteins Static seeding involves a cell suspension being directly
such as fibronectin, but also alter their functions such as deposited onto the scaffold surface and gradually allowing
proliferation, morphology, and matrix production (Hal- the cells to coat the scaffold, whereas dynamic seeding uti-
lab et al., 2001; Martin et al., 1995; Puckett et al., 2008; lizes any type of mechanical stimuli (e.g., stirring, shaking)
Saranya et al., 2011). to enhance the density and uniformity of cells on the surface
Surface coating of synthetic scaffolds is often required of the scaffold. In addition to delivering cells on the surface
to enhance the cell–scaffold interaction, as they often lack of a 3D scaffold, cells may be incorporated within the bulk
the biological signals that are required for cell attachment. of a scaffold. Encapsulation refers to a technique where cell
The coating process involves chemically treating the surface bodies are physically isolated from the surrounding environ-
to functionalize it with active domains that could be used ment while maintaining their activity and viability (Uludag
for modifications with bioactive molecules of choice, such et al., 2000). Hydrogels are utilized as the scaffold of choice
as ECM components that promote cell adhesion (collagen, for cell encapsulation, as they can provide a nontoxic,
fibronectin, and laminin), minerals to drive osteoconduc- hydrated environment for encapsulated cells to survive and
tion and osteointegration (calcium phosphate-based minerals proliferate. Cells are loaded into the hydrogel precursor solu-
such as hydroxyapatite), and drug molecules (Wu et al., 2014; tion before undergoing gelation, after which the hydrogel
 CHAPTER 2.6.3   Tissue Engineering Scaffolds 1319

can be implanted. The hydrated environment allows for persulfate/N,N,N′,N′-tetramethylethylenediamine (APS/

nutrients and signaling molecules from the body to diffuse TEMED) and APS/ascorbic acid, have been utilized to
into the hydrogel. Cells, in addition to being delivered in cross-link linear polymers with diacrylate-containing cross-
bulk hydrogels, can also be loaded into microspheres and linkers to form cell-laden hydrogels (Temenoff et al., 2003;
delivered into the defect site when encapsulated in a bulk Wang et al., 2006; Zhu and Ding, 2006). Photocross-link-
scaffold carrier. This method improves mass transfer of oxy- able systems are a subset of chemically cross-linked hydro-
gen and other nutrients to the cells, thereby enhancing the gels that require UV or visible light for activation. Polymers
viability of cells postimplantation (Hernández et al., 2010). such as PEG that are otherwise not photocross-linkable
Natural materials such as alginate and collagen (Chan et al., can be modified with acrylate groups, after which they can
2010; Tan and Takeuchi, 2007; Yu et al., 2010), as well as be cross-linked under visible or UV light with the help of
synthetic materials such as oligo(poly(ethylene glycol)fuma- photoinitiators (Burdick and Anseth, 2002; Sawhney et al.,
rate) (OPF) (Park et al., 2005) have been used for the fab- 1993).
rication of cell-laden hydrogel microspheres (Fig. [Link]). Chemically cross-linkable hydrogels that do not
Another method of cell delivery involves direct injec- depend on free radical initiators have also been devel-
tion of cell-laden hydrogel precursor solution into the body, oped. For instance, supramolecular self-assembly between
where it will fill in the exact size and shape of the defect poly(ethylene oxide) (PEO) and α-cyclodextrins (α-CD)
site and undergo gelation. These injectable systems allow for has been shown to result in the formation of a hydrogel
a minimally invasive delivery of cells to the site of inter- that is injectable and biocompatible (Li et al., 2003). Click
est, as they can be delivered using a syringe needle and thus chemistry, which refers to reactions that can be performed
bypass the need to perform an invasive surgery and suture in aqueous conditions and do not generate toxic byprod-
the scaffold into place. Such gelation can take place either ucts, has also been utilized to develop injectable systems as
chemically or physically, depending on the material of the cell carriers (Jiang et al., 2015a; Takahashi et al., 2013).
hydrogel. Injectable hydrogels that utilize chemical gela- Injectable hydrogels that undergo physical gelation do
tion often rely on free-radical initiators to start the reac- not rely on initiators or other chemical interactions for
tion between polymer chains that have reactive groups (e.g., hydrogel formation. Rather, they rely on changes in envi-
acrylates), or between the polymer chains and cross-linkers. ronmental cues such as temperature, pH, and ionic con-
Different combinations of initiators, such as ammonium centrations to undergo gelation. Thermogelling hydrogels

• Figure [Link] Scheme of the main applications of tissue-engineering scaffolds. (Protein structures
downloaded from [Link].)
1320 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

are a subset of injectable materials that will undergo revers- conditions, as biomolecules can be mixed with the aque-
ible gelation in response to temperature. One of the defin- ous precursor solution and be encapsulated as the solution
ing characteristics of these thermosensitive systems is their undergoes gelation. Such molecules can either be loaded
lower critical solution temperature (LCST), which is the into the bulk space of the hydrogel or covalently conjugated
temperature at which the system will undergo sol–gel tran- to the hydrogel backbone for a more controlled, sustained
sition. LCST can be tuned by adjusting the hydrophobic- delivery that does not depend solely on diffusion. In addi-
ity of the polymer. Ensuring that the LCST is near body tion, hydrogels can be designed so that they degrade in
temperature is desirable for tissue-engineering applications response to the physiological conditions of the surround-
(Kretlow et al., 2007). ing environment and simultaneously release encapsulated
biomolecules (Kost and Langer, 2012; Miyata et al., 2002).
Drug and Biomolecule Delivery For instance, pH-sensitive hydrogels have been extensively
developed and tested to control the release of GFs from the
Delivering drugs and bioactive molecules using scaffolds hydrogel (Garbern et al., 2010, 2011; Ishihara et al., 1984;
allows for a sustained and localized delivery, thus minimiz- Kim et al., 2009).
ing complications related to systemic delivery such as poor
localization of biomolecules at the target and low delivery Scaffold Materials
efficiency stemming from short half-lives of biological mol-
ecules (Florence and Jani, 1994). When drugs and biomole- Scaffold materials can range widely from synthetic scaffolds
cules are loaded inside a degradable scaffold, these molecules with little to no biochemical cues or factors incorporated to
will be gradually released and delivered to the adjacent tis- wholly biological systems possessing both innate and added
sue as the scaffold undergoes degradation over time. bioactive factors to guide tissue growth (Deforest et al.,
Drugs and biomolecules can be immobilized on scaffold 2009; Hubbell, 1995; Lutolf and Hubbell, 2005). As the
surfaces via covalent conjugation or physical adsorption field was initially being developed, the study of synthetic
(Sokolsky-Papkov et al., 2007). Physical adsorption relies biomaterials benefited greatly from the wealth of informa-
on noncovalent interactions between the substrate and the tion gained in the disciplines of chemical synthesis and
surface (e.g., ionic, hydrophobic, etc.), whereas covalent materials processing, and the field has only continued to
bonding requires reactive functional groups to be present evolve and grow since. Classifying these polymers can be
on both the molecule of interest and the surface of the scaf- challenging, but a popular method is by categorizing poly-
fold. This method often requires the surface to be treated mers as either polycondensation or polyaddition polymers.
with physical or chemical means to introduce such groups. This classification system, like all others, is imperfect but
Direct conjugation of proteins such as growth factors (GFs) provides a clear picture of the polymer end-product and is
also requires careful consideration when choosing which helpful as it organizes systems based on the polymer struc-
functional groups to modify, as poorly designed conjuga- ture (Odian, 2004). These two types of polymers include
tion schemes can negatively affect their bioactivity by dis- many important biomaterials including polyesters, polyure-
rupting the native folding state or blocking the access to thanes, polyamides, and polyacrylates (Table [Link]).
their active domains. In addition, conjugated factors will
not be internalized by cells unless the surface undergoes ero- Polycondensation Polymers
sion or degradation. Nevertheless, when well-executed, scaf-
folds covalently conjugated with GFs can show enhanced Polycondensation polymers are some of the most popular
bioactivity over delivery of soluble GFs directly to the tissue. polymers used in biomaterials applications and include poly-
This method of delivery also enables more careful control of esters, polyethers, polyurethanes, and more. These polymers
spatiotemporal distribution of factors on the surface of the often possess functional groups within the polymer chain
scaffold and a longer duration of the signals than when the backbone (i.e., esters, ethers, amides, etc.) (Odian, 2004)
signals are encapsulated within the scaffolds (Tayalia and and the polymerization methods used to form polyconden-
Mooney, 2009). sation polymers often involve the creation of low-molec-
Drugs and biomolecules can also be encapsulated in the ular-weight byproducts; in biomaterials, these byproducts
bulk space of the hydrogel during synthesis. Encapsula- are most often water or alcohol-containing groups. The
tion into solid, porous scaffolds is possible but significantly polymerizations used to create polycondensation polymers
more challenging than encapsulation in hydrogels due to are characterized by a drastic monomer conversion at the
the method of scaffold fabrication; for instance, synthesis beginning of the reactions due to the rapid formation of
of many porous synthetic scaffolds requires organic solvents oligomers, followed by a steady growth into polymers until
and exposure to harsh conditions such as temperature and all monomers are consumed. The system reflects this mono-
pressure, depending on which process is utilized to intro- mer conversion with a gradual steady increase in polymer
duce pores into the scaffold (Tayalia and Mooney, 2009). As molecular weight over time. In polyester-forming condensa-
a result, most biomolecules are incorporated after the scaf- tions, often a carboxylic acid- or alcohol-containing group
fold has been formed. Loading of drugs and biomolecules is the monomer and the system is usually acid-catalyzed.
in hydrogels, on the other hand, can be done under mild High temperatures and vacuum are often used to promote
 CHAPTER 2.6.3   Tissue Engineering Scaffolds 1321

TABLE
[Link]   Polymerization Methods for Creating Synthetic Biocompatible Polymers

Polymerization Method Characteristics Example Polymers

Polymerizations producing • Reaction exhibits rapid monomer conversion upon the Poly(ε-caprolactone) (PCL),
polycondensation start of the reaction poly(glycerol sebacate) (PGS),
polymers • Polymer molecular weight increases steadily over time polyurethane, polylactide
• Polymerizations often produce low-molecular-weight (PLA), polyglycolide (PGA),
byproducts so can be performed under vacuum poly(lactide-co-glycolide) (PLGA),
• Ring-opening polymerizations and click reactions are poly(dimethylsiloxane) (PDMS),
included poly(propylene fumarate) (PPF)
Polymerizations producing • Polymerization operates in distinct initiation, propagation, Propylene fumarate dimethacrylate
polyaddition polymers and termination steps (PFDMA) and diacrylate (PPF-DA),
• Reaction exhibits rapid polymer molecular weight growth poly(acrylamide), poly(ethylene
upon initiation of the reaction glycol) (PEG), ethylene glycol
• Monomer conversion progresses more steadily over time dimethacrylate (EGDMA),
• Ionic polymerizations and free-radical polymerizations are butanediol dimethacrylate (BDMA)
included

the polymerization and facilitate the removal of byprod- Click Reactions

ucts. These condensation techniques have been used for Click reactions are a subset of chemical reactions that can
decades and have produced condensation polymers such be carried out in aqueous conditions, at physiological tem-
as poly(propylene fumarate) (PPF) (Peter et al., 1999a,b), perature, and without cytotoxic initiators (Deforest et al.,
poly(glycerol sebacate) (PGS) (Wang et al., 2002), and 2009). These reactions are also highly specific and proceed
polyurethanes (Bachmann et al., 2001). Polyurethanes have in an orthogonal manner with high polymerization effi-
been used for tissue-engineering applications such as elec- ciency (Deforest et al., 2009; Tibbitt and Anseth, 2009).
trospun scaffolds (Kishan and Cosgriff-Hernandez, 2017; Click reactions encompass the well-known thiol-ene reac-
Nezarati et al., 2013) and in shape-memory implants for tion, which allows highly controlled spatial and temporal
embolic applications (Singhal et al., 2014). regulation of materials through photoinitiation (Deforest
et al., 2009). Click reactions can also be classified under
Ring-Opening Polymerization the umbrella of polycondensation polymers. Although the
formation of low-molecular-weight byproducts does not
Ring-opening polymerization is a common tool used to
always occur, the reaction follows the general trend of poly-
produce linear polymers. It is commonly used to produce
condensation polymers such as rapid monomer conversion
PCL (Dubois et al., 1991; Habibi et al., 2008; Hamitou
and a gradual increase in molecular weight, and the pres-
et al., 1977), among other notable polyester biopolymers
ence of functional groups in the polymer chain backbone.
such as PLA, polyglycolide (PGA), and the copolymer
Extensive work has demonstrated the power of click chemis-
PLGA (Gilding and Reed, 1979; Kaihara et al., 2007).
try in creating dynamic scaffolds that are responsive to both
Biodegradable sutures Dexon (PGA) and Vicryl (PLGA)
external stimuli (light, concentration gradient, mechanical
are approved for use in humans by regulatory authorities
stimulation, etc.) and to cells (Benoit et al., 2008; Brown
and have been safely used for decades (Bourissou et al.,
et al., 2018; Burdick and Anseth, 2002; Kloxin et al., 2009;
2004; Gilding and Reed, 1979). An extremely common
Sridhar et al., 2015). Additional work has been carried out
biocompatible polymer, PEG, can be produced through
to create biocompatible and bio-instructive scaffolding sys-
the ring-opening polymerization of an epoxide ring (Her-
tems (Arakawa et al., 2017; Gramlich et al., 2013; Jivan
zberger et al., 2016). Poly(dimethylsiloxane) (PDMS) is a
et al., 2016, 2018; Nimmo et al., 2011).
common polymer used in biological applications involv-
ing lithography which can also be produced through the
ring-opening polymerization of cyclic siloxane groups Polyaddition Polymers
(Rodriguez et al., 2007) and produces an optically trans-
parent polymer product (Ersen and Sahin, 2017). Many of Unlike polycondensation, polyaddition reactions do not
these ring-opening polymerizations are achieved through involve the formation of low-molecular-weight byproducts,
the initiation with an ionic group, or a coordination com- and the polymer backbone does not often contain functional
pound, the most common ones being Tin(II)-based (Gild- groups (Fried, 2014; Odian, 2004; Stevens, 1999). The
ing and Reed, 1979; Kaihara et al., 2007). Ring strain reactions to form polyaddition polymers include ionic and
contributes to reactivity so three- and four-membered free radical polymerizations. These reactions exhibit a rapid
rings are most reactive with cyclohexane groups being rela- increase in the molecular weight almost immediately upon
tively stable but still reactive given the correct conditions the initiation of the reaction while the monomer conversion
(Nuyken and Pask, 2013). progresses more steadily over time. Polyaddition reactions
1322 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

operate in distinct initiation, propagation, and termination the door for tissue-engineering scaffolds that can be created
steps, each with their own kinetic rate and one growing quickly to form robust scaffolding systems; polycondensation
reactive center that other monomers join as the polymer- polymers have been harnessed to create biocompatible scaf-
ization progresses. Some of the most notable examples of folds such as PCL, PLA, and PGA and PLGA which are com-
polyaddition polymers are propylene fumarate dimethacry- monly used in biodegradable sutures (Bourissou et al., 2004;
late (PFDMA) and diacrylate (PF-DA), both biocompatible Gilding and Reed, 1979). The field has expanded immensely
and mechanically robust bone tissue-engineering scaffold- and is expected to continue expanding in the coming years.
ing systems (Moglia et al., 2014; Timmer et al., 2003).

Ionic Polymerization Biological Polymers

Ionic polymerizations include both cationic and anionic reac- Biological polymers have emerged as attractive materials for
tions. Cationic polymerization systems are often initiated tissue engineering given their biocompatibility and biodegrad-
by a mineral acid, or a Lewis acid accompanied by a proton ability. Gelatin is a popular biological polymer that has been
source, which becomes the electrophile needed to begin the fabricated into a variety of scaffold systems; it is also highly bio-
reaction (Stevens, 1999). The method is attractive due to its compatible and contains the RGD peptide sequence known to
ability to produce “living polymerizations,” which has aided promote cell adhesion (Liu et al., 2017). Gelatin has also been
in the production of many commercially used block copoly- shown to demonstrate shear-thinning properties (Liu et al.,
mers (Stevens, 1999) and branched copolymers (Iatrou et al., 2017), which is very attractive for extrusion-based 3D print-
2002). These systems are often induced through the addition ing applications. Alginate is another popular biopolymer that
of a metal initiator and begin with a nucleophilic addition to is cross-linked via ionic cross-linking around divalent cations,
the monomer. The most notable biomaterial applications of most commonly Ca2+ (Lee and Mooney, 2012). This cross-
anionic polymerizations are the creation of star polymers of linking is reversible and has been harnessed to encapsulate
PEO (Gnanou et al., 1988; Peppas et al., 1999). single cells (Utech et al., 2015), and create stimuli-responsive
drug-delivery systems (Huebsch et al., 2014). Glycosamino-
Free Radical Polymerization glycans (GAGs) are another class of materials commonly used
Free radical polymerization is a rapid, highly effective tech- in tissue-engineering applications given their natural presence
nique widely used in the industry to produce commercial poly- in the ECM. Hyaluronic acid has been used for a variety of
mers such as poly(methyl methacrylate) (PMMA) (Charnley, tissue-engineering applications because of its natural presence
1960; Turner et al., 1981), poly(acrylamide), and many acry- in many wound-healing processes (Leach et al., 2003), as well
late and methacrylate terminated polyesters and polyethers as its ease for chemical modification and biodegradability
(Matyjaszewski and Xia, 2001; Stevens, 1999). Initiators for (Hubbell, 1995; Leach et al., 2003; Van Vlierberghe et al.,
free radical systems are diverse and include peroxides (Maty- 2011). Once degraded, the polymer has been shown to pro-
jaszewski and Xia, 2001), redox initiators (Hageman, 1985), mote angiogenesis (Leach et al., 2003). Sulfated GAGs such as
azo groups (Braun, 2009), and photoinitiators (Hageman, chondroitin sulfate and heparan sulfate can also sequester GFs
1985). Many peroxide initiators rely on temperature degra- to promote cell growth and proliferation (Rinker et al., 2018;
dation to generate free radical species for initiation (Moglia Van Vlierberghe et al., 2011).
et al., 2014), whereas redox systems can do so independent Decellularized tissues are a class of biomaterials that has
of temperature by including a reducing agent along with also been used to synthesize biological scaffolds. Once decel-
an oxidizing agent. Photoinitiators produce reactive species lularized, these tissues retain many of the bio-instructive cues
upon exposure to light of varying wavelengths depending on found in tissue that can promote native tissue integration and
the compound (Hageman, 1985). Azo compounds decom- defect repair (Badylak, 2004; Badylak et al., 2011; Cheng
pose in a range of temperatures so, depending on the reaction et al., 2014). Scaffolds derived from decellularized tissues such
requirements, a compatible azo initiator can be found (Braun, as muscle (Cheng et al., 2014; Merritt et al., 2010a,b; Smoak
2009). Some of the most notable work in free-radical polym- et al., 2019), lung (Nichols et al., 2013), and heart (Ott et al.,
erization has been the development of scaffolds consisting of 2008) have been documented. Further processing the decel-
polyester-based polymers, PPF (Peter et al., 1999a,b; Sikavit- lularized tissue (e.g., digestion with pepsin) for various appli-
sas et al., 2001), ethylene glycol dimethacrylate (EGDMA), cations such as electrospinning (Baiguera et al., 2014) and
butanediol dimethacrylate (BDMA) (Moglia et al., 2014), 3D printing (Pati et al., 2014) has also been explored.
and propylene fumarate dimethacrylate (PFDMA) (Moglia
et al., 2014; Sears et al., 2017; Whitely et al., 2017). Composites and Additives
Synthetic scaffolds offer a wealth of benefits to the field of
tissue engineering. The work done in the polymer engineer- Given the complexity of native tissue, composite and additive
ing and processing industry over the last several decades has biomaterial systems for tissue engineering are popular options
benefited the field immensely and brought forth technologies for encouraging regeneration (Temenoff and Mikos, 2000).
for the creation of highly monodisperse and biocompatible Tissues such as bone and cartilage are particularly popular
polymers. The formation of polyaddition polymers with vinyl options for composite biomaterials (Temenoff and Mikos,
monomers through free-radical polymerization has opened 2000; Venkatesan and Kim, 2014). Hydroxyapatite (HA) is a
 CHAPTER 2.6.3   Tissue Engineering Scaffolds 1323

natural calcium phosphate mineral found in bone and enamel, (Cao et al., 2009; Lee et al., 2011), and insulin-like growth
used in bone tissue engineering. However, due to their brittle- factor 1 (IGF-1) for a variety of applications (Chen et al.,
ness, ceramics such as HA are often combined with polymers 2009; Davis et al., 2006; Lee et al., 2011).
to create scaffolds with enhanced mechanical strength (Ven-
katesan and Kim, 2014). Polymers commonly used with HA Scaffold Fabrication Techniques
for bone tissue engineering include collagen, gelatin, PLA,
PLGA, PCL, and polyurethane. Composite scaffolds for carti- Scaffolds are one of the three main components of the tis-
lage often include cells and have been composed of polymers sue-engineering strategy to repair, regenerate, and remodel
such as thrombin and fibrin, as well as alginate, hyaluronic living tissues after an injury or disease (O’Brien, 2011).
acid, and oligo(poly(ethylene glycol) fumarate) (Park et al., Since the beginning of the tissue-engineering field, scaffolds
2007; Temenoff and Mikos, 2000). Another application of have shown a key role since they provide a 3D solid sup-
composite materials has been in neural tissue engineering. port for the attachment, proliferation, migration, and dif-
Composite fibers were used in neural regeneration applications ferentiation of the cells. Furthermore, they can also serve as
by electrospinning human glial cell-derived neurotrophic fac- reservoirs for the controlled release of bioactive molecules
tor (GDNF) inside a copolymer of ε-caprolactone and ethyl or drugs that can drive the regeneration process (Nair and
ethylene phosphate (Chew et al., 2007). Similarly, there have Laurencin, 2007). The field of tissue engineering has devel-
been other numerous documented examples of improved oped a wide range of processing techniques to fabricate
regenerative outcome for composite scaffolds incorporating porous scaffolds from one or more biodegradable polymers
transforming growth factor-β1 (TGF-β1) for cartilage repair, that are selected depending on the properties needed for the
vascular endothelial growth factor (VEGF) for angiogenesis intended application (Table [Link]).

TABLE
[Link]   Preparation Techniques for Tissue-Engineering Scaffolds, Requirements and Examples of Applications

Scaffold Fabrication
Techniques Requirements Applications
Rapid prototyping Must utilize inks that are easy to work with and Create 3D structures from computer models.
possess desired mechanical properties Stereolithograpy, selective laser sintering (SLS),
inkjet bioprinting, extrusion printing, bioplotting.
Often use amorphous and semicrystalline polymers
such as PCL, PLA, and PLGA. Poly(vinyl alcohol)
(PVA) and poly(propylene fumarate) (PPF) have also
been used
Electrospinning/ Relies on an electric field to aid in the deposition Fabricate microparticles via electrospraying or fibrous
electrospraying of the polymer solution onto a collecting plate mesh scaffolds via electrospinning. Have been used
so choosing a suitable polymer and solvent to generate scaffolds made from chitosan, gelatin,
carrier is required collagen, PCL, PLGA, polyurethanes, and more
Superstructure Depends on structural interactions to maintain Generate complex structures built on noncovalent
engineering scaffold integrity such as noncovalent, van interactions. Allows for reversibility, and it also
der Waals, hydrophobic, ionic, magnetic, and enables “self-healing” scaffolds to be created
electrostatic interactions so polymers must
be chosen that promote these interactions
Solvent casting, Must select a porogen not soluble in the solvent Generate porosity in scaffolds. Salt leaching is
particulate/porogen that the polymer is being cast in. Selecting a the most common porosity-inducing method.
leaching porogen of adequate size that also allows for Polymers such as sugar or paraffin have also been
interconnectivity is important as well used as porogens
Freeze-drying Must be able to withstand cold temperatures. Remove water and generate porosity when paired
The freezing rate impacts pore size with with annealing. Attractive for use with materials that
faster freezing rates producing homogeneous are temperature sensitive at higher temperatures
pore structures
Phase separation Requires immiscible phases to be chosen, and Create porous foams. Can also use thermally induced
consideration for compatibility of the phases phase separation to create porous structures with
with biological molecules must be considered high interconnectivity
Gas foaming Requires the polymer to be able to be saturated Generate highly porous structures by foaming dissolved
with CO2 CO2 gas. The CO2 interacts strongly with carbonyl
groups so PLA, PLGA, and PCL are commonly used.
Can also be used to decellularize tissues
1324 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

Rapid Prototyping offers very precise control over the microstructure design
and the obtained scaffolds usually hold high mechanical
Rapid prototyping or solid free form fabrication refers to a strength. The main drawbacks of this technique are the dif-
series of techniques that use polymeric materials by printing ficulty to alternate different materials in the construct and
layer-by-layer scaffolds from a 3D model created by com- the need for powdered materials with good flow properties
puter (Peltola et al., 2008). Compared to traditional biofab- to homogeneously deposit each layer (Mazzoli, 2013).
rication techniques, rapid prototyping can recreate complex
structures with high fidelity and reproducibility while
Electrospinning/Electrospraying
minimizing material loss (Bittner et al., 2018). Additional
advantages are the possibility to design patient-specific scaf- Electrospinning is a widely used technique to prepare nano-
folds and on-demand fabrication (Guvendiren et al., 2016). and/or microfibrous structures from polymeric solutions
Among various rapid prototyping techniques, extrusion using the action of an electric field (Lannutti et al., 2007;
printing, stereolithography (SLA), selective laser sintering Pham et al., 2006). The diameter and morphology of the
(SLS), digital light projection (DLS) and inkjet bioprinting resulting fibers that mainly define scaffold porosity can
have gained increased usage in fabricating tissue-engineered be modified with the polymer concentration, the voltage
scaffolds in the last decade (Ozbolat and Hospodiuk, 2016; applied, and the distance between the needle and the col-
Roseti et al., 2017). lector (Jiang et al., 2015b). Fiber diameter has a key role in
Extrusion printing is based on the melting of a poly- the micro- and macroscopic properties of the electrospun
meric admixture inside of a moving extruder arm and its scaffolds, including porosity and mechanical and biological
deposition on a printing platform through a nozzle using properties (Sisson et al., 2010; Unnithan et al., 2015). A wide
pneumatic or mechanical dispersers (Zein et al., 2002). range of both natural and synthetic polymers as well as their
Extrusion printing has become an attractive platform for blends have been used for electrospinning (Khajavi et al.,
the preparation of highly precise porous scaffolds for tis- 2016). In particular, core–shell coaxial electrospinning has
sue engineering because of its resolution, reproducibility, been explored as a method to prepare nanofibrous scaffolds
and fidelity to the predesigned model (An et al., 2015). In that encapsulate drugs or other bioactive molecules such as
particular, extrusion printing has been shown to be suit- GFs in the core (Yoon et al., 2018; Zhang et al., 2013). Melt
able for the fabrication of porous scaffolds prepared with a electrospinning is another variation of electrospinning, where
wide range of compositions including thermoplastics and the polymer is melted in a syringe or cartridge and ejected
composite admixtures (Bittner et al., 2019; Diaz-Gomez toward a collector by applying an electric field. This method
et al., 2019). However, the incorporation of bioactive or of electrospinning does not require solvents and thus results
heat-sensitive biomolecules in the aforementioned meth- in lower toxicity and fewer postprocessing steps compared
ods is limited due to the high temperatures and pressures to conventional electrospinning. Furthermore, when a mov-
involved in the extrusion process. A cell-friendly alternative able collector plate is used, it is possible to fabricate intricate
to the high temperatures involved in the extrusion printing porous structures from a template created using a software
is direct ink writing or cold temperature 3D bioprinting. A (Brown et al., 2016; Hochleitner et al., 2015).
wide range of natural polymers, including polysaccharides
such as alginate or chitosan, and proteins such as collagen,
Superstructure Engineering
gelatin, or elastin, among others, have been used as bioinks
to prepare cell- or GF-laden scaffolds for tissue engineering Self-assembly is considered a bottom-up autonomous
(Chung et al., 2013; Das et al., 2015; Echave et al., 2017; arrangement of components into certain patterns or struc-
Jammalamadaka and Tappa, 2018; Radhakrishnan et al., tures driven by short-distance interactions (noncovalent,
2017). van der Waals, hydrophobic interactions, ionic, magnetic,
SLA and DLS use a light beam or an image projection, or electrostatic). Using self-assembly, it is possible to control
respectively, to cure a polymer solution into a specific pat- the structure of the scaffolds at the molecular level to cre-
tern with high resolution (Dean et al., 2012; Melchels et al., ate highly complex structures that closely mimic the prop-
2010). After each layer is cured, the platform moves up, erties and architecture of the native tissues. Furthermore,
and the process is repeated until the fabrication is com- since self-assembly is a reversible process, most of the scaf-
pleted. SLA has become an alternative to extrusion-related folds developed using this technique present unique self-
approaches since shear-stress mechanisms are not involved healing properties that make them very attractive when used
(Lin et al., 2013). However, the need for photoinitiators that in locations that could present mechanical perturbations.
are potentially cytotoxic limits the use of this approach for Self-assembly scaffolds with diverse architectures have been
cell-laden tissue-engineering applications (Manapat et al., developed using protein-based strategies such as chemically
2017). SLS is another layer-by-layer technology that uses or biologically synthesized peptides. Self-assembly peptides
a high-energy laser to fuse a thin layer of powder particles are usually prepared as injectable hydrogels that self-assemble
together. The resolution of the printed object is restricted by in situ as a result of pH or temperature (Hosseinkhani et al.,
the precision of the laser to sinter the powder, and depends 2013). The main advantages of using peptides for the prepa-
on the laser power and speed, layer thickness, and laser ration of self-assembly scaffolds are their high biocompatibil-
focusing radius, among others (Shirazi et al., 2015). SLS ity and biodegradability. Furthermore, slight modifications
 CHAPTER 2.6.3   Tissue Engineering Scaffolds 1325

in the peptide sequences result in macromolecular changes scaffolds’ structural properties: fast cooling rates usually lead
that affect the properties of the scaffold and can be used to to more homogeneous pore structures, while lowering the
tune the mechanical and functional properties of the result- freezing temperature decreases the mean pore size. Anneal-
ing constructs (Habibi et al., 2016). However, since most of ing (increasing the temperature of the frozen material to
the compositions are hydrogels, their use is limited to soft tis- promote the growth of ice crystals) has also been described
sues, which remains as a challenge along with cost and scal- as a strategy to increase the mean pore size of scaffolds
ability (Dutta et al., 2017; Khan and Tanaka, 2017). (Pawelec et al., 2014; Raeisdasteh Hokmabad et al., 2017).
In general, freeze-drying only allows for the preparation of
Solvent Casting, Particulate/Porogen Leaching simple (planar) 3D structures, but complex structures have
been successfully prepared by controlling the molds and the
Solvent-casting particulate leaching (SCPL) is a simple and processing parameters (Brougham et al., 2017). Pore orien-
cost-effective method to prepare highly porous polymeric tation can be defined by controlling the growth speed and
scaffolds. Insoluble porogens such as salts are dispersed in orientation of the ice crystals, resulting in unidirectional
a polymer solution, and following a solvent evaporation porous scaffolds (Deville, 2010). Pore size can also be con-
step, a porogen–polymer composite is formed that is fur- trolled by the polymer concentration and molecular weight
ther washed to remove the porogen particles (Mikos et al., (Qian and Zhang, 2011).
1994). SCPL allows for the preparation of scaffolds with An alternative to freeze-drying is emulsion freeze-drying
a porosity higher than 90% and pore size up to 500 μm which has attracted attention due to its ability to prepare scaf-
(Prasad et al., 2017). Furthermore, it is relatively easy to folds with highly specific pore surface area. The method is
control the pore architecture (pore size, interconnectivity, based on the dissolution of a polymer solvent and its further
porosity) of the obtained scaffolds by the selection of ade- emulsification by the addition of a nonsolvent. The obtained
quate polymers, porogens, and their concentrations. Poro- emulsion is poured into a mold and quenched at low temper-
gen leaching, however, has been reported to result in poor ature prior to freeze-drying to remove the solvent and non-
interconnectivity and heterogeneous pore shapes. Porogen– solvent (Sultana and Wang, 2008, 2012). PVA is an attractive
polymer blends such as sodium chloride and PEG have polymer to be processed using these approaches since it
therefore been investigated to introduce a dual-leaching physically cross-links when submitted to freeze/thawing, thus
process, which could significantly increase the interconnec- avoiding the use of potentially cytotoxic cross-linkers (Nie
tivity of pores while maintaining a homogeneous pore size et al., 2012). The biodegradation rate of the scaffolds can also
distribution (Thadavirul et al., 2013). The main drawback be controlled by modifying the ratio of polymers included in
of SCPL is the use of organic solvents that could lead to the composition (Sultana and Wang, 2012).
cytotoxicity and also limit the incorporation of bioactive
molecules. Moreover, the mechanical properties of scaffolds Phase Separation
prepared with this technique are below the requirements of
load-bearing tissues (Janik and Marzec, 2015). In order to Phase separation can be achieved with the addition of a
increase the mechanical properties of the scaffolds, gel cast- nonsolvent, which yields scaffolds with heterogeneous pore
ing has become an interesting alternative. A green strategy structures. This option is less attractive for tissue engineer-
used to produce polymeric, ceramic, and composite porous ing scaffolds that generally require uniform porous struc-
scaffolds, gel casting consists of a preparation of an aqueous tures. A second approach is the thermally induced phase
polymeric solution that is foamed and polymerized in situ separation (TIPS) method, which is based on the decrease
through chemical reactions to prevent structure collapse. of thermodynamic stability of a polymeric solution with
The obtained structures are dried and, if ceramic materials small changes in the temperature of the system resulting in
are used, sintered (Montanaro et al., 2018). Scaffolds pro- a phase separation process which produces a polymer-rich
duced using gel casting showed high interconnectivity and phase and a polymer-lean phase. After the removal of the
dense pore walls, with high mechanical strength and porosi- polymer-lean phase by evaporation or lyophilization, the
ties over 90% (Baino et al., 2015; Siqueira et al., 2017). polymer-rich phase becomes a highly interconnected porous
structure with homogeneous pore size distributions (Lu
Freeze-Drying et al., 2013; O’Brien, 2011). By controlling process vari-
ables such as polymer concentration and molecular weight,
Freeze-drying is a simple approach for the preparation of solvent/nonsolvent ratio, and the cooling path, it is possible
highly porous polymeric scaffolds via freezing and sublima- to obtain scaffolds that have a wide range of morphologies
tion of an aqueous or organic polymeric solution using low and microporous structures. Since pores are formed in the
temperature and pressure. Since the process does not involve paths created by the solvent extraction, high interconnec-
high temperatures, it allows heat-sensitive components to tivity is easily achieved, which makes this technique very
be included into the scaffold. The structure of the scaffold attractive for cell spreading and migration (Wei and Ma,
can be tuned by adjusting the polymer concentration and 2004). Various synthetic and natural polymers have been
molecular weight, the viscosity of the polymer solution, processed alone or in combination with others to prepare
and solvent type. Other processing parameters such as the highly porous scaffolds with controlled structures, including
cooling rate and the freezing temperature strongly affect the gradients (Mannella et al., 2015; Pavia et al., 2008).
1326 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

Gas Foaming/Supercritical Fluid Processing environment. The scaffold degradation rate can be mea-
sured over any period of time by weight loss after incubation
Gas foaming is a solvent-free method that uses the expansion in an environment that mimics in vivo or accelerated in vivo
effect of a gas to fabricate highly porous scaffolds. CO2 is conditions. Drug release profiles from a bulk scaffold can be
the most used gas since it is safe, reusable, and cost-efficient. measured by using high-performance liquid chromatogra-
CO2 also reaches a supercritical state at mild temperature phy (HPLC), which can measure the concentration of drugs
and pressure (31.1°C and 73.9 bar) (Picchioni, 2014). In this that have been eluted from the scaffold to the surrounding
process, the polymer is saturated with CO2, which reduces media at specific time points (Haesslein et al., 2006; Shi
its glass transition temperature through a plasticizing effect. et al., 2010). For applications that require cell encapsula-
Once equilibrated, controlled depressurization results in the tion, biocompatibility can be measured indirectly by mea-
nucleation and expansion of the CO2, forming pores in the suring the viability of encapsulated cells, or by culturing the
material as it is evacuated (García-González et al., 2015). The cells with the leachable byproducts of the scaffold.
size and density of the pores can be regulated by adjusting Standards for characterizing scaffolds and biomaterials
the temperature, pressure, and depressurization rate. Dual for tissue-engineering applications have been developed by
pore populations, generally in the nano- and microscale, can standards organizations such as ASTM and International
also be achieved by modification of the depressurization rate Organization for Standardization (ISO) (Vo and Trachten-
during the process (Ji et al., 2012). Blends of two or more berg, 2014). In addition to characterization standards for
materials can be used to control the foaming process or the scaffolds in vitro, methods of assessing constructs in vivo
porous micro- and macrostructure of the obtained scaffolds have been developed (Trachtenberg et al., 2015). These
(Moghadam et al., 2017; Salerno et al., 2009). Due to its guides are application-specific and provide detailed guide-
mild processing temperature, biological molecules such as lines on techniques and procedures that are required to test
GFs have been incorporated in polymeric scaffolds without any given scaffold or biomaterial. Characterization tech-
affecting their biological activity (Diaz-Gomez et al., 2016; niques discussed in previous paragraphs should therefore be
Hile et al., 2000). conducted on the designed scaffolds according to the estab-
lished standards that correspond to the scaffolds’ potential
Scaffold Characterization Techniques application. This will also be important for future clinical
translation of such scaffolds (Table [Link]).
Scaffold characterization involves the characterization of the
material utilized to fabricate the scaffold and the bulk prop- Cell-Incorporated Scaffolds
erties of the obtained constructs. Material characterizations
involve methods to assess the purity and structure, such as Biomaterial scaffolds are ideal carriers for cell delivery (Ricles
nuclear magnetic resonance (NMR), mass spectrometry et al., 2016; Truong et al., 2015). Research has shown that
(MS), gas/liquid chromatography (GC/LC), and additional cell delivery alone results in cell death and low cell retention
spectroscopic methods such as Raman and infrared. Gel at the site of delivery (Chen et al., 2012; Ricles et al., 2016;
permeation chromatography (GPC) is widely used to mea- Vo et al., 2016). However, when encapsulated within a bio-
sure the molecular weight and polydispersity of synthesized material carrier, particularly those decorated with cell-specific
polymers. Thermal properties of polymers are measured or ECM-specific markers and factors, cell viability is improved
using differential scanning calorimetry (DSC) and thermo- and controlled differentiation may be induced (Na et al., 2007;
gravimetric analyzer (TGA). DSC will quantify glass tran- Nguyen and West, 2002; Ricles et al., 2016). Hydrogels are
sition and melting temperatures (Tg and Tm, respectively) a particularly popular biomaterial for cell encapsulation given
(Mikos et al., 1994), as well as LCST of thermoresponsive their highly swollen nature, their ability to be patterned with
polymers; TGA will measure the thermal stability of a given moieties, and to allow the diffusion of oxygen and nutrients
polymer and is widely used to characterize ceramics. to cells within the scaffolds (Nguyen and West, 2002; Ricles
Physical properties of synthesized scaffolds can be quan- et al., 2016; Truong et al., 2015; Vo et al., 2016). These cell-
tified via multiple methods. Microscopic techniques such encapsulated systems may be injectable, allowing the cell-laden
as scanning electron microscopy (SEM) and atomic force scaffold to fill the defect site (Ricles et al., 2016), or printed
microscopy (AFM) are frequently used to observe the sur- into their desired geometry (Billiet et al., 2014; Kolesky et al.,
face morphology of a scaffold (Ma et al., 2007). Of the two 2014). Cell-laden scaffolds also allow for multiple cell types to
techniques, AFM has a higher resolution and can accurately be incorporated into a scaffold, which is important in mimick-
quantify nanoscale topographical details. SEM or simple ing the complexity of native tissue (Xu et al., 2013), including
light microscopy can be used also to measure the size and interfacial gradient units of tissue such as the osteochondral
shape of microparticles. Mechanical properties such as com- unit at the bone/cartilage interface (Mohan and Detamore,
pressive or tensile moduli can be quantified by directly plac- 2014; Yang et al., 2017).
ing the scaffold under appropriate stress and measuring the Perfusion seeding is another technique for cell incorporation
stress-strain curve. In the case of thermosensitive hydrogels, in the scaffolds (Zhao and Ma, 2005). Stem cells may be seeded
a thermomechanical analyzer (TMA) can be used to con- onto scaffolds through perfusion-based methods and have been
duct mechanical testing under a temperature-controlled reported to maintain stemness in the case of MSCs (Zhao and
 CHAPTER 2.6.3   Tissue Engineering Scaffolds 1327

TABLE
[Link]   Description of the Characterization Techniques for Tissue-Engineering Scaffolds

Polymer/Scaffold Characterization
Property Technique Description
Chemical Nuclear magnetic Provides highly specific characterization of polymer structure and molecular
characterization resonance (NMR) weight. Most commonly used NMR techniques are 1H-NMR and 13C-NMR
spectroscopy with 1H-NMR being the most common for polymers for tissue-engineering
applications
Fourier transform Is used to characterize scaffolds and polymers alike. Spectra possess unique
infrared (FTIR) fingerprint regions for common bonds such as amides, carbonyls, and more to
spectroscopy characterize structures
Gas/liquid Separates and analyzes polymers that can be vaporized without decomposing.
chromatography Liquid chromatography (often high-performance liquid chromatography or
(GC/LC) HPLC) consists of a chromatography column and a detection tool such as a
UV spectrometer that helps separate and identify products as they are eluted
through the column
Raman Aids in the characterization and identification of polymers by looking at the spectra
spectroscopy to identify fingerprint regions for common chemical bonds, similarly to FTIR
Mass/molecular Mass spectrometry Ionizes the product and calculates molecular weight based on time of flight to a
weight (MS) detector. Provides highly specific mass values
characterization
Gel permeation Characterizes molecular weights based on well established PEG or PMMA
chromatography standards. Can characterize hydrophilic and hydrophobic polymers. Can also
(GPC) be paired with mass spectrometry for added accuracy
Thermal properties Differential scanning Determines the glass transition temperature (Tg) and the melting temperature (Tm)
characterization calorimetry (DSC) of the polymer
Thermogravimetric Determines the thermal stability of the polymer over a range of temperatures
analysis (TGA)
Morphology/surface Scanning electron Aids in determining surface features of polymer scaffolds such as mesh size or
characterization microscopy (SEM) porosity
Atomic force Is used to image polymers and compounds at single-atom resolution. Can also be
microscopy (AFM) utilized to determine mechanical properties of scaffolds at single bond resolution
Mechanical properties Rheology Measures mechanical properties for viscoelastic materials. Can determine the
characterization point of gelation for scaffolds
Tensile testing Is used to determine the tensile properties of a polymer or scaffold materials
Compressive testing Determines the compressive properties of a polymer or scaffold material

Ma, 2005). Perfusion-based seeding has also been used to vas- Conclusions
cularize scaffolds in 3D printed systems (Miller et al., 2012).
Bioreactor culture is closely related and often follows perfusion- The field of materials development for tissue engineering
based seeding. This allows for the diffusion of nutrients to cells has advanced greatly in recent years. Scaffolds can be created
(Cartmell et al., 2003; Saini and Wick, 2003). It also allows the as carriers for cells, drugs, and bioactive factors for diverse
cells within the scaffold to be placed under shear forces during tissue engineering, drug delivery, and wound-healing appli-
culture to maintain mechanical stimulation to improve viabil- cations. These scaffolds are often biodegradable via ester
ity (Cartmell et al., 2003), induce differentiation of bone cells hydrolysis or enzymatic degradation and can be composed
(Gomes et al., 2003) and endothelial vascular cells (Yamamoto, of synthetic or natural polymers. Using a wide variety of
2004), and create better disease models in the case of Ewing fabrication techniques such as rapid prototyping, electro-
sarcoma (Santoro et al., 2015). Tissues such as cartilage, bone, spinning, solvent casting/porogen leaching, gas foaming,
and heart valves require mechanical stimulation for their func- and more, robust and porous scaffolds can be created for a
tion (Cartmell et al., 2003; Hoerstrup et al., 2000; Saini and variety of applications and their features and properties can
Wick, 2003), and engineered tissue quality is greatly enhanced be tailored depending on their intended application. Many
for such tissues with mechanical stimulation via the shear forces challenges still remain in the field of materials development
seen in bioreactor culture (Bancroft et al., 2002; Holtorf et al., for tissue engineering, but ongoing work in the field shows
2005a,b; Sikavitsas et al., 2003). promise for significant developments in the near future.
1328 SEC T I O N 2. 6 Applications of Biomaterials in Functional Tissue Engineering

CASE STUDY: MATRIX-ASSISTED AUTOLOGOUS CHONDROCYTE TRANSPLANTATION (MACT)

Multiple reparative surgical techniques have been developed ACI, where a type I/III collagen membrane is used as a cell-
over the years to treat articular cartilage lesions. Microfracture, seeded scaffold to deliver in vitro-expanded autologous chon-
or marrow stimulation, refers to a technique where the drocytes to the defect site.4 The membrane is customized to
subchondral bone beneath the defect is penetrated using fit the size of the defect site before being sealed in place using
a microneedle to allow the flow of blood and bone marrow fibrin glue.
into the site. The formation of a blood clot, which contains MACT also presents certain challenges. Expansion of chon-
progenitor cells from the marrow, gradually leads to drocytes in vitro has been shown to lead to dedifferentiation;
regeneration of the tissue. Another option available to patients is studies have shown that when chondrocytes are cultured on a
osteochondral autograft or allograft, where cartilage pieces from stiff, 2D substrate, they gradually lose the ability to produce type
either low weight-bearing regions of the patient’s (autograft) II collagen and glycosaminoglycans even after the first pas-
or a donor’s (allograft) joints are harvested and transferred to sage.5 Cell expansion is also a time-consuming process, and
the defect site. These surgical techniques, however, present the number of chondrocytes required for transplantation, and
certain challenges. For instance, microfracture has been shown therefore the overall culture period, will depend on the size of
to be linked with formation of fibrous cartilage,1 osteochondral the defect. Nevertheless, MACT is one of the first FDA-regulated
autograft with donor site morbidity,2 and allograft with immune tissue-engineered therapies for clinical usage.
response and loss of cell viability.2 1Kreuz et al., 2006. Osteoarthritis and Cartilage. [Link]
In the late 20th century, a cell-based approach to treating [Link].2006.05.003.
osteochondral defects was developed. This technique, called 2Haene et al., 2012. J. Bone Joint Surg. [Link]
autologous chondrocyte implantation (ACI), involves small carti- 02010.
lage biopsies being taken from the patient to harvest chondro- 3Brittberg et al., 1994. N. Engl. J. Med. [Link]

cytes that are then expanded in vitro and delivered directly into 9410063311401.
the defect site as a suspension.3 Matrix-assisted autologous 4Cherubino et al., 2003. J. Orthop. Surg. [Link]

chondrocyte transplantation (MACT) is the second-generation org/10.1177/230949900301100104.

5Benya et al., 1982. Cell. [Link]

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Chapter Exercises Chapter Exercise Answers
1. You and your team are designing a material that can offer 1. Part 1:
three different degradation profiles both in vitro and in (a) 25% PCL, 75% 50:50 PLGA
vivo. The tissue engineering scaffolds will be composed of (b) 50% PCL, 50% 50:50 PLGA
blends of PLGA and PCL and will be porous. (c) 75% PCL, 25% 50:50 PLGA

Given the following scaffold formulations, which would you The greater the content of PCL, the slower the degradation
recommend for (a) the fastest degradation profile, (b) interme- profile as PCL is more hydrophobic and highly crystalline.
diate degradation, and (c) slowest degradation, and why?
• 25% PCL, 75% 50:50 PLGA Part 2:
• 50% PCL, 50% 50:50 PLGA (a) 50% PCL, 50% 25:75 PLGA
(b) 50% PCL, 50% 50:50 PLGA
• 75% PCL, 25% 50:50 PLGA
(c) 50% PCL, 50% 75:25 PLGA
Similarly, for the following formulations, which would you
The greater the content of PLA, compared to PLGA, the
recommend for (a) the fastest degradation profile, (b) interme-
slower the degradation rate given the increased hydropho-
diate degradation, and (c) slowest degradation, and why?
bicity and crystallinity of PLA.
• 50% PCL, 50% 75:25 PLGA   
• 50% PCL, 50% 25:75 PLGA 2. Extrusion based printing would be recommended as that
• 50% PCL, 50% 50:50 PLGA system for 3D printing is compatible with many biologi-
   cal polymers, as well as crosslinking systems that do not
2. A company wants to 3D print mesenchymal stem cells require exposure to high temperatures and harsh initia-
into a commercial tissue engineering scaffold system. tors.
Which 3D printing method would you recommend for 3. A variety of cell types could be chosen. Induced pluripo-
them to use and why? tent stem cells (iPSCs) would be a good choice and a
3. You are designing a tissue engineering scaffold for pancreatic hydrogel would be the best material for the intended
islet cell regeneration. Which cell type would you choose for application given the material mechanical properties
your scaffold and why? For the tissue engineering scaffold, being the closest to native pancreatic tissue.
would you choose (a) a hydrogel, (b) a calcium phosphate 4. The best seeding method for the situation would be
cement, or (c) an electrospun PCL mesh? Why? perfusion-based seeding of the Ewing Sarcoma cells. The
4. You have an electrospun tissue engineering scaffold that you system would also necessitate bioreactor culture in order
intend to use as a cancer model for Ewing Sarcoma to study to better recreate the in vivo environment when deter-
the efficacy of chemotherapeutics in vitro. Which cell seed- mining chemotherapeutic efficacy.
ing method would you use to seed your scaffold? Why? 5. 1H-NMR can be used to characterize the polymer chem-
5. You have synthesized a new polyaddition polymer and need ical structure and either APC/GPC can be used to char-
to characterize the chemical structure and molecular weight. acterize the molecular weight.
Which characterization techniques would you use? 6. Gas foaming would be a very good option as CO2 is read-
6. You and your team want to create a highly porous PCL ily soluble in PCL and gas foaming does not require heat-
scaffold with both nano- and microporosity, encapsulating ing the mixture to high temperatures that would denature
growth factors that cannot be dissolved in an organic sol- or disrupt the bioactivity of any growth factors incorpo-
vent. Which scaffold preparation method should you and rated into the system.
your team choose and why?   
  

1334.e1