Fisher John P. e.a. (ed.) Tissue Engineering

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7-8 Tissue Engineering

TABLE 7.6 Cytokines and Their Effects

Cytokine Effect

IL-1, TNF-α, INF-γ , IL-6 Mediate natural immunity

IL-1, TNF-α, IL-6 Initiate nonspeciﬁc inﬂammatory responses

IL-2, IL-4, IL-5, IL-12, IL-15, and TGF-β Regulate lymphocyte growth, activation, and differentiation

IL-2 and IL-4 Promote lymphocyte growth and differentiation

IL-10 and TGF-β Down-regulate immune responses

IL-1, INF-γ , TNF-α, and MIF Activate inﬂammatory cells

IL-8 Produced by activated macrophages and endothelial cells

Chemoattractant for neutrophils

MCP-1, MIP-α, and RANTES Chemoattractant for monocytes and lymphocytes

GM-CSF and G-CSF Stimulate hematopoiesis

IL-4 and IL-13 Promote macrophage fusion and foreign body giant cell formation

TABLE 7.7 Mechanisms of Immune Mediated Responses

Type Immune mechanism

TYPE I Anaphylactic IgE antibodies, produced by B cells, affect the immediate release of

basophilic amines and other mediators from basophils and mast cells

followed by the recruitment of other inﬂammatory cells

TYPE II Cytotoxic Formation and binding of IgG and IgM to antigens on target cell surfaces that facilitate

phagocytosis of the target cell or lysis of the target cell by activated complement components

TYPE III Immune complex Circulating antigen–antibody complexes activate complement

whose components are chemotactic for neutrophils that release enzymes

and other toxic mediators leading to cellular and tissue injury

TYPE IV Cell-mediated Sensitized T lymphocytes release cytokines and other mediators that lead to

(delayed) cellular and tissue injury

is the paracrine effect and an example is when IL-7 produced by bone marrow stromal cells promotes

the differentiation of B-cell progenitors in the bone marrow. The third way is when the cytokine affects

many cells systemically. This is the endocrine effect and an example is when circulating IL-1 and TNF-α

produce the acute-phase response during inﬂammation.

This brief and very limited overview of the humoral and cellular mediated immune responses due

to space limitations, now provides a basis for understanding mechanisms of immunologic tissue injury,

which is also called hypersensitivity reactions. Hypersensitivity reactions can be initiated either by the

interaction of antigen with humoral antibody or by cell-mediated immune mechanisms. Table 7.7 lists the

four types of hypersensitivity reactions together with a very brief description of their immune mechanism.

Type I hypersensitivity (anaphylactic) is generally deﬁned as a rapidly developing immunologic reaction

occurring within minutes after the combination of an antigen with antibody bound to mast cells or baso-

phils in individuals previously sensitized to the antigen. Type II hypersensitivity (cytotoxic) is mediated by

antibodies directed toward antigens present on the surface of cells or other tissue components. Three dif-

ferent antibody-dependent mechanisms may be involved in this type of reaction: complement-dependent

reactions, antibody-dependent cell-mediated cytotoxicity, or antibody-mediated cellular dysfunction.

Type III hypersensitivity (immune complex mediated) reaction is induced by antigen-antibody com-

plexes that produce tissue damage as a result of their capacity to activate the complement system. Type IV

hypersensitivity (cell-mediated) reactions are initiated by speciﬁcally sensitized T lymphocytes. This reac-

tion includes the classic delayed-type hypersensitivity reaction initiated by CD

+ T cells and direct cell

cytotoxicity mediated by CD

+T cells. Immunologic reactions that occur with organ transplant rejection

also offer insight into potential immune responses to tissue-engineered devices. Mechanisms involved

in organ transplant rejection include T cell-mediated reactions by direct and indirect pathways and

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Inﬂammatory and Immune Responses 7-9

antibody-mediated reactions. Immune responses may be avoided or diminished by using autologous or

isogeneic cells in cell/polymer scaffold constructs. The use of allogeneic or xenogenic cells incorporated

into the device require prevention of immune rejection by immune suppression of the host, induction

of tolerance in the host, or immunomodulation of the tissue-engineered construct. The development of

tissue-engineered constructs by immunoisolation using polymer membranes and the use of nonhost cells

have been compromised by immune responses. In this concept, a polymer membrane is used to encapsu-

late nonhost cells or tissues thus separating them from the host immune system. However, antigens shed

by encapsulated cells were released from the device and initiated immune responses [12,16,17].

Although exceptionally minimal and superﬁcial in its presentation, the previously discussed humoral

and cell-mediated immune responses demonstrate the possibility that any known tissue-engineered con-

struct may undergo immunologic tissue injury. To date, our understanding of immune mechanisms and

their interactions with tissue-engineered constructs is markedly limited. One of the obvious problems is

that preliminary studies are generally carried out with nonhuman tissues and immune reactions result

when tissue-engineered constructs from one species are used in testing the device in another species.

Ideally, tissue-engineered constructs would be prepared from cells and tissues of a given species and

subsequently tested in that species. Whereas this approach does not guarantee that immune responses will

not be present, the probability of immune responses in this type of situation is markedly decreased.

The following examples provide perspective to these issues. They further demonstrate the detailed

and in-depth approach that must be taken to appropriately and adequately evaluate tissue-engineered

constructs or devices and their potential adverse responses.

Babensee et al. [18,19] have tested the hypothesis that the biomaterial component of a medical device,

by promoting an inﬂammatory response can recruit antigen-presenting cells (APCs, e.g., macrophages

and dendritic cells) and induce their activation, thus acting as an adjuvant in the immune response to

foreign antigens originating from the histological component of the device. Utilizing polystyrene and

polylactic-glycolic acid microparticles and polylatic-glycolic scaffolds together with their model antigen,

ovalbumin, in a mouse model for 18 weeks, Babensee et al. demonstrated that a persistent humoral

immune response that was Th2 helper T cell dependent, as determined by the IgG1, was present. These

ﬁndings indicated that activation of CD

+T cells and the proliferation and isotype switching of B-cells had

occurred. A Th1 immune response, characterized by the presence of IgG2a was not identiﬁed. Moreover,

the humoral immune responses for all three types of microparticles were similar indicating that the

production of antigen-speciﬁc antibodies was not material chemistry-dependent in this model. Babensee

suggests that the presence of the biomaterial functions as an adjuvant for initiation and promotion of the

immune response and augments the phagocytosis of the antigen with expression of MHC class II and

co-stimulatory molecules on APCs with the presentation of antigen to CD

+T cells.

Cleland et al. [20] have shown the signiﬁcance of the use of appropriate animal models in character-

izing protein/biodegradable polymer constructs. Utilizing biodegradable PLGA microspheres containing

recombinant human growth hormone (rhGH), they used Rhesus monkeys, transgenic mice expressing

hGH and normal control (Balb/C) mice in their in vivo studies. The Rhesus monkeys were used for serum

assays in determining growth hormone release as well as tissue responses to the injected microcapsule

formulations. Placebo injection sites were also used and a comparison of the injection sites from rhGH

PLGA microspheres and placebo PLGA microspheres demonstrated a normal inﬂammatory and wound

healing response with a normal focal foreign body reaction. To further examine the tissue response and

potential for immune reactions, transgenic mice were used to assess the immunogenicity of the rhGH

PLGA formulation. Transgenic mice expressing a heterologous protein have been previously used for

assessing the immunogenicity of sequence or structural mutant proteins [21]. With the transgenic anim-

als, no detectable antibody response to the rhGH was found. In contrast, the Balb/C control mice had a

rapid onset of high titer antibody response to the rhGH PLGA formulation.

Immunotoxicity is any adverse effect on the function or structure of the immune system or other

systems as a result of an immune system dysfunction. Adverse or immunotoxic effects occur when

humoral or cellular immunity needed by the host to defend itself against infections or neoplastic

diseases (immunosuppression) or unnecessary tissue damage (chronic inﬂammation, hypersensitivity

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7-10 Tissue Engineering

or autoimmunity) is compromised. In vivo responses indicating immunotoxicity include histopathological

changes, humoral responses, host resistance, clinical symptoms, and cellular responses (T cells, natural

killer cells, macrophages, and granulocytes). The inﬂammatory response considered to be immunotoxic

is persistent chronic inﬂammation. It is this persistent chronic inﬂammation that is of concern as immune

granuloma formation and other serious immunological reactions such as autoimmune disease may occur.

In biological response evaluation, it is important to discriminate between the short-lived chronic inﬂam-

mation that is a component of the normal inﬂammatory and healing responses vs. long-term, persistent

chronic inﬂammation that may indicate an adverse immunological response.

Immunosuppression may occur when antibody and T cell responses (adaptive immune response)

are inhibited. A potentially signiﬁcant consequence of this type of response is more frequent in seri-

ous infections resulting from reduced host defense. Immunostimulation may occur when unintended

or inappropriate antigen-speciﬁc or nonspeciﬁc activation of the immune system is present. From a

tissue-engineering perspective, antibody and cellular immune responses to a foreign protein may lead to

unintended immunogenicity. Enhancement of the immune response to an antigen by a biomaterial with

which it is mixed ex vivo or in situ may lead to adjuvancy that is a form of immunostimulation. This effect

must be considered when biodegradable controlled release systems are designed and developed for use as

vaccines. Autoimmunity is the immune response to the body’s own constituents that are considered in

this response to be autoantigens. An autoimmune response, indicated by the presence of autoantibodies

or T lymphocytes that are reactive with host tissue or cellular antigens may, but not necessarily, result in

autoimmune disease with chronic, debilitating and sometimes life-threatening tissue and organ injury.

Direct measures of immune system activity by functional assays are the most important types of tests for

immunotoxicity [22–25]. Functional assays are generally more important than tests for soluble mediators,

which are more important than phenotyping. Functional assays include skin testing, immunoassays

(e.g., ELISA), lymphocyte proliferation, plaque-forming cells, local lymph node assay, mixed lymphocyte

reaction, tumor cytotoxicity, antigen presentation, and phagocytosis. As with any type of test for biological

response evaluation, immunotoxicity tests should be valid and have been shown to provide accurate,

reproducible results that are indicative of the effect being studied and are useful in a statistical analysis.

This implies that appropriate control groups are also included in the study design.

References

[1] Anderson, J.M., Biological responses to materials, Ann. Rev. Mater. Res., 31, 81, 2001.

[2] Anderson, J.M., Mechanisms of inﬂammation and infection with implanted devices, Cardiovasc.

Pathol., 2, 33S, 1993.

[3] Cotran, R.Z., Kumar, V., and Robbins, S.L., Eds., Inﬂammation and repair in Pathologic Basis of

Disease, 6th ed., W.B. Saunders, Philadelphia, 1999, p. 50.

[4] Gallin, J.L. and Snyderman, R., Eds., Inﬂammation: Basic Principles and Clinical Correlates, 3rd ed.,

Raven Press, New York, 1999.

[5] Babensee, J.E. et al., Host responses to tissue engineered devices, Adv. Drug Del. Rev., 33, 111, 1998.

[6] Anderson, J.M., Multinucleated giant cells, Curr. Opin. Hematol., 7, 40, 2000.

[7] Henson, P.M., The immunologic release of constituents from neutrophil leukocytes: II. Mechanisms

of release during phagocytosis, and adherence to nonphagocytosable surfaces, J. Immunol., 107,

1547, 1971.

[8] McNally, A.K. and Anderson, J.M., Beta1 and beta2 integrins mediate adhesion during macrophage

fusion and multinucleated foreign body giant cell formation, Am. J. Pathol., 160, 621, 2002.

[9] McNally, A. and Anderson, J.M., Interleukin-4 induces foreign body giant cells from human

monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to

morphological variants of multinucleated giant cells, Am. J. Pathol., 147, 1487, 1995.

[10] Brodbeck, W.G. et al., Inﬂuence of biomaterials surface chemistry on apoptosis of adherent cells,

J. Biomed. Mater. Res., 55, 661, 2001.

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Inﬂammatory and Immune Responses 7-11

[11] Janeway, C.A. et al., Immunobiology 5: The ImmuneSystem in Health and Disease, Garland Publishing

Inc., New York, 2001, p. 1.

[12] Babensee, J.E. et al., Host response to tissue engineered devices, in Advanced Drug Delivery Reviews,

Vol. 33, Elsevier, Amsterdam, 1998, p. 111.

[13] Lefell, M.S., Donnenberg, A.D., and Rose, N.R., Eds., Immunologic Diagnosis of Autoimmune

Disease, CRC Press, Boca Raton, FL, 1997, p. 111.

[14] Cohen, I.R. and Miller, A., Eds., Autoimmune Disease Models, A Guidebook, Academic Press,

New York, 1994.

[15] Anderson, J.M. and Langone, J.J., Issues and perspectives on the biocompatibility and immun-

otoxicity evaluation of implanted controlled release systems, J. Control. Release, 57, 107,

1999.

[16] Brauker, J.H. et al., Neovascularization of synthetic membranes directed by membrane microarchi-

tecture, J. Biomed. Mater. Res., 29, 1517, 1995.

[17] Brauker, J. et al., Neovascularization of immuno-isolation membranes: the effect of membrane

architecture and encapsulated tissue, Cell Transplant., 1, 163, 1992.

[18] Matzelle, M.M. and Babensee, J.E., Humoral immune responses to model antigen co-delivered with

biomaterials used in tissue engineering, Biomaterials, 25, 295, 2004.

[19] Babensee, J.E., Stein, M.M., and Moore, L.K., Interconnections between inﬂammatory and immune

responses in tissue engineering, Ann. NY Acad. Sci., 961, 360, 2002.

[20] Cleland, J.L. et al., Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA)

microspheres provide a long lasting effect, J. Control. Release, 49, 193, 1997.

[21] Stewart, T.A. et al., Transgenic mice as a model to test the immunogenicity of proteins altered by

site-speciﬁc mutagenesis, Mol. Biol. Med., 6, 275, 1989.

[22] Rose, N.R., de Mecario, E.C., Folds, J.D., Lane, H.C., and Nakamura, R.M., Eds., Manual of Clinical

Laboratory Immunology, ASM Press, Washington, DC, 1997.

[23] Smialowicz, R.J. and Holsapple, M.P., Eds., Experimental Immunotoxicology, CRC Press, Boca

Raton, FL, 1996.

[24] Burleson, G.R., Dean, J.H., and Munson, A.E., Eds., Methods in Immunotoxicology, Wiley-Liss,

New York, 1995.

[25] Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, R., Eds., Current

Protocols in Immunology, Greene Publishing Associates and Wiley Interscience, New York, 1992.

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Polymeric Scaffolds

for Tissue Engineering

Applications

Diana M.Yoon

John P. Fisher

University of Maryland

8.1 Introduction.............................................. 8-1

8.2 Natural Polymers for Scaffold Fabrication ............. 8-1

Polysaccharides • Polypeptides

8.3 Synthetic Polymers for Scaffold Fabrication ........... 8-5

Polyesters • Other Synthetic Polymers

8.4 Scaffold Design Properties .............................. 8-10

Fabrication • Micro-Structure • Macro-Structure •

Biocompatibility • Biodegradability • Mechanical Strength

8.5 Summary ................................................. 8-12

References ....................................................... 8-12

8.1 Introduction

The incorporation of polymeric scaffolds in tissue regeneration occurred in the early 1980s, and it con-

tinues to play a vital role in tissue engineering [1–3]. The function of a degradable scaffold is to act as

a temporary support matrix for transplanted or host cells so as to restore, maintain, or improve tissue.

Scaffolds may be created from various types of materials, including polymers. There are two main classes

of polymers, based upon their source: natural or synthetic. Polymeric scaffolds may be used to support a

variety of cells for numerous tissues within the body. The design of a polymeric scaffold plays a signiﬁc-

ant role in proper cell growth. Therefore, several important properties must be considered: fabrication,

structure, biocompatibility, biodegradability, and mechanical strength. This review will discuss natural

and synthetic polymers, as well as the properties that scaffolds exhibit.

8.2 Natural Polymers for Scaffold Fabrication

There are two major classes of natural polymers used as scaffolds: polypeptides and polysaccharides

(Table 8.1, Figure 8.1). Natural polymers are typically biocompatible and enzymatically biodegradable.

The main advantage for using natural polymers is that they contain bio-functional molecules that aid

the attachment, proliferation, and differentiation of cells. However, disadvantages of natural polymers

do exist. Depending upon the application, the previously mentioned enzymatic degradation may inhibit

8-1

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8-2 Tissue Engineering

TABLE 8.1 Properties of Degradable Natural Homopolymers That Have Been Utilized in the Fabrication of Tissue

Engineering Scaffolds

Natural Curing Primary degradation Primary degradation

polymer method method products References

A Agarose Entanglement Enzymatic agarases Oligosaccharides [4,8]

B Alginate Cross-linking Alginate lyases Mannuronic acid and guluronic

acid

[4,10–13]

C Hyaluronic acid Entanglement Enzymatic

hyaluronidase

β(1–4) linked glucuronic acid

and glucosamine

[4,8,21,22]

D Chitosan Cross-linking Enzymatic chitosanase Glucosamines [4,8,22,25,26]

E Collagen Cross-linking Enzymatic collagenase

or lysozyme

Various peptides depending on

sequence

[8,31–33]

F Gelatin Entanglement Enzymatic

collagenases

Various peptides depending on

sequence

[4,8,13,22]

G Silk Entanglement Proteolytic proteases Various peptides depending on

sequence

[43]

function. Further, the rate of this degradation may not be easily controlled. Since the enzymatic activity

varies between hosts, so will the degradation rate. Therefore it may be difﬁcult to determine the lifespan

of natural polymers in vivo. Additionally, natural polymers are often weak in terms of mechanical strength

but cross-linking these polymers have shown to enhance their structural stability.

8.2.1 Polysaccharides

8.2.1.1 Agarose

Agarose is a polysaccharide polymer extracted from algae (Table 8.1, Figure 8.1). Its molecular struc-

ture contains an alternating copolymer linkage of 1,4-linked 3,6 anhydro-α-l-galactose and 1,3-linked

β-d-galactose [4]. Agarose is water-soluble due to the presence of hydroxyl groups. Two agarose chains

interact with each other through hydrogen bonding to form a double helix. Atlow temperatures the agarose

chains form a thermally reversible gel. Thus, at high temperatures agarose gels may be made water-

soluble. Many of the properties of agarose gels, particularly strength and permeability, can be adjusted

by altering the concentration of agarose [4]. For example, low concentrations of agarose lead to a highly

porous entangled structure with limited mechanical strength. Agarose in its native state is enzymatically

degradable by agarases.

For tissue culture systems, agarose hydrogels are widely investigated because they permit growth of cells

and tissues in a three-dimensional suspension [5]. Agarose gels have been found to maintain chondrocytes,

the predominant cell type in cartilage, in culture for 2 to 6 weeks [6]. Furthermore, agarose hydrogels

embedded with chondrocytes allow the expression of type II collagen and proteoglycans [5,7]. These results

show promise for using agarose gels in cartilage repair. Additionally, agarose gels have been investigated

as a matrix for nerve regeneration [8,9]. Dorsal root ganglia (DRG), a neural cell, has been encapsulated

in agarose hydrogels. The inﬂuence of increasing porosity and incorporating charged biopolymers (e.g.,

chitosan) produced an increase in neurite extension of DRG isolated from chicks [9].

8.2.1.2 Alginate

Alginate is a naturally derived water-soluble polysaccharide obtained from algae (Table 8.1, Figure 8.1)

[4]. It is a polyanion composed of two repeating monomer units: β-d-mannuronate and α-l-guluronate

[10,11]. Alginate is readily degraded at the β(1–4) linkage site, located in between the monomer units,

by alginate lyases [10]. The physical and mechanical properties of alginate are dependent on the length

and proportion of the guluronate block within the chain. Due to alginate’s polyelectrolytic nature, it

readily forms into an ionotropic cross-linked hydrogel when exposed to divalent ions, the most common

being calcium ions [12,13]. Furthermore, alginate gels may be solubilized when these cations are removed

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Polymeric Scaffolds 8-3

COO

–

COO

–

COO

–

COO

–

COOH

HN C

NNN

OH CH

NHCOCH

(b)

(a)

(c)

(f)

(e)

(g)

(d)

Agarose

Alginate

Hyaluronic acid

Chitosan

Collagen*

Gelatin*

Silk**

FIGURE 8.1 Repeating structural unit of degradable natural homopolymers under investigation as scaffold materials

for tissue engineering applications. (*Collagen and gelatin have variable sequences, but are predominately glycine,

proline, and hydroxyproline. **Silks have a glycine rich sequence, with glycine repeating every second or third amino

acid residue. The variable residues are primarily alanine or serine.)

or sequestered. This ease in gel reversibility allows alginate to be especially useful for studies requiring

the retrieval of encapsulated cells. Alginate does not contain cellular recognition proteins, limiting cell

attachment to the natural polymer. By cross-linking alginate with poly(ethylene glycol)-diamine the

mechanical properties of alginate can be controlled and even improved [4].

A variety of different cells have been found to maintain their morphology in alginate scaffolds

[10,14–17]. The adhesion of ﬁbroblasts in alginate sponges was found not to be affected by the porosity

of the scaffold [14]. Chondrocytes embedded in alginate proliferated and expressed type II collagen [18].

Hepatocytes, the functional cell in liver, were also found to grow in alginate scaffolds. When hepatocytes

were seeded in three-dimensional porous alginate, albumin was secreted, indicating proper cell function

[19]. Other types of cells studied in alginate include cardiomyocytes, rat marrow cells, and Schwann

cells [10,17,20].

8.2.1.3 Hyaluronic Acid

Hyaluronic acid (hyaluronan, HA) is a naturally occurring polysaccharide with a β(1–3) linkage of two

sugar units: d-glucuronate and N-acetyl-d-glucosamine (Table 8.1, Figure 8.1). Hyaluronic acid is an

abundant glycosaminoglycan within the extracellular matrix of tissues that promotes early inﬂammation

critical for wound healing [21]. Furthermore, hyaluronic acid is nonimmunogenic and nonadhesive,

making it an attractive option for biomedical applications. In dilute concentrations hyaluronic acid has a

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8-4 Tissue Engineering

random coil structure that becomes entangled as its concentration increases [22]. Some disadvantages of

hyaluronicacid are that it is highly water-solubleand it degrades rapidly by enzymes, such as hyaluronidase.

However, when fabricated into a hydrogel, hyaluronic acid is often more stable [4,8,21].

Currently, an ester derivative of hyaluronic acid has been used as a tissue engineering scaffold [8,23,24].

Adipose precursor cells proliferated and differentiated in HA sponges [23,24]. Additionally, HA has been

used for osteochondral repair by incorporating mesenchymal progenitor cells, which differentiate into

osteoblasts and chondrocytes [8]. Furthermore, the interaction of chondrocytes embedded in HA has

resulted in the expression of collagen type II, forming a tissue similar to native cartilage [23].

8.2.1.4 Chitosan

Chitosan is a partially deacetylated derivative of chitin, found in the exoskeleton of crustaceans and insects

(Table 8.1, Figure 8.1). It is a linear polysaccharide composed of β(1–4) linked d-glucosamine with

randomly dispersed N-acteyl-d-glycosamine groups [4,22,25,26]. Less than 40% of chitosan is composed

of the N-acteyl-d-glycosamino group, which makes its molecular structure similar to glycosaminoglycans

[4,8]. Chitosan is easily degraded by enzymes such as chitosanase and lysozyme. Chitosan is catonic

with semi-crystalline properties of a high charge density. These attributes allow chitosan to be insoluble

above pH 7 and fully soluble below pH 5. At high pH solutions, chitosan can be gelled into strong ﬁbers

[26]. Furthermore, hydrogels can also be formed by either ionic bonding or covalent cross-linking, using

cross-linking agents such as glutaraldehyde [4].

Chitosan is a well-deﬁned matrix and as a porous scaffold, hepatocytes were found to maintain a

rounded morphology when cultured within chitosan scaffolds [27]. Results showed an increase in albumin

secretion as well as urea synthesis, vital metabolic activities of liver cells [27]. Additionally, cross-linking

chitosan gels with glutaraldehyde showed increased urea formation by hepatocytes [28]. Furthermore,

chitosan has shown promise as an orthopaedic scaffold. When a sponge form of chitosan was seeded

with rat osteoblasts, alkaline phosphatase production was detected, as well as bone spicules, indicating

initial bone formation [29]. Also, chitosan scaffolds were found to support chondrocyte attachment and

expression of extracellular matrix proteins [30].

8.2.2 Polypeptides

8.2.2.1 Collagen

Collagen is a major component of structural mammalian tissues (Table 8.1, Figure 8.1). Currently, 19

different types of collagen have been found, with the most abundant being type I collagen [31]. Collagen

is composed of three polypeptide chains that intertwine with one another to form a triple helix. The

polypeptide chains have a repeating sequence of glycine-X–Y, where X and Y are most often found to

be proline and hydroxyproline. The chains are held together through hydrogen bonding of the peptide

bond in glycine and an adjacent peptide carbonyl group [32]. The presence of adhesion domains allows

collagen to display an attractive surface for cell attachment [31]. Some drawbacks in collagen are its high

variability due to the numerous forms. Collagen is enzymatically biodegradable, and has a tendency to

degrade quickly, limiting its mechanical properties. Cross-linking collagen with glutaraldehyde decreases

its degradation rate [33].

Collagen naturally promotes healing wounds by promoting blood coagulation [31]. These attributes

allow collagen to be used as a scaffold for cells within the epithelium, such as ﬁbroblasts and keratino-

cytes [34–36]. Type II collagen is suitable for attaching chondrocytes and aiding in their proliferation and

differentiation [37]. Additionally, rat hepatocytes attached onto collagen–chitosan scaffolds secreted albu-

min, which conﬁrms cell function [38]. Furthermore, corneal keratocytes cultured on collagen sponges

synthesized proteoglycans, indicating corneal extracellular matrix formation [39].

8.2.2.2 Gelatin

Gelatin is a collagen derivative acquired by denaturing the triple-helix structure of collagen into

single-strand molecules (Table 8.1, Figure 8.1). It is water-soluble, and entangles easily to form into a

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Polymeric Scaffolds 8-5

gel through changes in temperature [4,13]. Gelatin is broken down enzymatically by various collagenases.

The primary form of a gelatin scaffold is a hydrogel. Stabilization of gelatin hydrogels occurs through

chemical cross-linking with agents such as glutaraldehyde [22].

Gelatin has been used to support cells for orthopaedic applications. Rat marrow stromal osteoblasts

encapsulated in gelatin microparticles retained their phenotype [40]. Also, porous gelatin scaffolds sup-

ported human adipose derived stem cell attachment and differentiation into a variety of cell lineages.

These constructs expressed a chondrogenic phenotype, indicated by the expression of hydroxyproline and

glycosaminoglycans, both of which are present in the extracellular matrix of cartilage [41]. Furthermore,

nonorthopaedic cell types, such as respiratory epithelial cells and cardiocyocytes, have been shown to

attach and form rounded morphologies in gelatin scaffolds [17,42].

8.2.2.3 Silk

Silks are ﬁbers that have a protein polymer basis (Table 8.1, Figure 8.1) [43]. There are two main sources

of silks: spiders and silkworms. The primary structure of silks is a repetitive sequence of proteins that

are glycine-rich. The secondary structure of silks is a β-sheet, which allows silks to exhibit enormous

mechanical strength and is the primary reason for its strong interest as a scaffold. Most types of silks

undergo slow proteolytic degradation by proteases, such as chymotrypsin [43]. A drawback for using

silks is the potential formation of a granuloma as well as an allergic response. There has been a positive

attachment and growth response of ﬁbroblasts on ﬁbroin silks. And recently, osteoblasts seeded on silks

have displayed bone growth characteristics [44,45].

8.3 Synthetic Polymers for Scaffold Fabrication

Polymers that are chemically synthesized offer several notable advantages over natural-origin polymers.

A major advantage of synthetic polymers is that they can be tailored to suit speciﬁc functions and thus

exhibit controllable properties. Furthermore, since many synthetic polymers undergo hydrolytic degrad-

ation, a scaffold’s degradation rate should not vary signiﬁcantly between hosts. The most common

synthetic polymers are polyesters. Other types include polyanhydrides, polycarbonates, and polyphos-

phazenes (Table 8.2, Figure 8.2). A signiﬁcant disadvantage for using synthetic polymers is that some

degrade into unfavorable products, often acids. At high concentrations of these degradation products,

local acidity may increase, resulting in adverse responses such as inﬂammation or ﬁbrous encapsulation

[8,46,47].

8.3.1 Polyesters

8.3.1.1 Poly(glycolic) Acid

Poly(glycolic) acid (PGA) is a linear aliphatic polyester in the family of poly(α-hydroxy esters) (Table 8.2,

Figure 8.2). It is formed by ring-opening polymerization of cyclic diesters of glycolide [46]. Due to

the crystallinity of glycolide, PGA is a highly crystalline polymer that has a high melting point (185 to

225

◦

C) and low solubility in organic solvents [47,48]. Further, PGA is hydrophilic and undergoes bulk

degradation, often leading to a sudden loss in mechanical strength. PGA degrades hydrolytically into

glycolic acid, which is metabolized in the body. An unfortunate attribute of glycolic acid is that at high

concentrations it lowers the pH of the surrounding tissue, causing inﬂammation and possible tissue

damage [46].

Poly(glycolic) acid polymers have been investigated as a scaffold to support various types of cell growth.

Bovine chondrocytes seeded on PGA scaffolds in vitro expressed sulfated glycosaminoglycans 25% more

than native cartilage [49]. In vivo results using a mouse model were comparable to these in vitro results.

Other studies seeded myoﬁbroblasts and endothelial cells on a PGA ﬁber matrix. These cells persisted

and expressed elastic ﬁlaments and collagen, demonstrating the potential use of PGA scaffolds for heart

valve engineering [50]. Hepatocytes have also attached to PGA and initiated albumin synthesis [51]. Some