Fisher John P. e.a. (ed.) Tissue Engineering
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22
Cartilage Tissue
Engineering
Fan Yang
Jennifer H. Elisseeff
Johns Hopkins University
22.1 Introduction.............................................. 22-1
Prospects for Tissue Engineering
22.2 Cells ....................................................... 22-2
Chondrocytes • Mesenchymal Stem Cells • Other Cell
Sources
22.3 Scaffolds .................................................. 22-4
Natural Polymers • Synthetic Polymers
22.4 Mechanical Stimuli ...................................... 22-6
22.5 Growth Factors........................................... 22-7
22.6 Zonal Organization ...................................... 22-7
22.7 Conclusion ............................................... 22-8
References ....................................................... 22-8
22.1 Introduction
This chapter provides a review of important developments in the field of cartilage tissue engineering, with
an emphasis on articular cartilage. Clinical significance of cartilage repair is discussed and the necessity of
using tissue engineering approach to solve this problem is presented. Structure, function, and biochemical
componentsof cartilage are described. This chapter will compareand contrast the various options available
for cartilage tissue engineering. Four major components of tissue engineering in relation to cartilage are
discussed including cells, scaffolds, growth factors, and mechanical stimuli.
Cartilage is a highly specialized connective tissue consisting of dispersed chondrocytes embedded in
a rich extracellular matrix [1]. The matrix, primarily composed of proteoglycans, collagen, and water,
is directly responsible for the unique functional properties of cartilage and provides shape, resilience, and
resistance against compression and shear [2,3]. Despite the relatively simple structure, cartilage has little
capacity for repair and regeneration due to many factors such as lack of vascularity and progenitor cells,
a sparse and highly differentiated cell population, and a slow matrix turnover [4–6].
There are three types of cartilage: hyaline cartilage, fibrocartilage, and elastic cartilage. Each type of
cartilage has different functional properties and hence different biochemical contents. Hyaline cartilage is
rich in Type II collagen and proteoglycans and is found mainly on the articular surface of joints where it
serves as a shock absorber. Elastic cartilage consists of elastic fibers, not exclusively collagen. The elastic
fibers give this type of cartilage the ability to be deformed and return to shape. Examples of elastic cartilage
22-1
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22-2 Tissue Engineering
include external ear, epiglottis, and upper portion of larynx. Fibrocartilage is rich in Type I collagen and
is found in tissues that are subject to tensile forces such as the intervertebral disk.
More than 1 million surgical procedures in the United States involve cartilage replacement. Conven-
tional therapies include drilling and debriding, autologous cartilage transplantation, and implantation of
artificial polymers or metal prostheses [7]. Drilling and debriding may provide immediate relief of symp-
toms but usually result in the production of a fibrocartilaginous tissue with poor long-term outcome.
Transplantation can be an effective treatment for patients with small cartilage defects, but its application
is limited due to insufficient donor tissue supply, donor site morbidity, and technical difficulties to shape
host cartilage into desired, delicate three-dimensional shapes. Artificial prostheses can be a source of infec-
tion, unwanted protein deposition, and immune responses without being able to adapt to environmental
stresses.
22.1.1 Prospects for Tissue Engineering
Given the clinical significance of cartilage injury and the poor outcomes of the conventional therapies out-
lined above, investigators have been motivated to seek other approaches for improving cartilage treatment.
One possible solution is to create new cartilage using tissue engineering strategies. Tissue engineering is
an interdisciplinary area that combines biology, material sciences, and clinical medicine to regenerate
or restore tissue structure and function that is lost due to trauma, disease, or congenital abnormalities.
Cartilage contains only one cell type and has no blood supply. This relatively simple cartilage structure
has allowed significant progress in the area of cartilage tissue engineering [8,9].
22.2 Cells
Cells are the building blocks of tissues and the fundamental source of extracellular matrix that forms
tissues. Identification and creation of a suitable cell population is the first critical step toward cartilage
tissue engineering and often requires a large number of cells. Numerous cell types have been explored
including chondrocytes [10,11], bone-marrow derived mesenchymal stem cells (MSCs) [12,13], stem
cells isolated from adipose tissue [14], from muscle [15], and embryonic stem cells [16,17]. All these
cell types have been shown to exhibit a chondrogenic potential under appropriate culture conditions.
When choosing the optimal cell type, it is important to consider the cell proliferative capacity, phenotype
stability, and immunogenicity.
22.2.1 Chondrocytes
Chondrocytes, the major cell type that is present in differentiated cartilage, is the most obvious cell option
for cartilage tissue engineering. Thus far, autologous chondrocyte transplantation (ACT) is the only Food
and Drug Administration (FDA) approved cartilage repair product in the United States. This procedure
uses in vitro expanded autologous chondrocytes, harvested from a biopsy, combined with a periosteal
membrane to repair a cartilage defect (Figure 22.1).
The ACT was first used in humans in 1987 and results of the first pilot study was published in 1994
[10]. Since then more than 950 patients have been treated with this technique with 2 to 10 years follow-
up [11]. Histology of the repair tissue shows that this technique gives stable long-term results with a
higher success rate in femoral condyle (84 to 90%) than in other locations (average 74%). Despite the
encouraging results that ACT has shown, several problems remain with the approach. First, the age
or disease state of the patient may limit the availability and function of desired chondrocytes. In vitro
expansion is also limited due to replicative senescence that chondrocytes exhibit. More importantly,
once removed from their extracellular environment and expanded in monolayer, chondrocytes would
rapidly lose their differentiated phenotype, characterized by transforming into a fibroblastic morphology,
decreasing expression of Type II Collagen and aggrecan with an increase in Type I Collagen expression
[18,19]. Studies have shown that chondrocyte phenotype can be retained or reexpressed by suspending
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Cartilage Tissue Engineering 22-3
Lesion
Biopsy of healthy
cartilage
Periosteal flap
taken from
medial tibia
Periosteal flap
sutured over lesion
Injection of
cultured chondrocytes
under flap into lesion
Suspension of
2.6 × 10
6
–5× 10
6
cells
Enzymatic digestion
Cultivation for 11–21 days
(10-fold increase in number of cells
Trypsin treatment
FIGURE 22.1 Flow diagram for ACT articular chondrocyte harvest and implantation. (From Brittberg, M.,
Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., and Peterson, L. N.Engl.J.Med.1994; 331: 889–895. With
permission.)
chondrocytes in a three-dimensional environment such as agarose gel [20], alginate beads [21], and
collagen gel [22]. Optimal combinations of monolayer and three-dimensional cultures for cartilage tissue
engineering are currently being investigated [23]. Challenges remain for patients who do not have healthy
cartilage where autologous cell transplantation cannot be used and alternate cell sources need to be
explored.
22.2.2 Mesenchymal Stem Cells
Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiate into a vari-
ety of connective tissue cells including bone [24], cartilage [25], and adipose tissue [12] both in vitro and
in vivo. MSCs are present in various tissue types during human development and are prevalent in adult
bone marrow. As a readily available source, MSCs can be isolated, expanded in culture, and differentiated
into chondrogenic cells under appropriated tissue conditions. Numerous studies have shown that trans-
forming growth factor-beta (TGF-β) plays an important role in inducing undifferentiated MSCs into
the chondrogenic pathway. In the presence of TGF-β, MSCs will gradually transform from a fibroblastic
morphology to a mature chondrocyte morphology, accompanied by a production of cartilage-specific
extracellular matrix proteins including Type II collagen, glycoaminoglycan, and proteoglycans. Unlike
osteogenesis, which can be induced in monolayer culture, chondrogenesis of MSCs can only be achieved
in micromass pellet culture [25] or high density suspension in a three-dimensional environment [26].
This close cell–cell contact requirement resembles the natural environment of embryonic cartilage devel-
opment, which suggests that MSCs are undergoing a similar pathway. These results indicate that successful
tissue-engineered cartilage repair based on MSCs can be achieved by recapitulating aspects of embryonic
tissue formation. Studies so far demonstrated the great potential of using MSCs for cartilage regeneration
as an alternate resource, which may eliminate the need harvest cartilage from the patient. More in vivo
studies on cartilage regeneration using MSCs will occur as they reach clinical application.
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22-4 Tissue Engineering
22.2.3 Other Cell Sources
The discovery of human embryonic stem cells (hESCs) capable of indefinite replication and the ability
to differentiate into all somatic cells of the body brings optimism that great progress will be made to
cell transplantation therapies [27,28]. Theoretically, hESCs can be used to generate any desired cell or
tissue for the treatment of degenerative diseases. So far, very few studies have been performed inducing
hESCs into chondrogenic lineage [16,17]. Challenges with hESCs include potential of undesired growth,
difficulty to achieve homogeneous differentiation in vivo, and related ethics issues. Other potential cell
sources were found by Mizuno [29], who demonstrated that human dermal fibroblasts seeded onto Type I
collagen sponges containing demineralizd bone matrix can be stimulated to produce a cartilaginous tissue
expressing Type II collagen. Also, Lorenz [30] recently reported that human adipose tissue derived cells
can also be induced into chondrogenic pathway in vitro.
22.3 Scaffolds
Although some approaches to cartilage tissue engineering use cells alone (i.e., ACT), most studies indicate
that the scaffold is helpful for promoting desired cell phenotypes and regeneration of cartilage. Ideally,
the scaffold serves as a structural support for cells at the early stage of tissue development and degrades
into nontoxic products at a rate related to the formation of a functional extracellular matrix. Numer-
ous scaffolds and matrices have been explored for cartilage tissue engineering including both natural
and synthetic polymers. Natural polymers are usually porous, biocompatible, and can degrade without
immunogenicity. Problems related to naturally derived scaffolds include batch-to-batch quality variance
and weak structural properties. By contrast, synthetic polymers provide a more consistent quality and
more control over the material properties such as degradation rate, mechanical properties, and surface
biology. When choosing a scaffold, tissue engineers must decide which genre of materials, or combination,
is more appropriate depending on the specific purpose and location of use.
22.3.1 Natural Polymers
As a major component of cartilage extracellular matrix, collagen was the first natural material explored as
scaffold for cartilage tissue engineering. Collagen can be easily extracted from tissues such as tendon and
can be readily processed into multiple forms including sponges or meshes. Collagen is liquid at 4
◦
C
and can be polymerized by raising the temperature. Further crosslinking after gelation introducesextended
degradation times and greater mechanical strength [31]. Chondrocytes have been shown to maintain
their morphology and phenotype when seeded in collagen gels [32–34]. Grande et al. [35] showed that
collagen sponges stimulate collagen synthesis while bioabsorbable polymer such as polyglycolic acid (PGA)
enhanced proteoglycan synthesis.
Hyaluronan comprises 1 to 10% of the cartilage glycosaminoglycan (GAG) and plays an important role
in many biological processes such as cell attachment, hydration, and matrix synthesis. HYAFF® 11 (Fidia
Advanced Biopolymers, Abano Terme, Italy) is a semisynthetic derivative of Hyaluronan by esterification
of carboxyl groups of the glucuronic acid with benzyl alcohol. This processing greatly improves the
originally poor ductility of hyaluronan, enabling HYAFF® 11 to be processed into multiple forms such as
fibers, membranes, or microspheres. HYAFF and ACP, two hyaluronan-based biomaterials with different
degradation profiles, were tested as chondrogenic delivery vehicles for rabbit MSCs and compared with a
well-characterized porous calcium phosphate ceramic delivery vehicle [36]. Results showed that HYAFF
11 sponges incorporated twice as many cells and produced a 30% increase in the relative amount of bone
and cartilage per unit area.
Agarose and alginate are examples of natural materials that are based on linear polysaccharide polymers
that are extracted from seaweed [37]. These polymers are highly water-soluble due to a large number of
hydroxyl groups. Agarose gels can be easily formed by pouring a warm liquid solution of the polymer
into a mold, which gels as the solution cools to room temperature. Benya et al. [38] demonstrated that
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Cartilage Tissue Engineering 22-5
dedifferentiated rabbit articular chondrocytes can survive and redifferentiate during suspension culture
in firm agarose gels. Recently, it has also been shown that dynamic pressure transmission through agarose
gel was complete and immediate, which validates the use of agarose gels in studies employing dynamic
pressurization in cartilage tissue engineering [39].
Alginate is a block copolymer of alternating residues of M (d-mannuronic acid) and G (l-guluronic
acid). Alginate forms a gel in the presence of divalent ions such as calcium or magnesium. Alginate
has been widely used as scaffold in cartilage tissue engineering because its solubility, gelation rate, and
mechanical properties can be adjusted by the G/M ratio in the polymer backbone. Hauselmann showed
that human chondrocytes cultured in alginate formed a similar matrix to native human articular cartilage
[40]. When exposed to reduced oxygen tension (5%), chondrocytes encapsulated in an alginate gel has
been shown to upregulate aggrecan and Type II collagen gene expression while downregulating Type I
collagen expression [41].
22.3.2 Synthetic Polymers
No single existing polymer can meet the demands of all tissue-engineering applications, given the unique
biochemical and mechanical requirements for different tissues or sites of implantation. Poly(lactic acid,
PLA), PGA, and poly(lactic-co-glycolic acid, PLGA) are among the earliest synthetic polymers that were
investigated for cartilage tissue engineering [7]. Originally designed for use as biodegradable surgical
sutures, these polyesters can form highly porous lattice structures that allow cell encapsulation and
proliferation with effective nutrient and waste transfer. A number of cell types, such as chondrocytes and
bone-marrow derived MSCs have been seeded [42,43] in these polyester materials and cartilage matrix
production have been achieved both in vitro and in vivo. Most studies involve implantation of cell-seeded
polymer constructs into subcutaneous pockets in nude mice. Grande has shown neocartilage formation
when implanted a chondrocyte-PGA construct into the rabbit knee defects [44]. PGA and PLA scaffolds
are also biodegradable via nonspecific hydrolytic cleavage at the ester linkage site. Although polyester
scaffolds have been widely used, they too have drawbacks such as poor cell attachment, difficulty in
molding, and mild inflammatory reaction once implanted in vivo.
Injectable hydrogels are a genre of scaffolds that can be administered to fill defects of any shape and
polymerize in situ with precise temporal and spatial control in a minimally invasive manner. Sims ini-
tially investigated the utilization of polyethylene oxide, a linear polyether-(CH
2
CH
2
O)
n
as encapsulating
polymer scaffolds for delivering large numbers of isolated chondrocytes via injection [45]. New cartilage
formation was observed as early as 6 weeks, with specimens exhibiting a white opalescence and histological
characteristics consistent with those of hyaline cartilage.
Moreover, polyethylene oxide gels can be modified by adding acrylate end group that can potentially
allow the transformation from a liquid to a solid with a defined structure in the presence of ultraviolet
light. Elisseeff demonstrated that this transdermal photopolymerization technique can be used to implant
the chondrocyte-seeded scaffolds, allowing simple exposure of the skin surface to light to induce gelation
of scaffolds injected subcutaneously in nude mice [46,47] (see Figure 22.2). Williams et al. [26] has
further shown the feasibility of applying photopolymerizing poly(ethylene glycol)-based hydrogels for
chondrogenesis of adult goat MSCs, in the presence of growth factors. Pluronics is a commercially
available copolymer of poly-(ethylene oxide) and poly-(propylene oxide) (Pluronics; BASF Corporation;
Mount Olive, NJ). Pluronics forms a gel through physical crosslinks by hydrophobic group association at
body temperature [48]. The main limitation of hydrogels for cartilage tissue engineering is the relatively
weak mechanical properties when compared to other solid fibrous scaffolds. Mechanical properties of
hydrogels may be improved by creating composite materials that incorporate a solid and hydrogel.
Ideally, polymers used in tissue engineering should be not only biocompatible, but also bioresponsive
and biodegradable. The common feature of early polymers in biomedical applications was their biological
inertness. Research in the past decade has been focusing more on the development of bioresponsive
polymers that can respond to the local biological and physical microenvironment and elicit a controlled
action and reaction. To enhance the interaction between the scaffold and the encapsulated cells, various
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22-6 Tissue Engineering
Chondrocytes harvested from
calf femoropatellar groove
Polymer solution
Polymer exposed to light
Four injection sites
beneath dermis of
nude mouse
Polmer–cell suspension
FIGURE 22.2 Schematic of procedure for cartilage tissue engineering by using transdermal photopolymerization
depicting (1) isolation of bovine chondrocytes from the femoropatellar groove and combination with polymer (10 to
20% poly[ethylene oxide]-dimethacrylate) to form (2) a polymer/chondrocyte suspension. The polymer/chondrocyte
suspension is subsequently (3) injected subcutaneously on the dorsal surface of a nude mouse, and (4) photopoly-
merized by placement of the mouse under an ultraviolet A lamp for 3 min. (From Elisseeff, J., Anseth, K., Sims, D.,
McIntosch, W. et al. Plast. Reconstr. Surg. 1999; 104: 1014–1022. With permission.)
biological signals such as cell adhesion peptides (i.e., RGD — arginine–glycine–aspartic acid), and growth
factors have been incorporated into scaffolds. Enhanced cell attachment and extracellular deposition was
achieved using scaffolds with biological activity [49–52]. As tissue mass increases and more extracellular
matrix is deposited, it is also very important for the scaffold to degrade in a similar rate to allow effective
diffusion and space for the newly formed tissue. Also, biodegradable scaffolds can play an important role
as delivery vehicles for sustained growth factor release [53].
22.4 Mechanical Stimuli
Cartilage, especially articular cartilage, is a tissue that is accustomed to mechanical stimulation. During
joint loading, articular cartilage undergoes compression and a plowing motion, and chondrocytes imbed-
ded in the cartilage thus experience direct compressive deformation, shear, and hydrostatic pressure [54].
To understand the mechanotransduction pathway, various bioreactors have been designed to mimic the
conditions necessary for functional cartilage tissue engineering. Freed and Vunjak-Novakovic [55] have
demonstrated that dynamic stimuli is critical for cartilage matrix production by chondrocytes seeded on
a PGA scaffold. Under static conditions, cell growth rates on the PGA scaffolds decreased due to limited
diffusion caused by increased cell mass and new matrix deposition. Therefore, to engineer cartilage tissue
that is clinically useful in size, it is essential to improve the in vitro culture conditions [42,56,57]. Carver
and Heath [58] examined the effect of intermittent pressure on chondrocytes seeded on PGA scaffolds
using a semiperfusion bioreactor. It was found that intermittent pressure increased the GAG content to
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Cartilage Tissue Engineering 22-7
native levels but the compressive modulus of the construct, though increased, did not reach the native
level. This suggested that although ECM synthesis has been increased, matrix organization may be incom-
plete. Therefore, a current challenge in engineering functional cartilage tissue using bioreactor approach
includes not only upregulation of ECM synthesis, but also its retention in the matrix and reorganization to
restore the native-level mechanical function. More work is still needed to determine the type of mechanical
forces, amplitude, and duty cycle that would induce the chosen cell type to form a functional cartilage
tissue.
22.5 Growth Factors
Research advances in the fields of embryonic development, molecular and cellular biology have identified a
variety of proteins that can affect cell chondrogenic differentiation and phenotypic expression, including
transforming growth factor TGF-βs, insulin-like growth factor (IGF-1), bone morphogenetic proteins
(BMPs), fibroblast growth factors (FGFs), and epidermal growth factor (EGF) [59]. These molecules
have a broad range of activities, including proliferation induction, increasing the synthesis and deposition
of cartilage extracellular matrix by chondrocytes, and inducing chondrogenesis by MSCs. Transforming
growth factor (TGF-β) superfamily, including TGF-βs 1–3 and insulin-like growth factor-1 (IGF-1),
stimulate GAG synthesis while chondrocyte synthesis of collagen II is strongly stimulated by IGF-1.
It has also been demonstrated that MSC cultures exposed to TGF-βs show increased cell density, nodule
formation, GAG, and Type II collagen synthesis [26,60].
The majority growth factor studies have been in vitro and focused on simply adding a soluble growth
factor to the medium [26]. However, for clinical application, a continuous and stable effect of growth
factors is a challenge. This challenge may be overcome by several possible approaches. Growth factors can
be tethered to the scaffold by chemical modification and being released as the scaffold degrades [51]. They
can also be encapsulated in polymer microspheres to achieve a sustained release [61]. The major limitation
of both the above methods is that growth factors are proteins that can easily denature and it is challenging
to retain biological activities during relative harsh chemical synthesis or microsphere suspension process.
Advances in gene transfer technology provide another novel alternative to achieve sustained and loc-
alized presentation of growth factors or gene products [62]. A variety of cDNAs have been cloned to
stimulate biological processes that could promote chondrogenesis. Direct injection of a vector or genetic-
ally modified cells into the defect is conceptually the simplest approach to gene transfer. Besides the growth
factors mentioned above, this method can also be applied to deliver chondrogenesis-related transcrip-
tional factors such as Sox-9, signal transduction molecules such as SMADS [63]. Since these molecules
function intracellularly and cannot be delivered to cells in a soluble form, gene transfer is a unique method
for translation of these technologies in a clinical setting.
22.6 Zonal Organization
Articular cartilage is comprised of three zones that exhibit significant differences in their metabolic
rate, matrix synthesis, and response to mechanical stimuli [54]. As opposed to current tissue engineer-
ing strategies where chondrocytes from all zones are seeded homogeneously, isolation of chondrocyte
subpopulations from specific cartilage zones and their organization into a scaffold to mimic the native
organization may provide engineered cartilage with enhanced structure and functional properties [64].
The superficial zone has a relatively low glycoaminoglycan content and compressive modulus, which facil-
itates the conformation of superficial zone to the opposing interface and allows stress distribution under
compressive conditions. Klein isolated chondrocytes from the superficial and middle zones of immature
bovine cartilage and cultured in alginate for 1 to 2 weeks. Analysis indicated that chondrocyte subpopu-
lations play an important role in determining the structure, composition, metabolism, and mechanical
function of tissue-engineered cartilage constructs [65]. Sharma has demonstrated the feasibility of using
multilayered photopolymerizing hydrogel to recreate the zonal organization of articular cartilage [66].
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22-8 Tissue Engineering
Chondrocytes from the upper, middle, and lower zones of bovine articular cartilage were isolated and
photoencapsulated in a trilayered poly(ethylene oxide)-diacrylate, PEODA, hydrogel. Cell tracking and
viability assay showed that cells remained in their respective polymerized layer and cell viability was com-
parable throughout all three layers. Biochemical analysis further demonstrated that distinct metabolic
characteristics are maintained when the chondrocytes are cultured in the layers, suggesting that this mul-
tilayer culture system may provide a native tissue mimicking environment that promotes regulatory signal
molecules exchange.
22.7 Conclusion
Advances in tissue-engineering research provided an alternative possible therapeutic approach to restore
lost tissue and function. Many researchers have focused on cartilage tissue engineering with encouraging
results that will continue to motivate research efforts to engineer cartilage tissue with biological and
mechanical properties similar to native tissue. The zonal organization of cartilage can be recreated using
layered scaffolds, allowing a better mimic of the native cartilage tissue. Further development of cartilage
tissue engineering is also closely related to the advances in polymer science to synthesize bioactive and
biodegradable polymer with suitable biocompatibility. More research is required to achieve a sustained
delivery of biological signals such as growth factors and transcriptional factors to promote tissue devel-
opment in vivo. Identifying and providing suitable mechanical stimuli may also help engineering tissue
to produce cartilage-specific biochemical compositions in an organized manner and hence acquire the
mechanical properties that would meet the physiological needs of clinical application.
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