Bio-Medical Materials and Engineering 20 (2010) 127–133 127

DOI 10.3233/BME-2010-0624
IOS Press

Introduction to tissue engineering and

application for cartilage engineering
N. de Isla a,c,∗ , C. Huseltein a,c , N. Jessel d , A. Pinzano a,c , V. Decot a,b,c , J. Magdalou a,c ,
D. Bensoussan a,b and J.-F. Stoltz a,b,c
a
CNRS, Faculté de Médecine, Vandoeuvre-lès-Nancy, France
b
CHU de Nancy, Unité de Thérapie Cellulaire et Tissulaire, Brabois, Vandoeuvre-lès-Nancy, France
c
Nancy Université, Université Henri Poincaré, Nancy, France
d
INSERM, Strasbourg, France

Abstract. Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and mole-
cular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western
Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general compo-
nents are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for
transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreo-
graph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs
or tissues.
Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development,
grafting and safety studies.
Keywords: Tissue engineering, cartilage, mechanobiology, mesenchymal stem cells

1. Main parameters in tissue engineering

1.1. How to choose the cells?

For tissue engineering, it seems better to choose an autologous cell to regenerate a tissue. But, these
cells are not easy available and are frequently in pathological state. This explains the increasing interest
for stem cells. Embryonic stem cells seem the most interesting cells due to their multipotentiality. In
adult however, stem cells (bone marrow, umbilical cord, amniotic fluid, . . . ) have more limited poten-
tialities and their isolations, is not an ethical problem and is generally easier. At least, fetal stem cells
are precursor cells of the hematopoietic system, although their ability to participate to the formation of
other tissues is much more unlikely. We must also recall the current expectations in the development of
new therapies via the introduction of genes in cells to counteract a given deficiency [1].

1.2. Preparation of the cells: Engineering approaches

After choosing the cells, their multiplication in culture in an adapted bioreactor or in a biomaterial
must be developed without any modification of their properties. Thus, it is necessary to have culture
*
Address for correspondence: N. de Isla, UMR CNRS 7561, Faculté de Médecine, 54500 Vandoeuvre, France. Tel.: +33 383
683457; E-mail: [email protected].

0959-2989/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved
128 N. de Isla et al. / Introduction to tissue engineering and application

mediums (composition, specific growth factors, . . . ) compatible with the cells of interest, with a GMP
process. Moreover, the use of optimized mechanical forces is generally a prerequisite in the process of
developing in vitro biotissues (concept of mechanobiology and tissue remodelling).

1.3. Grafting

One other problem to solve is the local implantation with or without biomaterial. It is important to note
that clinical applications of biotherapies are strongly controlled in Western Countries. Harvesting cells
or tissues of human origin can only be realized in health centers accredited by Public Authorities (in
France, different regulation laws described the procedure of authorization related to preparation, storage
and use of cells and tissues). The European Regulating Authorities are very strict on the nature of the
clinical trials and on the characteristics of the patients to treat.

1.4. Safety studies

Before grafting, different points must be precised:

• What type of grafting is planned for the patients? (what is the severity degree of the pathology to
consider, i.e., for instance, the early phase or after stabilization?. . . ) [2].
• What is the benefit for the patient?
• Site of the graft or injection.
• Clinical evaluation: fundamental phase which must validate the graft through studies on total or
partial tissue restoration.
• Immunosuppression needed.
• Possible side effects.

2. Cartilage functions and repair

Cartilage is a mesenchymal tissue composed of a cell (chondrocyte), extracellular matrix (ECM) mole-
cules, and water. Chondrocytes represent the cellular component and represent 1–2% of cartilage tissue
volume. The cartilage ECM is comprised of collagen fibers supporting glycoproteins and proteogly-
cans – which have a protein core associated with glycosaminoglycan molecules such as hyaluronic acid
(HA) and chondroitin sulfate. The tissue fluid, mainly water, contributes to the mechanical properties of
cartilage and provides nutrition and exchange with synovial fluid in the joint and with extracellular fluid
of other adjacent tissues.
There are three types of cartilage that are distinguished by their molecular components in the ECM,
their anatomic location, and their function. Hyaline cartilage has a white glassy appearance and is found
primarily in the joint. Its ECM is composed of water, proteoglycans, and type II collagen. Hyaline carti-
lage functions to provide stable movement with minimal friction. It demonstrates an excellent ability to
provide resistance to compression and distribute loads – adapting to the load-bearing force as it changes
over time. Elastic cartilage is distinguished by the presence of elastin in the ECM. Elastin cartilage pro-
vides a structural function; represented by the support it provides the airway and the external ear. Lastly,
fibrocartilage has a higher proportion of type I collagen in the matrix. Fibrocartilage is found at end
 N. de Isla et al. / Introduction to tissue engineering and application 129

of tendons and ligaments in apposition to bone providing tensile strength and countering compression
and shear forces. In contrast to fibrocartilage, hyaline and elastic cartilage have a perichondrium, which
channels a limited blood supply and contains a source of progenitor cells.
All types of cartilage are distinguished by a limited ability for intrinsic repair after sustaining dam-
age. This is due, in part, to a lack of vascularity, the sparse cellularity of native chondrocytes, and the
inflammatory response [3].
Osteoarthritis frequently arises from cartilage damage due to sports injury, trauma and/or simply
overuse. The resultant degeneration of cartilage combined with its inability to self-repair leads to further
degradation of the joint.
Current therapies for cartilage repair are inadequate for restoring form and function. Cartilage grafts
suffer from many problems including limited donor tissue availability, donor site injury, scarring, and
pain. Similarly, allogeneic and alloplastic implants have a high risk of infection, graft resorption, and
structural failure. The implant materials do not integrate into the host tissue and have a limited life-
time. In addition, implantation generally requires invasive surgery. In many cases the procedure leaves a
poor esthetic result. Therefore, there is a large opportunity for new therapies such as cartilage engineer-
ing [4,5].
To day the only FDA approved cellular-based therapy for cartilage defects uses differentiated chon-
drocytes [6–8]. Autologous cells are harvested from a biopsy and expanded ex vivo to create a large
number of cells for transplantation. While autologous expanded chondrocytes have a low risk of im-
mune rejection, they have a tendency to dedifferentiate (lost their phenotype) in vitro [9].
A second cellular paradigm for engineering tissue is the use of undifferentiated progenitor cells that
have the ability to proliferate and differentiate into the target tissue [10]. Embryonic cells are pluripotent
and theoretically have an unlimited life span and unlimited potential to become any differentiated cell in
the body. However, there is some controversy about using these cells due to their origin and procurement.
Furthermore, there are concerns about the potential immunogenicity and oncological transformation of
these cells.
Adult stem cells are a second group of undifferentiated cell. These multipotent cells are found in adult
tissue, such as bone marrow, adipose tissue, umbilical cord, . . . . Their physiological role is concerned
by their regenerative capabilities of injured tissue. MSCs derived from bone marrow have received sig-
nificant attention in regenerative medicine for their ability to differentiate into numerous cells lineages
and form tissues including cartilage and bone.
To create distinct tissue types, specific controls on the induction and maintenance of stem cell differ-
entiation is imperative. The mechanisms of induction, stability, and permanence of differentiation and
proliferation remain to be elucidated. The use of scaffolds and biological signalling molecules will play
an important role in the signalling mechanisms of stem cells.
Biological factors that guide cellular differentiation include soluble biochemical signals, transfection
of gene vectors, ECM molecules, environmental factors like mechanical compression, and cell–cell in-
teractions. A combination of these factors can promote cellular differentiation and proliferation. It should
be emphasized at this time that many signals, signalling pathways and the rationale behind physiological
design remain to be elucidated.
The influence of mechanical forces on cell function in vitro has been demonstrated on engineering
cartilage, bone, smooth muscle and also many other tissues. In cartilage production, dynamic mechanical
stresses on chondrocytes and MSCs promote differentiation and increased matrix production [11].
130 N. de Isla et al. / Introduction to tissue engineering and application

3. Parameters for cartilage engineering

3.1. The cells

Numerous cell types have been used including chondrocytes, bone-marrow derived mesenchymal stem
cells (MSCs), stem cells isolated from adipose tissue, from bone marrow, fetal cord and also embryonic
stem cells. 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.
Chondrocytes
Chondrocytes, the major cell type that is present in differentiated cartilage, is the most obvious cell
option for cartilage tissue engineering. Autologous chondrocytes transplantation (ACT) is the only Food
and Drug Administration (FDA) approved cartilage repair product in the United States but several prob-
lems remain with the approach. In vitro expansion is also limited due to replicative senescence that
chondrocytes exhibit. More importantly, once removed from their extracellular environment and ex-
panded 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. Studies have shown that chondrocyte phenotype can be
retained or reexpressed by suspending chondrocytes in a three-dimensional environment such as agarose
gel, alginate beads and collagen gel [12].
Mesenchymal stem cells
Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiate into a
variety of connective tissue cells including bone, cartilage, and adipose tissue both in vitro and in vivo
[13,14]. MSCs are present in various tissue types during human development [15–17] and are preva-
lent in adult bone marrow [18]. As a readily available source, MSCs can be isolated from bone marrow
or fetal cord (Wharton jelly) [19] expanded in culture, and differentiated into chondrogenic cells un-
der appropriated tissue conditions. Numerous studies have shown that transforming 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 pro-
teins including Type II Collagen, glycoaminoglycan and proteoglycans [20].
These results indicate that successful tissue-engineered cartilage repair based on MSCs can be
achieved by recapitulating aspects of embryonic tissue formation [21]. 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.
Other cell sources
Theoretically, hESCs can be used to generate any desired cell or tissue for the treatment of degenera-
tive diseases. So far, very few studies have been performed inducing hESCs into chondrogenic lineage.
Challenges with hESCs include potential of undesired growth, difficulty to achieve homogeneous differ-
entiation in vivo, and related ethics issues. Other potential cell sources were found by Mizuno [22], who
demonstrated that human dermal fibroblasts seeded onto Type I Collagen sponges containing deminer-
alied bone matrix can be stimulated to produce a cartilaginous tissue expressing Type II Collagen. Also,
Lorenz recently reported that human adipose tissue derived cells can also be induced into chondrogenic
pathway in vitro [23].
 N. de Isla et al. / Introduction to tissue engineering and application 131

3.2. Scaffolds

Tissue engineering scaffolds are designed to provide a three-dimensional (3D) environment to sup-
port and direct cellular processes in their migration, proliferation and differentiation toward functional
tissue [24]. The scaffold provides mechanical stability and surface chemistry to orchestrate biological
signals and influence cellular adhesion and function. Scaffolds must be biocompatible with host tissue,
porous enough to allow nutrients to reach cells, and ultimately degrade without toxic effects as tissue
develops.
Biological scaffolds include natural collagen, alginate, HA and acellular dermis [25–27]. Biological
scaffold potentially allow for better regulation of cell adhesion and matrix production of the resident
cells. However, biological scaffolds have a risk of contamination or immune reaction than synthetic
scaffolds. On the other hand, synthetic materials are created de novo from molecules such as glycolic
acid or lactic acid polymers, provide more precise control over the structural properties, mechanical
properties and rates of resorption.

3.3. Mechanical stimuli

Cartilage, especially articular cartilage, is a tissue that is accustomed to mechanical stimulation. Dur-
ing joint loading, articular cartilage undergoes compression and a plowing motion, and chondrocytes
imbedded in the cartilage thus experience direct compressive deformation, shear, and hydrostatic pres-
sure. To understand the mechanotransduction pathway, various bioreactors have been designed to mimic
the conditions necessary for functional cartilage tissue engineering. Freed and Vunjak-Novakovic [28]
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.

3.4. Growth factors

Variety of proteins 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). These molecules have
a broad range of activities, including proliferation induction, increasing the synthesis and deposition
of cartilage extracellular matrix by chondrocytes, and including chondrogenesis by MSCs. Transform-
ing growth factor (TGF-β) superfamily [29], 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.
The majority growth factor studies have been in vitro and focused on simply adding a soluble growth
factor to the medium. However, for clinical applications, a continuous and stable effect of growth factors
is a challenge.

4. Conclusion

The regeneration of cartilage is and will remain a challenge for the development of cell therapy, tissue
engineering and gene therapy. However, to this day many problems remain to be solved:
132 N. de Isla et al. / Introduction to tissue engineering and application

• Technical problems regarding the definition of supports (scaffolds), cells used and stability and
culture medium. In particular, the impact of the biomaterial used remains to be defined.
• Legal issues with respect to the different regulations in the USA, Europe, etc.
Biocartilage can be introduced via cell implantation, biocartilage transplantation or gene therapy.
Complementary scientific approaches remain to be developed. Nevertheless, current knowledge permits
a certain optimism for the future.

Acknowledgement

This work was partly supported by an ANR grant (Cartispray), “Région de Lorraine, CUGN and
Conseil Général de Meurthe-et-Moselle”.

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