Biomaterials 21 (2000) 431}440

Review: tissue engineering for regeneration of articular cartilage

Johnna S. Temeno! !, Antonios G. Mikos!,",*
!Department of Bioengineering, Rice University, 6100 Main, Houston, TX 77005-1892, USA
"Department of Chemical Engineering, Rice University, 6100 Main, Houston, TX 77005-1892, USA
Received 27 July 1999; accepted 30 September 1999

Abstract

Joint pain due to cartilage degeneration is a serious problem, a!ecting people of all ages. Although many techniques, often surgical,
are currently employed to treat this a%iction, none have had complete success. Recent advances in biology and materials science have
pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the
biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair
methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and consider-
ations in scale-up of cell}biomaterial constructs are summarized. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Cartilage repair; Cartilage regeneration; Cartilage tissue engineering; Cell transplantation; Bioreactors; Chondrogenesis

1. Introduction to compression and the ability to distribute loads that

Joint pain is a major cause of disability in middle-aged
and older people [1]. Pain usually results from degener-
ation of the joint's cartilage due to primary osteoarthritis
or from trauma causing loss of cartilage [1]. As many as
36 million Americans su!er from some form of arthritis
[2] and cartilage damage from sports injury is also com-
mon. It has been shown that up to 16% of injuries to the
knee cause intra-articular bleeding [3,4]. Since cartilage
shows very little tendency for self-repair, these injuries
are maintained for years and can eventually lead to
further degeneration (secondary osteoarthritis) [4].
Given the debilitating nature of severe joint pain,
scientists and surgeons have tried for decades to repair or
regenerate lost cartilage, but there has been little success
due to the complex properties of the tissue and its essen-
tial function in the body. Hyaline cartilage, the type of
cartilage found in joints, provides stable movement with
less friction than any prosthetic replacement, and can
alter its properties in response to di!erences in loading.
Although appearing to be a simple, avascular matrix,
hyaline cartilage possesses properties such as resistance
* Correspondence address: Department of Bioengineering, Rice Uni-
versity, 6100 Main, MS 142, Houston, TX 77005-1892, USA. Tel.:
#713-285-5355; fax: #713-285-5353.
E-mail address: mikos@rice.edu (A.G. Mikos)
cannot be fully replaced by any other tissue or device
designed to date [5,6].
This review will provide an overview of cartilage anat-
omy and types of injury, as well as focus on current
strategies for cartilage repair in the knee joint, including
promising new strategies involving tissue engineering.
The knee, with two distinct articulating surfaces is a con-
dylar-type joint. Both bones, the femur and the tibia, that
comprise the joint are covered with a layer of hyaline
cartilage at the joint surface. Immediately below this is
the periosteal covering of the bone. The synovial mem-
brane encircles the joint, thereby forming a barrier to
retain the synovial #uid in the knee. This #uid provides
lubrication and nutrients for the cartilage since no blood
vessels penetrate into the tissue from the subchondral
bone [7].
Although only hyaline cartilage is found in the knee,
two other kinds, "brocartilage and elastic cartilage, are
seen in other areas of the body. All three are composed of
chondrocytes and extracellular matrix macromolecules.
Elastic cartilage forms the ear and nose and is character-
ized by the presence of elastin in the extracellular matrix
(ECM). Fibrocartilage has a higher proportion of col-
lagen in the ECM than hyaline cartilage and is found at
the ends of tendons and ligaments in apposition to bone.
Hyaline cartilage has a white, glassy appearance, and
unlike "brocartilage, shows no macroscopic evidence of
"bers [8].

0142-9612/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 1 3 - 6
432 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
2. Composition and anatomy of articular cartilage
2.1. Chondrocytes
In humans, chondrocytes represent only about 1% of
the volume of hyaline cartilage but are essential since it is
these cells that replace degraded matrix molecules to
maintain the correct size and mechanical properties of
the tissue. Thus, microscopically, the cells' endoplasmic
reticulum and Golgi apparatus are prominent. In addi-
tion, many contain lipid and glycogen stores and
secretory vesicles. Some chondrocytes have cilia that
extend from the cell into the ECM and are believed to
play a role in sensing the mechanical environment of the
cell, since chondrocytes are known to modify matrix
properties in response to loading [5].
Chondrocytes originate from mesenchymal stem cells
(MSCs) found in the bone marrow in mature individuals.
During embryogenesis, the MSCs start to di!erentiate
into chondrocytes and secrete a cartilaginous matrix.
During this time, the cells continue to divide. They pass
through various lineage states and eventually the chon-
drocytes in the central zone, located next to what will
soon be bone, enter the "nal stage of development and
become hypertrophic chondrocytes, producing proteins
that are important in calci"cation of the matrix. Other
chondrocytes (on the periphery) secrete collagen and
matrix molecules in the right proportions to produce
hyaline cartilage. Mature articular chondrocytes, unable
to proliferate, appear rounded and are completely en-
cased in matrix [5,9].
2.2. Extracellular matrix
2.2.1. Collagen
Collagen types II, VI, IX, X, and XI are found in
articular cartilage, although type II accounts for 90}95%
of the collagen in the matrix. Type II collagen has a high
amount of bound carbohydrate groups, allowing more
interaction with water than some other types. Types IX
and XI, along with type II, form "brils that interweave to
form a mesh. This organization provides tensile strength
as well as physically entrapping other macromolecules.
Although the exact function of types IX and XI are
unknown, type IX has been observed to bind super"cially
to the "bers and extending into the inter-"ber space to
interact with other type IX molecules, possibly acting to
stabilize the mesh structure. Type X is found only near
areas of the matrix that are calci"ed [6,10].
2.2.2. Proteoglycans
Proteoglycans are composed of about 95% polysac-
charide and about 5% protein. The protein core is asso-
ciated with one or more varieties of glycosaminoglycan
(GAG) chains. GAG chains are unbranched polysaccha-
rides made from disaccharides of an amino sugar and
another sugar. At least one component of the disaccharide
has a negatively charged sulfate or carboxylate group, so
the GAGs tend to repel each other and other anions while
attracting cations and facilitating interaction with water.
Hyaluronic acid, chondroitin sulfate, keratan sulfate, der-
matan sulfate and heparan sulfate are some of the GAGs
generally found in articular cartilage [5,10,11].
There are both large aggregating monomers and
smaller proteoglycans present in articular cartilage. The
aggregating proteoglycans, or aggregans, are composed
of monomers with keratan sulfate and chondroitin sul-
fate GAGs attached to the protein core. In most aggre-
gan molecules, link proteins connect many (up to 300) of
these monomers to a hyaluronic acid chain. Aggregans
"ll most of the inter"brillar space of the ECM and are
thought to be responsible for much of the resilience and
stress distribution in articular cartilage through their
ability to attract water. There are no chemical bonds
between the proteoglycans and collagen "bers; aggrega-
tion prevents di!usion of the proteoglycans out of the
matrix during joint loading [5,6,10].
The smaller proteoglycans include decorin, biglycan
and "bromodulin. They have shorter protein cores and
fewer GAG chains than their larger counterparts. Unlike
aggregans, these molecules do not a!ect physical proper-
ties of the tissue, but are thought to play a role in cell
function and organization of the collagen matrix [5].
2.2.3. Noncollagenous proteins
In contrast to proteoglycans, glycoproteins have only
a small amount of oligosaccharide associated with the
protein core. These polypeptides help to stabilize the
ECM matrix and aid in chondrocyte}matrix interac-
tions. Both anchorin CII and cartilage oligomeric protein
anchor chondrocytes to the surrounding matrix. Other
noncollagenous proteins commonly found in most tis-
sues, such as "bronectin and tenascin, are also observed
in articular cartilage and are believed to perform similar
functions as the glycoproteins [5].
2.2.4. Tissue yuid
Tissue #uid is an essential part of hyaline cartilage,
comprising up to 80% of the wet weight of the tissue. In
addition to water, the #uid contains gases, metabolites and
a large amount of cations to balance the negatively
charged GAG's in the ECM. It is the exchange of this #uid
with the synovial #uid that provides nutrients and oxygen
to the avascular cartilage. In addition, the entrapment of
this #uid though interaction with ECM components pro-
vides the tissue with its ability to resist compression and
return to normal shape after deformation [5,10].
2.3. Zones of organization
Articular cartilage can be divided into four zones
(super"cial, transitional, middle and calci"ed) based on
 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
di!erences in matrix morphology and biochemistry. In
each zone, there are three distinct regions: the pericellular
region, the territorial region and the interterritorial re-
gion. The pericellular and territorial regions provide
means for chondrocyte attachment to the ECM and
protection of the cells during loading. The pericellular
region contains almost exclusively noncollagenous bind-
ing proteins. Part of the territorial region is composed of
collagen "brils that follow the pericellular envelope but
may extend to encompass two or more chondrocytes.
Farther away from the cells, the territorial collagen is less
aligned and "brils cross each other to form a basket
structure around the chondrocyte(s). The interterritorial
region is marked by an increase in "bril diameter and
orientation of "bers in a more parallel fashion. This
region is primarily responsible for the mechanical prop-
erties of the tissue [5].
2.3.1. Superxcial zone
The super"cial zone is the thinnest zone and is made of
two distinct layers. An acellular sheet of predominantly
collagen "bers (the lamina splendens) covers the joint.
Deep to this, the second layer is composed of #attened
chondrocytes with their long axes parallel to the articular
surface. The ECM in this area has more collagen and less
proteoglycan than the other zones. There are also large
amounts of "bronectin and water. This combination of
molecules imparts more tensile strength to this area of
the matrix, which could be useful in resisting shear from
the articulating surfaces. The super"cial zone also is
important for the compressive strength of the tissue and
possibly in isolation of cartilage from the immune system
[5,10].
2.3.2. Transitional zone
Occupying more volume than the super"cial zone, the
transitional zone includes chondrocytes that are spheri-
cal and contain synthetic organelles such as the endo-
plasmic reticulum, Golgi bodies and mitochondria. The
ECM in this area has larger collagen "brils, more pro-
teoglycan and less collagen and water than in the pre-
vious zone. The interterritorial "brils are aligned
obliquely or randomly to the articular surface, as op-
posed to parallel in the super"cial zone [5,10].
2.3.3. Middle (radial) zone
The middle zone is usually the biggest and has the
largest diameter collagen "brils, the most proteoglycan,
and the least water. The cells are rounded, like in the
transitional zone, but are stacked in columns perpendicu-
lar to the articulating surface. The territorial region of the
matrix encloses each column [5,10]. These cells show
high synthetic activity*ten times that of super"cial zone
chondrocytes [12]. Also, in this area, the orientation of
the interterritorial "bers changes again so that they are
433
now aligned perpendicular to the joint surface. The "bers
extend into the tidemark, a basophilic line of unknown
composition that indicates the beginning of calci"ed tis-
sue [5,10].
2.3.4. Calcixed cartilage zone
The thin calci"ed cartilage zone lies closest to the
subchondral bone and acts as a transition from soft
hyaline cartilage to bone. Signi"cant shear stress can be
produced in this area due to the interface between the soft
cartilage and much sti!er bone [6]. The chondrocytes
here are smaller, with almost no endoplasmic reticulum.
In some places, they seem to be completely surrounded
by calci"ed ECM, indicating that they have very little
metabolic activity [5,10].
3. Articular cartilage injury
There are three main types of cartilage injury: matrix
disruption, partial thickness defects, and full thickness
defects. Matrix disruption occurs from blunt trauma,
such as dashboard injuries in automobile accidents. The
ECM is damaged, but if the injury is not extreme, the
remaining viable chondrocytes will increase their syn-
thetic activity to repair the tissue. Partial thickness
defects demonstrate disruption of the cartilage surface
("ssures, etc.) but this does not extend to the subchondral
bone. Immediately following the injury, nearby cells be-
gin to proliferate, but for reasons that remain unclear,
cellular attempts to "ll the defect cease before it is re-
paired. Full thickness defects arise from damage that
transverses the entire cartilage thickness and penetrates
the subchondral bone. In this case, the defect is "lled
with a "brin clot and a classic wound healing response
ensues. With this type of injury, unlike the others,
there is access to a population of progenitor cells from
the bone marrow which can migrate to "ll the defect
[13,14]. These cells usually cause replacement of the
"brin clot with tissue intermediate between hyaline
and "brocartilage. This tissue is usually less sti! and
more permeable than native cartilage, which could
contribute to its eventual degradation over a period of
months [14].
Several factors may explain why cartilage demon-
strates very little capability for self-repair after injury.
Chondrocytes are not required to proliferate to maintain
cartilage, as in some other tissues (i.e. skin), and mature
chondrocytes have a relatively low metabolic activity [5].
Except in the case of full-thickness defects, there is no
direct access to progenitor cells (such as are found in
bone marrow) and the resident chondrocytes may be
impeded by the ECM from migrating to "ll the defect
[13,15]. In addition, the proteoglycans in the ECM can
prevent cell adhesion, further undermining the native
repair process [15,16].
434 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
4. Treatments for articular cartilage defects
4.1. Symptom management
Due to the di$culties in natural repair of cartilage,
clinicians have turned to other methods to return func-
tionality of the joint. If cartilage damage is severe, symp-
toms are managed using resection or other surgical
techniques. Resection of articular cartilage is often fol-
lowed by implantation of a prosthesis in the joint [4].
Relief of pain can also be provided by an osteotomy,
which removes part of one of the bones in an articulating
surface to decrease loading of the joint. This also gives
the surgeon an opportunity to bring regions of the joint
with remaining cartilage in contact with each other or to
correct misalignments due to previous degeneration [1].
These techniques are often employed after other methods
have failed [4].
4.2. Cartilage restoration
In patients with smaller lesions, attempts are made to
restore cartilage to the joint surface through a variety of
methods. These include implanting replacement tissue
grafts, or employing techniques that encourage the native
repair process [4].
4.2.1. Tissue grafts
Cartilage can be replaced either with small plugs from
a less weight bearing region of the joint (autografts) or
with a full allograft. Autografts are limited by the small
amount of cartilage available in the body for transplanta-
tion to other sites. Usually, this cartilage is taken from
the patella, the femoral condyle or the proximal "bula
[1,4,17]. While this procedure does seem to decrease pain
in many patients (70% good results at two to "ve years
[11]), the e!ects of damage to the donor site must be
considered. There is also concern that tissue from a less
weight bearing region will not be able to withstand the
forces imparted at the joint surface [15]. Allografts have
resulted in a 95% survival rate at "ve years, making them
a useful treatment. However, fresh grafts have been seen
to induce an immune response once implanted [1,4,17].
Frozen grafts may reduce this response as well as provide
more time for screening of the donor tissue for pathogens,
but freezing can reduce tissue viability [15].
Another option is the use of periosteal or perichondrial
grafts. For the former, periosteal tissue is taken from an
adjacent area and placed in the cartilage defect with the
belief that the undi!erentiated periosteal cells will be
induced to form chondrocytes in their new environment.
This technique has been used for more than a decade in
the United States with somewhat encouraging results,
depending on exact placement of the periosteal tissue in
the defect and other factors [4,8]. A similar procedure
can be done with perichondrium from the rib, but more
chondrogenesis was observed using periosteal grafts than
perichondrial grafts [4]. Furthermore, there was up to
70% failure of these grafts at "ve years due to ossi"cation
of the perichondrial-derived tissue [3,18]. Although these
procedures have proven to be e!ective for some patients,
and remove the problem faced in other autografts of
tissue damage due to alteration of position in the joint,
they do result in donor site morbidity and can require
a second incision to obtain the graft tissue [4].
4.2.2. Cartilage regeneration
In an e!ort to eliminate the need for a donor site, and
because of concerns associated with allogeneic and auto-
geneic implants, many attempts have been made to heal
or regenerate existing cartilage, rather than replace it.
The proposed techniques have focused on either enhanc-
ing the intrinsic regenerative properties of the tissue or
transplanting extra chondrocytes to form more tissue.
Unfortunately, neither of these has been completely suc-
cessful, especially in older populations [1,4].
4.2.2.1. Regeneration enhancement. The most common
treatment of cartilage degeneration is to penetrate the
subchondral bone, either by abrasion or drilling. This, in
essence, creates a full-thickness defect. A clot forms over
the bone surface which, if left unloaded, can provide
a sca!old for migration of MSCs and their eventual
di!erentiation into chondrocytes and osteocytes [19].
However, results of this method are mixed: the tissue
formed ranges from no cartilage to "brocartilage to hya-
line cartilage, depending on the patient. Even when hya-
line-like cartilage is created, the mechanical properties
and durability are less than that of the original tissue.
Due to the large variability in outcomes, the best that can
be concluded is that this method has some chance of
helping and little chance of harming the patient [1,3,4].
Less invasive techniques to restore cartilage include
treatment with lasers or electrical stimulation, decreased
joint loading in conjunction with continuous passive
motion, or the use of pharmacological agents to stimu-
late chondrocytic activity (see [4] for a more complete
review of each technique). Passive motion shows con-
siderable regeneration for small defects (less than 3 mm),
and has been found to enhance other procedures for
cartilage healing [1,4,14]. Injections of various pharma-
cologics, such as growth factors (transforming growth
factor-b1, insulin-like growth factor-1 and bone mor-
phogenic proteins) and corticosteroids, have been attem-
pted as a means to increase chondrocyte proliferation
and matrix deposition. However, corticosteroids have
produced ambiguous results and growth factors can also
stimulate the formation of osteophytes that can be quite
painful. Therefore, more research should be done to
determine which pharmacologics should be administered
and in what doses before this treatment will be e!ec-
tive [4].
 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
4.2.2.2. Cell transplantation
Cell suspensions. Another option is transplantation of
chondrocytes or undi!erentiated cells to restore lost tis-
sue mass. An optimal approach would be to take a small
biopsy of cells (progenitor or di!erentiated chondrocytes)
expand the number of cells, and then return them to the
defect once the proper mass has been generated. Im-
plantation of individual chondrocytes has been re-
searched since 1968, but the success rate has been less
than 40%, due to the problem of retaining the cells in the
defect for a length of time that would allow them to begin
to produce matrix [20,21].
However, recently, a surgical method has been de-
veloped which uses a periosteal #ap sutured over the
defect as a barrier beneath which cultured autologous
chondrocytes are injected. The cells remain in the defect
and this technique has shown promise in human trials
conducted over the past 12 years in Sweden and the
United States. A small biopsy of autologous tissue is
obtained during arthroscopic evaluation of the joint and
is cultured for approximately three weeks, resulting in
a 10}12-fold increase in cell number. During the surgical
procedure, the periosteum (cambium layer toward the
defect) is attached with sutures and "brin glue to healthy
cartilage near the lesion and the cultured cells are injec-
ted beneath it [18,22,23].
In the initial study, 14 of 16 patients with defects in the
femoral condyle had good results after 2 yr, while healing
in patellar defects was less encouraging (two of seven had
good outcomes) [22]. Since that time, further studies
have corroborated these results. A US study begun in
1995 reports graft failure in only 7% of 70 patients and
indicates that this procedure may hold promise to repair
joints with multiple lesions [3]. In a long-term study, 30
of 31 patients maintained good results over 5}10 yr in
Sweden [18].
Despite these encouraging results, this procedure,
although clinically available in the US and other
countries, should not be regarded as a panacea. As
indicated above, the best results are found for healing
lesions in the femoral condyle [3] and the need for a
periosteal #ap requires a donor site. Interestingly,
although short-term (three months) studies with rabbits
reported healing similar to that found in humans [24],
long-term results in canines have not been as promising.
Although initially the procedure described above
caused cartilage regeneration in canine knees, this was
not permanent, and 12}18 months after surgery, the
cartilage began to degrade [25]. In another study [26],
at 12}18 months, no di!erence was observed between
the healing of control defects, defects with addition of
a periosteal #ap alone, or with a #ap and autologous
chondrocytes. Because the canine cartilage repair
process seems very similar to that of the human [27],
explanations for these discrepancies are yet to be
determined.
435
Cells in scawolds. Alternatively, instead of tissue #aps,
highly porous sca!olds may be used to maintain di!eren-
tiated cells in a given area. This design is optimal because
it signi"cantly reduces donor site morbidity and, in addi-
tion to simply providing a boundary for retention of cells,
the sca!old also acts as a substrate to which the anchor-
age-dependent chondrocytes can adhere [28]. When cul-
tured two-dimensionally, such as in Petri dishes, these
cells dedi!erentiate, assuming a more #attened appear-
ance and producing Type I instead of Type II collagen.
However, when grown in three dimensions, chondrocytes
maintain their di!erentiated phenotype and function. In
this way, sca!olds can encourage the proliferation of
chondrocytes without sacri"cing important functions to
dedi!erentiation [29]. Sca!olds have also been used
without seeded chondrocytes as a technique for regenera-
tion enhancement by encouraging cell migration [30,31],
but these studies are not reviewed here. Please refer to the
recent article by Hunziker [15] for further treatment of
this subject.
Various sca!old materials have been tested, including
both naturally derived and synthetic polymers. (For
more complete listings, see [11,14,15]). This review will
not discuss all of these materials, but rather focus on
those that have been most extensively used in tissue-
engineered constructs. Although recent work with hy-
aluronic acid-based carriers is promising [32,33], the
natural polymer that has received the most attention is
collagen. In 1983, it was found that chondrocytes main-
tain di!erentiated phenotype and GAG production for
six weeks in collagen gels [34]. Since that time, re-
searchers have continued to show encouraging results
from use of these sca!olds. A key advantage is that,
because collagen can be recognized by cellular enzymes,
it can be remodeled and degraded to provide space for
the growing tissue [35]. Collagen matrices have also been
found to have the proper molecular cues to stimulate new
collagen production by transplanted cells as compared
with other sca!old types [36]. Caplan et al. reported that
when MSCs were seeded into collagen gels implanted in
osteochondral defects in rabbits, embryogenesis was
recapitulated and both bone and hyaline cartilage were
formed, although mechanical properties of the regen-
erated tissue were signi"cantly less than normal values,
and there was some evidence of degeneration after 24
weeks [37,38].
Although preliminary results are promising for natu-
rally derived polymers, concerns about the feasibility of
"nding large amounts of these materials for clinical ap-
plications, as well as assurance of pathogen removal has
prompted other researchers to investigate the use of
synthetic polymers. These materials can be easily mass-
produced and their properties can be tailored for speci"c
applications. This includes creating degradable polymers,
which like collagen, allow room for tissue growth in
the construct and eliminate the need for a second surgery
436 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
to remove the implant. There is also the possibility
that growth factors or other pharmacologics could be
included in the sca!old to encourage certain cellular
responses, such as proliferation or di!erentiation.
However, few of these polymers have been FDA ap-
proved for use in humans [28].
Much current research has focused on chondrocyte
interaction with those biodegradable polymers that are
FDA approved: poly(glycolic acid) (PGA), poly(L-lactic
acid) (PLLA) and their copolymer, poly(DL-lactic-co-
glycolic acid) (PLGA) [36,39}46]. They are poly(a-hy-
droxy esters) and are degraded by hydrolysis. PLLA is
more hydrophobic and less crystalline than PGA and
degrades at a slower rate [28]. Firm hyaline-like cartilage
has been observed after six weeks when undi!erentiated
perichondrial cells were seeded onto PLLA meshes and
implanted in the femoral condyles of rabbits [42,43].
Similar cartilage morphology was found using PGA por-
ous non-woven sca!olds seeded with bovine chon-
drocytes and cultured in vitro for 12 weeks. In this case,
mechanical properties such as compressive modulus and
aggregate modulus were similar to that of normal bovine
cartilage [44]. Both types of degradable polyester tended
to increase proteoglycan synthesis compared to a col-
lagen sca!old [36]. Cell growth was approximately twice
as high initially (less than two months) on PGA than
PLLA matrices when seeded with bovine chondrocytes,
but after six months, total cellularity was found to be
similar. Initial di!erences were attributed to the fact that
PLLA degrades much more slowly and so left less space
for cell proliferation [45]. PLLA has been found to be
less toxic to human chondrocytes than PGA in studies
maintaining a constant pH over 12 days [46].
A main disadvantage of all of these sca!old materials is
that they require an operation for implantation. This has
led to the development of polymers that can be injected
along with cells and then cross-linked in situ to form
matrices. Several investigators are exploring the option
of combining "brinogen and thrombin to form a degra-
dable "brin mesh that can be used to support chon-
drocytes [47}49]. When the cell}"brinogen}thrombin
mixture was injected into defects in horses, more GAGs,
aggregan and type II collagen were present in the new
tissue at eight months than in defects that were left
untreated [49]. Alginate and bovine articular chon-
drocytes, injected into nude mice and crosslinked with
calcium, also formed cartilage, but studies longer than 12
weeks have not been conducted to determine if there is
any long-term tissue degradation [29,50,51]. Some forms
of alginate have been found to be immunogenic, as evi-
denced by increased lymphocyte number and presence of
anti-alginate antibodies [52]. A further concern about
the biocompatibility of the material is that, according to
Cao et al., there is more in#ammatory response to al-
ginate than to injectable synthetic materials [53]. Syn-
thetic polymers, such as poly(ethylene oxide) (PEO) or
copolymers of ethylene and propylene oxide P(EO-co-
PO), have also been tested as injectable matrices for
chondrocyte transplantation. Sims et al. reported that 12
weeks after subcutaneous delivery of bovine chon-
drocytes in a PEO gel into nude mice, cartilage that was
biochemically similar to natural bovine cartilage had
been produced [54]. In a comparative study, when
seeded with autologous chondrocytes and injected in
pigs, the P(EO-co-PO) copolymer was found to produce
more cartilage-like tissue than either PGA sca!olds or
alginate hydrogels. In this case, however, it must be noted
that the chondrocytes were of aural rather than articular
origin [53].
Types of cells used. Certain polymer properties, such
as degradation time, may be dictated by the type of cells
to be used in the sca!olds. As evidenced in the above
examples, both fully di!erentiated chondrocytes and pro-
genitor cells have been employed to produce hyaline-like
cartilage. Chondrocytes are very easy to obtain, but it has
been found that as the cells are cultured for longer peri-
ods before implantation, the cartilage formed is increas-
ingly "brous in nature [29]. Also, according to Caplan
et al., chondrocytes implanted in osteochondral defects in
rabbits did generate cartilage, but a subchondral plate
was only observed if undi!erentiated MSCs were used
[20,38]. MSCs can be expanded many-fold with little
e!ect on the tissue that is eventually formed and have
been shown to di!erentiate into both bone and cartilage
cells, making them prime candidates for transplantation
in tissue-engineered constructs [38,55}57].
With either type of cell chosen, good sca!old seeding is
required for the formation of functional cartilage. Seed-
ing density is important since it has been found that cells
seeded too sparsely result in incomplete "lling of the
sca!old, leading to the possibility of "brous ingrowth
which would adversely a!ect the properties of the tissue
generated [58,59]. In addition, surface tension can pre-
vent liquid media with cells from entering some synthetic
sca!olds, such as PGA and PLLA. Therefore, these
meshes must be pre-wet with alcohol which is then dis-
placed by media to obtain seeding deep within the con-
struct [28]. Other techniques to improve seeding include
dipping the sca!olds in poly(L-lysine) or type II collagen
to improve initial cell attachment [46]. If materials that
set in situ are used, in addition to good cell seeding, the
cytotoxicity of the reagents and temperature change dur-
ing setting must be minimized to promote cartilage
formation in the construct [28,60].
5. Bioreactors for chondrocyte and cartilage tissue growth
In the past ten years, there have been signi"cant ad-
vances in growing cartilage with properties similar to
those found in vivo, but to transfer this technology to
a clinical setting, scale-up remains a large problem. Both
 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
methods to mass-produce the needed biomaterials, and
large-scale production of cells must be developed. Be-
cause synthesis is very speci"c to the material chosen
(natural vs. synthetic), only large-scale expansion of cells
is reviewed here.
It is estimated that to resurface an entire joint
would require an engineered cartilage implant of the
size 5 cm in diameter (1}5 mm thick), while current
laboratory techniques test constructs only approximately
5 mm in diameter [61]. High-density cell cultures
can be used to prevent chondrocytic dedi!erentiation
that often occurs in two-dimensional cultures [62,63],
but bioreactors present numerous advantages for
growing large amounts of tissue quickly [64]. These
advantages include that bioreactors provide uniform
mixing and precise control over mass transfer rates,
facilitating maintenance of nutrient levels and pH. In
addition, shear stress can be easily monitored in many
types of reaction vessels [64]. It has been found that
shear stress can have a signi"cant impact on the morpho-
logy and thus the mechanical properties of cartilage, so
regulation of this parameter is essential for quality
control of the product [61]. Using process control tech-
niques, the #ow rates in the reactor can be modulated to
maintain important parameters (i.e. nutrient concentra-
tion) over time as the construct becomes less permeable
due to matrix deposition and increased chondrocyte
number [64].
5.1. Spinner yasks
One of the simplest bioreactor designs is the spinner
#ask. Typically, sca!olds seeded with chondrocytes are
attached to needles hanging from the stopper of the #ask.
Media is added to cover the sca!olds and mixing is
maintained with a magnetic stir bar in the bottom of the
#ask. Media is changed every few days to insure high
nutrient concentration. Studies by Freed et al. showed
that constructs cultured in spinner #asks were larger and
contained more cells after "ve weeks than those grown in
Petri dishes [65].
5.2. Microcarriers
To culture individual cells (not seeded in sca!olds) in
a bioreactor, microcarrier suspensions can be used. Vari-
ous types of microcarrier beads, such as collagen or
dextran, have been tested. These carriers are suspended
in a reactor with continuously stirred media and an
inoculum of chondrocytes is added. As in the spinner
#asks, the media is exchanged every few days. Cell doubl-
ing times were shown to be around two to three days in
these bioreactors as compared up to "ve days in Petri
dishes, suggesting that the increased mass transfer in the
bioreactor encouraged cell growth. In addition, the
microcarrier suspension appeared to enhance mainten-
437
ance of the di!erentiated chondrocytic phenotype
[64,66].
5.3. Perfusion culture
While the above types of bioreactors show favorable
results, there is concern that the mechanical mixing may
induce unwanted shear gradients in the reactor vessel. To
eliminate this possibility, microcarriers can be placed in
#uidized bed or air-lift reactors where mixing is achieved
by #uid recirculation rather than impeller motion [64].
Another simple design to improve mass transfer while
eliminating mechanical mixing is the perfusion culture
system. Sca!olds are seeded with cells statically and
subsequently hooked to a perfusion system that uses
a peristaltic pump to deliver a constant #ow of media to
the culture chamber containing the constructs. The me-
dia then exits through another port that empties into
a waste vessel. Good matrix formation and mature chon-
drocytic phenotype was observed after two weeks in
culture [67] and, after nine days, the cellularity in test
constructs was higher than static controls [68]. In
a long-term study, tissue with histological and mechan-
ical characteristics similar to native tissue was evident
after 50 days of perfusion [69]. Similar experiments using
marrow stromal cells indicate that perfusion enhances
growth and function over two weeks [70]. However,
currently, this system is only useful for three-dimensional
expansion of cells in sca!olds.
5.4. Rotating-wall bioreactors
A promising bioreactor design that can be used with
either microcarriers or sca!olds without the concern of
mechanical mixing is the rotating-wall vessel reactor
originally designed by NASA to simulate the e!ects of
microgravity. The most common type is the slow turning
lateral vessel (STLV). The reactor is comprised of two
concentric cylinders. The stationary inner cylinder has
a membrane that allows gas exchange while the outer
cylinder, made from a non-permeable material, rotates.
The space between the two cylinders is perfused continu-
ously with media. Pre-seeded polymer constructs or
chondrocytes and microcarriers are placed inside the
reactor and are kept in a state of constant free-fall by
adjusting the speed of rotation of the outer cylinder so
that the centrifugal force just balances the force of gravity
and the #uid drag on the objects inside. Mixing occurs
due to the small amount of unavoidable settling which
creates movement of the sca!old/microcarrier relative to
the media [61,65].
With microcarrier suspensions, the STLV provided an
environment that encourages aggregation of chon-
drocytes to form tissue. This may be a means to grow
larger sections of cartilage that could be placed directly
into a defect without the use of an implanted matrix to
438 J.S. Temenow, A.G. Mikos / Biomaterials 21 (2000) 431}440
retain the cells [71]. Using sca!olds, when grown in the
STLV for "ve weeks, cell}polymer constructs showed
more GAG production than those placed in spinner
#asks [65]. In further experiments, the STLV constructs
were found to have 68% as much GAG, 33% as much
collagen and similar cellularity to natural cartilage [72].
However, while these constructs demonstrated better
mechanical properties than those grown in other condi-
tions (static and modi"ed spinnner #ask), the equilibrium
modulus, dynamic sti!ness and streaming potential were
only 20}25% as high as fresh cartilage explants [73].
In order to improve the quality of cartilage produced,
researchers have begun to determine optimal operating
parameters for the STLV bioreactor. When rates of oxy-
gen exchange were varied, it was discovered that, al-
though cartilage is generally regarded as a hypoxic tissue,
higher rates of oxygen tension resulted in larger tissue
constructs from pre-seeded polymer sca!olds. These con-
structs also showed higher amounts of ECM synthesis
than those grown at a lower oxygen concentration [74].
However, the STLV may be replaced by a larger rotat-
ing-wall vessel, the high aspect ratio vessel (HARV), to
create the large constructs needed for joint resurfacing.
Because of the size and design di!erences, the HARV has
"ve times as much surface area for gas exchange per unit
of reactor volume as the STLV. This increase in surface
area may be especially important given the previous
"ndings relating construct size to oxygen tension. This
vessel has been used to culture constructs up to 10 cm in
diameter [61].
5.5. Further development
Many in vitro studies have shown that mechanical
forces greatly in#uence the development of cartilage (see
[75] for a review of the e!ects of mechanical forces) [75].
Recently, researchers have begun to combine the above
culture systems with external mechanical stimuli to im-
prove the quality of the cartilage produced. Carver et al.
recently described a perfusion system which applies inter-
mittent hydrostatic pressure to chondrocytes imbedded
in polymer sca!olds. The pressurized sca!olds demon-
strated cartilage development with higher GAG content
after "ve weeks than in samples that were not exposed to
pressure [76].
6. Conclusions
The recent work with bioreactors holds promise that
large numbers of cells can be grown e$ciently and quick-
ly, pushing cell transplantation to the forefront of new
solutions for the problem of articular cartilage injury.
However, many questions remain before tissue engineer-
ing techniques can be used to mass produce cartilage
replacements. Further studies must be conducted to "nd
an optimal polymer for use in the sca!old. Also, the type
of cells (di!erentiated or progenitor) that are best for this
application has yet to be decided. It is the interaction of
these two parameters (material and cells), along with the
possible addition of growth factors and/or mechanical
stimulation to in#uence cellular development, which will
determine the ultimate success of the implant.
As these issues are considered, long-term results are of
utmost concern: no current experimental strategy has
succeeded in consistently producing cartilage that shows
no signs of degeneration after one year. Structure at these
late time points is also extremely important. None of the
studies reviewed here have created a tissue that fully
regenerates the natural zonal organization of articular
cartilage, even after several months in vivo. A "nal hurdle
to full cartilage regeneration is the integration of any
newly formed tissue with the existing cartilage. Without
this integration, forces that occur at the interface could
have deleterious long-term e!ects on the replacement
tissue.
Despite considerable work that must be done to opti-
mize parameters for formation of tissue-engineered
cartilage, because this tissue is not vascularized and in-
corporates only one cell type, it is likely the next tissue
after skin to be successfully regenerated. Further devel-
opment of techniques for chondrocyte transplantation
would be an incredible advance in the "eld, providing
hope for many patients that must endure chronic joint
pain due to failure of conventional methods to restore
cartilage function.
Acknowledgements
The work on skeletal tissue engineering has been
supported by the National Institutes of Health (R29-
AR42639, R01-AR44381, and R01-DE13031). J.S. Te-
meno! acknowledges the "nancial support of a Whitaker
Foundation Graduate Fellowship.
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