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In Vitro Engineering of Human Autogenous Cartilage

URSULA ANDERER and JEANETTE LIBERA

ABSTRACT

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in

vitroengineered cartilage, such as prevention of immune response and structural as well as functional

integration to surrounding tissue, we established a three-dimensional cell culture system without adding

exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology

and protein expression was analyzed using histological and immunohistochemical investigations on spheroid

cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids

were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the

synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific

mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior

of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids

quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics

to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human

serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim

of cells remained on the surface. These results show that the spheroid culture allows for the first time the

autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to

human serum. (J Bone Miner Res 2002;17:14201429)

Key words: tissue engineering, cartilage, autologous chondrocyte transplantation, in vitro, three-

dimensional

INTRODUCTION

SELF-REPAIR OF human hyaline cartilage does not occur.

Therefore, cartilage injuries initiate a progressive deg-

radation that eventually results in osteoarthritis.(13) An

accepted approach for the regeneration of hyaline cartilage

after traumatic cartilage damage is the autologous chondro-

cyte transplantation.(46) However, the in vitro engineering

of three-dimensional hyaline cartilage tissue with the re-spective structure and function is still a challenge for carti-

lage repair in contrast to using cell suspensions as trans-

plants. For three-dimensional in vitro engineering, cell-

seeded scaffolds have been tested. The in vitro culturing of

chondrocytes in the presence of growth factors on various

three-dimensional scaffolds resulted in the maintenance of

the cartilage-specific phenotype.(79) In animal models,

these cell-seeded scaffolds allowed a formation of repair

tissue similar to hyaline cartilage.(8,10,11) Unfortunately, the

repair is often accompanied by considerable fibrocartilage

formation.(12,13) Further demands on in vitro engineered

tissues are the integration into native tissue and the tissue

formationadapted resorption of scaffolds. However, nei-ther the integration of these cell-seeded scaffolds into sur-

rounding host cartilage, nor predictable resorption of the

scaffold polymers, has been optimized for application in

patients.(14)

Few approaches have been advanced to overcome these

problems by renouncing any scaffolds. The generation ofThe authors have no conflict of interest.

co.don AG, Molecular Medicine, Biotechnology, and Tissue Engineering, Teltow, Germany.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 17, Number 8, 2002 2002 American Society for Bone and Mineral Research

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cartilage-like structures was observed when human chon-

drocytes isolated from osteoarthritic hips were cultured in a

gyratory shaker.(15) However, to maintain the differentiated

phenotype, the addition of growth factors was necessary.

The aim of this study was to engineer three-dimensional

hyaline human cartilage that has the capacity to integrate

with native cartilage and prevent immune responses. For

that purpose, we established an autologous spheroid system

to culture human chondrocytes without adding xenogenous

serum, growth factors, or scaffolds, considering that several

growth factors and scaffolds are not permitted for use in

humans. Only human serum was added to the cell culture

medium. In this autologous spheroid system, cells form

three-dimensional aggregates and generate their own extra-

cellular matrix that is similar to the natural matrix of hyaline

cartilage. These in vitro engineered cartilage tissues adhere

to and integrate into native tissue. We further show that the

addition of fetal calf serum (FCS) or antibiotics to culture

medium delays or even inhibits the engineering of cartilage-

like tissue in vitro. Our methodology describes, for the first

time, the in vitro engineering of three-dimensional autoge-

nous cartilage that seems suitable for the treatment of car-tilage lesions, degenerative changes in cartilage, and phar-

maceutical test systems.

MATERIALS AND METHODS

Chondrocyte culture

Articular cartilage was obtained from human articular

condyles in volunteer patients undergoing knee surgery.

Cartilage (60 100 mg) was minced and digested in a 50-ml

Falcon tube using 20 25 U/mg collagenase type II (Bio-

chrome, Berlin, Germany) at 37C for 8 h in a gyratory

shaker (110 rpm). Isolated cells were washed and resus-

pended in culture medium with the addition of FCS (n 2),autologous serum (n 3), or pooled human serum (n 6)

from separate human volunteers. No growth factors, cyto-

kines, or other supplements were added. Chondrocytes were

seeded in Falcon culture flasks (75 cm2, 15 ml medium) and

maintained at 37C in a humidified atmosphere and 5%

CO2. Medium was changed twice weekly. After reaching

confluence, the cells were trypsinized using trypsin-EDTA

(PAA-Laboratories GmbH, Colbe, Germany) and cultured

in larger Falcon flasks (225 cm2, 35 ml medium). Experi-

ments were performed with chondrocytes between the sec-

ond and seventh monolayer passage. For generation of

spheroids, chondrocytes were seeded in hydrogel-coated

96-well plates. For hydrogel coating, agarose was melted in

cell culture medium (2% wt/vol) and pipetted into the wells.After a jelling time of 2 h at room temperature, cell sus-

pensions (1 105 and/or 2 105 cells/well in 250 l

medium) were added. Starting with 1 105 and 2 105

cells/well should hint at a possible change in size and

quality of cell aggregates. Cell aggregates of the appropriate

experimental set-ups were analyzed after 5 days, 2 weeks,

and 1, 2, and 3 months (n 2). After aggregation of

chondrocytes, 210 single spheroids were transferred into

one well, allowing coalesce of spheroids. For co-culture,

single spheroids were placed on cartilage tissue of isolated

human femoral condyles (medial and lateral, 3.5 2 0.8

cm) from volunteer patients (n 3 patients) undergoing

knee replacement surgery because of osteoarthritis. Con-

dyles with adherend spheroids were surrounded and covered

with medium (50 ml) and cultured in petri dishes under

standard conditions. After different time points (45 minutes,

3 weeks), condyles were frozen and histologically analyzed.

To assess cell growth and cell differentiation in the presence

of antibiotics, chondrocytes and spheroids were cultured in

the addition of penicillin (100 U/ml) and streptomycin (100

mg/ml).

Proliferation analysis

Cell proliferation was assessed by BrdU incorporation

into monolayer (incorporation time: 6 h, 24 h) and aggre-

gated cells (incorporation time: 6 h, 24 h, 2 days) using a

cell proliferation kit (Amersham, Freiburg, Germany).

Three samples per time point were analyzed.

Histological analysis

To analyze chondrocytes in the monolayer culture, cell

suspensions were added to glass slides in appropriate dishes.

The cells adhere and proliferate directly on the glass sur-

face. Slides were washed twice in phosphate buffered saline

(PBS) andfixed in methanol/acetone (1:2) at 20C for 10

minutes. Spheroid specimens were embedded in Tissue-Tek

(Miles, Naperville, IL, USA), snap-frozen in liquid nitro-

gen, and cut into 5- to 7-m sections using a cryomicrotome

(Microm, Walldorf, Germany). Sections were mounted on

pretreated slides (Superfrost Plus; Menzel Glaser, Braun-

schweig, Germany), air dried, and fixed in concentrated

acetic acid:ethanol (1:20) at room temperature (RT) for 20

minutes. Hematoxylin/eosin (HE), safranin O, and Goldner-

trichrome staining were performed on serial sections ofspheroids and monolayer cells directly grown on slides

using standard histochemical techniques.

Immunohistochemical analysis

Collagen and S-100 antigen(16) expression was assessed

on serial sections of snap-frozen spheroids and monolayer

slides using the avidin biotin complex (ABC) method

(DAKO, Hamburg, Germany). As primary antibodies, poly-

clonal rabbit antisera recognizing human types I and II

collagen (1:30 and 1:15; Novo Castra, Newcastle upon

Tyne, UK) and S-100 protein (1:100, DAKO) were used. To

avoid nonspecific binding of the antibodies, slides were first

incubated with tris(hydroxymethyl)aminomethane (TRIS)buffer containing 5% normal porcine serum and 0.1% bo-

vine serum albumin (BSA) at RT for 30 minutes. Incubation

with primary antisera followed at 4C in a humidified cham-

ber for 12 h. After three washes in TRIS, bound primary

antibody was detected using the DAKO ESAB System

AP, with fuchsin as the substrate for alkaline phosphatase

(DAKO). Cell nuclei were counterstained with hematoxy-

lin.

Control procedures paralleled each step. TRIS was ap-

plied to the sections instead of the primary antibodies as a

1421IN VITRO ENGINEERING OF HUMAN AUTOGENOUS CARTILAGE

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control for the secondary antibody. Articular cartilage-bone

sections were used as respective controls for the specificity

of the primary antibodies against collagen type I and II. For

the S-100 antiserum, a neuroblastoma cell line (SK-N-SH;

American Type Culture Collection, Rockville, MD, USA)

andfibroblasts were used as positive and negative controls,

respectively.

RESULTS

Spheroid formation

For in vitro engineering of three-dimensional hyaline

cartilage tissue, we investigated the differentiation of hu-

man chondrocytes in aggregate culture. After seeding chon-

drocytes in hydrogel-coated wells, cells aggregated to form

a disc measuring 900-1200 m in diameter after 1 day.

During the next 2 weeks, discs rounded and became more

compact, with diameters ranging from 350 to 500 m (Figs.

1A and 1B). Coalescence of several spheroids was initiated

by an active migration of surface cells of adjacent aggre-

gates (Fig. 1B). Through 8 days, remodeling of the aggre-gates resulted in a more homogenous spherical structure,

and gaps between aggregates werefilled (Fig. 1C). Separate

from the ability to merge, aggregates also migrated and

contacted tissue culture flasks or glass slides (Fig. 1D).

Spheroid morphology and differentiation

During the initial 2 weeks, spheroid chondrocytes pro-

duced high amounts of acidic mucopolysaccharides, indi-

cated by the green intercellular matrix on Goldner-stained

sections (Fig. 2B). Aggregates cultured in medium with

autologous and pooled serum revealed a homogenous dis-

tribution of intact spherical cells in the interior and flattened

cells on the surface (Figs. 2A, 2B, and 3A3C). Viable cellsand round nuclei in the interior of spheroids suggest that

sufficient nutrient supply is available for all cells. As indi-

vidual aggregates merged, an adhesion zone consisting of

several flattened cell layers between two aggregates was

still observable after 24 h in close contact (Fig. 2A, arrow-

head). However, by 5 days, these cells had achieved a

histotypical morphology similar to that of internal cells

(Fig. 2A, arrow).

Chondrocytes in monolayer culture loose the expression

of collagen type II and the cartilage specific intracellular

protein S-100(16) completely during their third or fourth

passage, corresponding with a high expression of collagen

type I (data not shown). However, when chondrocytes of the

second to the seventh monolayer passage were cultured asspheroids, a reexpression of collagen type II and S-100

occurred (Figs. 3A and 3B). This chondrocyte differentia-

tion was accompanied by the loss of cell proliferation,

FIG. 1. Chondrocyte aggregates cultured in autologous serum after

different times. (A) Ball-shaped aggregate after 4 days. (B) Merge of

three 16-day-old aggregates after 2 days: cells stretching from one

aggregate to another (arrow). (C) Merged aggregates from B, 8 days

later: former gap is filled with cells (arrow). (D) Cell emigration from

aggregate to an artificial surface after 2 days. (AC) Live cell aggre-

gates and (D) immunohistochemistry of collagen type I.

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analyzed by BrdU incorporation into cell nuclei (data not

shown). In the presence of autologous serum, a weak ex-

pression of collagen type II in the outer cell layer of sphe-

roids was detectable after 1 week. After 6 weeks, a weak

expression of collagen type II was also observed in the

interior (Fig. 3A). In medium supplemented with allogenous

pooled human serum, reexpression of collagen type II in the

outer cell layer was delayed and not as high as in autologous

culture. Collagen type II was detectable only after the third

week in culture (data not shown). The S-100 reexpression

was generally similar to that of collagen type II under

autologous and human pooled serum conditions (Fig. 3B).

In contrast to the high expression of collagen type I in all

cells in monolayer culture, a reduced expression or com-

plete loss of collagen type I was found in the interior cells

of the aggregate (Fig. 3C). Throughout the time course ofthree-dimensional culture, and independent of serum type,

the outer cell layers expressed the highest amount of colla-

gen type I. The expression of collagen type I was higher

than that of collagen type II.

Under prolonged culture conditions, the in vitro gener-

ated tissues more closely resembled the typical features of

in vivo cartilage: solid and elastic aggregates with a bright

white surface (Fig. 4A). Cryosections revealed flattened

cells on the surface (Figs. 4B and 4E) and an increased

amount of intercellular matrix in the core of spheroids (Figs.

4B 4D). This core area stained positive with safranin O,

affirming the synthesis and extracellular deposition of hya-

line cartilage specific proteoglycans (Fig. 4B). Cell number

per given matrix area varied with the depth in the aggregate.In the core area, cells were widely separated by matrix, and

the cell number per given matrix area was approximately

twice that of native cartilage (Figs. 4B and 4F). Nearer the

surface, the number of cells per matrix area increased and

was twice that of the core. In contrast to the randomly

distributed cells in the core area of aggregates, cells on both

sides of a merging zone were oriented vertical to the contact

area (Fig. 4B). This orientation is known for native articular

cartilage where chondrocytes are oriented perpendicular to

the tidemark.

Influence of antibiotics and FCS on cell condition

Usually, antibiotics are added to cell culture medium. In

the presence of penicillin and streptomycin in chondrocyte

monolayer cultures with autologous serum, we observed a

prolongation of culture time to reach confluence compared

with the absence of antibiotics. Furthermore, the aggrega-

tion of chondrocytes was delayed for up to 5 days (data not

shown). During thefirst 4 weeks in aggregation culture, the

expression of cartilage-specific proteins and the cell density

were similar to aggregates cultured in the absence of anti-

biotics. However, there is no augmentation in the expression

of collagen type II, S-100, and acidic mucopolysaccharides

in aggregated chondrocytes up to 3 months (data not

shown). This parallels the still high and unchanged cell

density in spheroids cultured in the presence of antibioticsobserved after 3 months (data not shown).

While growing human chondrocytes in monolayer and

aggregate culture in addition of FCS, several differences

were apparent. In the monolayer, the proliferation rate of

chondrocytes decreased (data not shown). Furthermore,

when cells were transferred in aggregate culture, the

aggregation of cells was delayed significantly. By 7 days,

a central hole in the aggregate was present, and surface

cells were more densely packed (Fig. 5A). During the

first 6 weeks in aggregate culture with FCS, no quanti-

tative differences in the expression of collagen type I and

polysaccharides compared with autologous cultured ag-

gregates were found (data not shown). However, theexpression of collagen type II and S-100 protein was

delayed and reduced with only a weak expression after 4

weeks (Fig. 5B, shown for collagen type II). After 3

months in FCS culture, the inner part of the aggregates

was disintegrated and crumbled. HE staining revealed the

absence of viable cells. Only outer cells retained the

typical morphology and expressed type I collagen and

S-100 (Figs. 5C and 5D). However, cells and matrix were

loosely organized, and a shedding of the outer cells was

detected (Figs. 5C and 5D).

FIG. 2. Histological analysis of chondrocyte (seven passages in monolayer) aggregates cultured in human pool serum for 16 days. (A) HE

staining: difference in cell morphology in the inner (spherical cells) and outer part ( flattened cells) of the aggregate. Flattened cells in the adhesion

zone after 1 day (arrowhead) and spherical cells after 5 days (arrow). Gap between two aggregates (two arrowheads) was filled with cells after

5 days (two arrows). (B) Goldner-stained sections of aggregates from A.


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Aggregate attachment

To assess the integrative capacity of in vitro engineered

tissues, a cartilage-explant spheroid co-culture system was

used. After only 45 minutes, multiple focal adhesion points

were formed connecting the in vitro generated tissue with

native cartilage (Fig. 6A). Surface cells of the 3-week-old

aggregates in the contact area had already changed their

morphology fromflattened to spherical cells. However, the

cartilage on the opposing side of the aggregate retained its

flattened surface cells (Fig. 6A). Over the course of 3 weeks

in culture, the spheroids became more flattened. By active

migration, aggregate cells were widely distributed on the

surface of the degenerated cartilage. The cells not only

migrated on the native cartilage surface but also synthesized

new matrix (Fig. 6B). The migratory capacity enabled chon-

drocytes to integrate in surface fissures in addition to cov-

ering the surface (Fig. 6B). No active invading growth of

spheroid derived cells was observed. The newly formed

matrix on the cartilage explant surface is characterized by

stronger HE staining (Fig. 6B). This layer shows a higher

cell density than native tissue, indicating the cartilage form-

ing capacity of spheroids.

DISCUSSION

This study shows the potential to use a three-dimensional,

autologous in vitro culture system to engineer human artic-

ular cartilage. The formation of three-dimensional cartilage-like tissue was achieved without using any scaffolds. The

only supplement to culture medium was patient-specific

serum. No growth factors or other additives were used to

induce chondrogenic differentiation and maintain long-term

stability of the tissue constructs. The engineered tissue

constructs attach, migrate, and integrate with native tissues,

thereby meeting important requirements for tissue recon-

struction and/or regeneration.

For generating cartilage in vitro that typifies not only the

morphology but also sustains the physiological behavior of

chondrocytes, it is necessary to culture the cells in a three-

dimensional arrangement.(17,18) It is known that a three-

dimensional arrangement leads to a specific cell shape and

environmental conditions determining gene expression andbehavior of cells.(17) The present work shows that a close

three-dimensional contact of human spherical chondrocytes

enables them to arrange themselves in three-dimensional

cell aggregates, to synthesize cartilage-specific proteins and

matrix components, and to deposit the components in the

intercellular space (Fig. 4). This behavior parallels the nat-

ural process of chondrogenesis: aggregation of chondropro-

genitor cells followed by the synthesis of a cartilaginous

extracellular matrix.(19) Therefore, it is obvious that the

cellular environment and cell shape of human chondrocytes

in aggregate culture is responsible for this histotypical or-

ganization.

A multitude of studies have been done using different

ways to culture chondrocytes in three-dimensional systems.First of all, various scaffold materials were used to create a

three-dimensional system (e.g., agarose, alginate, collagen,

fibrin glue, polyglycolic acid [PGA], and polylactide acid

[PLA]).(2026) All these cell-seeded scaffolds have the

growing of cells in a three-dimensional environment accom-

panied by a regaining or maintenance of cartilage-specific

features in common. However, with respect to clinical ap-

plication, naturally occurring xenogenous and allogenous

materials (e.g., type I collagen, hyaluronic acid, or fibrin

glue) may be immunogenic and bear safety risks, and their

FIG. 3. Immunohistochemical analysis of chondrocyte (three pas-

sages in ML) aggregates cultured in autologous serum for 6 weeks. (A)

Collagen type II, (B) S-100, and (C) collagen type I cells were immu-

nolocalized on frozen sections counterstained with hematoxylin.


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application is partially forbidden by the Drug Act. One

attempt to resolve these problems is the use of atelocolla-

gen, where the antigenic determinants on the peptide chain

of type 1 collagen (telopeptide) are removed.(9,11) The phe-

notype of freshly isolated chondrocytes could be maintained

in the atelocollagen gel, and a cartilage-like tissue devel-

oped after implantation in rabbit and in human. However,

L-ascorbic acid is necessary to culture the cell-seeded scaf-

folds, and patients have to be tested concerning their allergic

reaction to atelocollagen. Furthermore, chondrocytes weredirectly seeded into the gel after isolation from the biopsy,

wherefore a rather large cartilage specimen has to be har-

vested from healthy cartilage tissue from the patient. Other

scaffolds are not biodegradable or are resorbed with a

greater time constant than cartilage regeneration (e.g., hy-

aluronic acid).(27) During this resorption process, polymer

scaffolds may produce harmful degradation products.(28)

Furthermore, the integration of the cell-seeded scaffold to

the adjacent normal cartilage is sometimes not shown or

often incomplete.(11,14,29)

With the aim to avoid scaffold materials for three-

dimensional culture, chondrocytes were grown in micro-

mass or high-density systems. However, cartilage-like mor-

phology and reexpression of cartilage-specific proteins

could only be maintained for up to 4 weeks and only in the

presence of transforming growth factor 1 (TGF-1), bone

morphogenetic protein (BMP)-2, or ascorbic acid.(3036)

Additionally, most of the growth factors are not permitted

for the processing of human cell based drugs. Using our

described autologous spheroid culture system, neither theaddition of scaffolds, growth factors, or cytokines nor phys-

ical manipulations were necessary to induce the formation

of stable cell aggregates and the specific chondrogenic

phenotype of cells.

The in vitro generated cartilage-like tissue is character-

ized by a time-dependent increased expression of collagen

type II, S-100, and cartilage-specific proteoglycans, paral-

leled by a reduction of the cell-matrix-ratio. This indicates

a progressive phenotypical differentiation of chondrocytes

and a potential for matrix maturation. The extracellular

FIG. 4. In vitro engineered hu-

man cartilage after 3 months cul-

tured in autologous serum. (A)

Live tissue (1.8 1.5 0.5

mm). (B) Safranin O staining:

cells in the core are widely sepa-

rated by extracellular matrix

mainly consisting of hyaline-

cartilage specific proteoglycans

(arrow). In the outer regions, the

matrix deposition is reduced (ar-

rowhead). (CE) Immunolocal-

ization of different proteins coun-

terstained with hematoxylin: (C)

collagen type II, (D) S-100 pro-

tein, and (E) collagen type I in the

outer region and the inner part of

the aggregate. (F) HE staining of

native cartilage.


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matrix deposition starts in the core of the spheroids

(Fig. 4B), resulting in a gradient of matrix deposition. This

initial pattern of matrix deposition is also observed in stud-

ies using chondrocyte-seeded scaffolds, where the gradient

is equalized in long-time culture.(18) Independent of culture

time, the locally different matrix production in spheroids is

paralleled by a heterogenous cell morphology comparable

with native cartilage tissue: round cells in deeper regions

andflattened cells in the outer zone.(37) This stable hyaline-like in vitro tissue morphology indicates optimized tissue

culture conditions.

Keeping in mind that tissue engineering based therapies

necessitate high amounts of cells, but only small biopsy

specimens with a low yield of cells are available, an aug-

mentation of cell number before a tissue engineering pro-

cess is unavoidable. Additionally, transferring a small

amount of freshly isolated chondrocytes directly into a

three-dimensional system leads to a proliferation stop (see

Results), resulting in an insufficient cell number and density

for tissue regeneration processes.(38) Expanding chondro-

cytes in monolayer culture results in the loss of their

cartilage-specific phenotype and matrix protein expression,

and a modified cell behavior (e.g., responsiveness to growthfactors).(3942) Our experiments also show that human

chondrocytes cultured as a monolayer shift their collagen

expression from type II to type I, paralleled by a loss of the

intracellular protein S-100. However, even after seven pas-

sages in monolayer culture, chondrocytes restored their

cartilage-specific phenotype after transferring into autolo-

gous three-dimensional culture. The reexpression of colla-

gen type II shows that the shift in collagen expression is

only a transient phenotype. Therefore, the monolayer cul-

ture seems to be a useful tool for cell expansion. (43)

To increase the size of in vitro cartilage-like tissue, the

initial cell number could be changed or the ability of ag-

gregates to coalesce could be used. From work done in

tumor spheroid cultures, it is known that nutrient diffusion

is not sufficient to maintain viability of core cells in sphe-

roids larger than 800-1000 m in diameter.(4446) For that

reason, this study focused on aggregates smaller than 800

m in height. When smaller aggregates were fused, the

fusion process was mediated by the flattened surface cells

(Fig. 2A). Morphological changes of chondrocytes accom-

panying the coalescence of spheroids indicate the potential

of the cells to adapt to changed environmental conditions.

Furthermore, the migration of outer spheroid chondrocytes

on artificial surfaces, as well as on native tissue, showed a

potentially capacity of spheroids to adhere and integrate

with appropriate structures such as cartilage. This integra-

tive property of spheroidal in vitro cartilage fulfills one of

the major challenges confronting in vitro engineered tis-

sues in regenerative medicine.

Standard cell culture procedures use FCS as medium

supplement because of the limited availability of human

autologous serum. After replacing autologous serum byFCS in the spheroid culture system, aggregates failed to

produce stabile compact spheroids with the chondrocyte-

specific phenotype. Neither long-time stability of aggre-

gates, viability of core chondrocytes, nor cartilage-specific

protein expression could be maintained (Figs. 5C and 5D).

It is assumed that the deviating composition and/or amount

of serum components(47) (e.g., in regard to growth factors,

hormones, or further xenogenous proteins) are responsible

for the altered aggregation, differentiation, and finally the

death of human chondrocytes (Fig. 5).

F IG. 5 . Chondrocyte aggre-

gates after different time points in

FCS culture. (A) Five-day-old

aggregate still with a hole in the

core. (B) Four-week-old aggre-

gate with only 1-week expression

of collagen type II in the interior.

(C and D) Immunolocalization of

different proteins in 3-month-old

aggregates. (C) S-100 protein

only expressed in the vital rimand (D) collagen type I expressed

in the vital rim and the merging

zone.


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Further standard supplements in cell culture systems are

antibiotics to suppress bacterial contamination. The addition

of antibiotics to autologous/allogenous culture medium al-

lowed cells in spheroids to survive, but a higher cell/matrix

ratio was displayed compared with those cultures main-

tained in the absence of antibiotics. This inhibited differen-

tiation of chondrocytes may be caused by the inhibition of

protein synthesis by streptomycin or an effect of penicillin

on extracellular matrix deposition.(48) Taking together, these

results show that in vitro three-dimensional explant or tissue

engineering studies using human cells or tissues may beoptimized by the presence of donor-specific serum and the

absence of antibiotics. However, the availability of donor-

specific serum is limited. Our results also showed that the

supplementation of cell culture medium with pooled human

serum is a suitable alternative for in vitro tissue engineering

studies.

Prior uses of the three-dimensional spheroid culture sys-

tem are well established in tumor biology, where cells are

cultured as multicellular tumor spheroids (MTS). MTS were

developed to maintain tumor cell physiology in vitro that is

altered in monolayer culture.(49,50) Important differences

between MTS and nontumorigenic spheroids are the prolif-

eration and invasion into neighboring tissue. Tumor cells in

the rim of the MTS continue to divide, resulting in a

continuous growth of spheroids. In contrast, proliferation of

human chondrocytes is inhibited when cultured as sphe-

roids. In contrast to MTS cells, cells from chondrocyte

spheroids do not invade adjacent tissue.(51) Results show

that chondrocytes from spheroids seem to have the capacity

to migrate on the surface of tissue explants and recover

osteoarthriticfissures.

This aggregate culture system is a very effective method

to generate in vitro cartilage-like tissue without using any

scaffold, growth factors, or further additives. Using the

aggregate culture technique supplemented only with autol-

ogous serum, chondrocytes formed a hyaline-like three-

dimensional cell-matrix arrangement. The in vitro engi-

neered tissues are characterized by a long-time stability and

show the capacity for integration with native tissue. Reach-

ing a size of approximately 1 mm, the in vitro tissues are

suitable for clinical use, pharmaceutical test systems, and

scientific studies.At this stage of our investigations, underlying mecha-

nisms of morphology and physiology of cells and matrix

maturation in spheroids are not known. Further studies

should clarify if the different environmental conditions of

surface/core cells and a gradient of nutrients, oxygen, and

metabolics within the spheroids are responsible for this

cartilage engineering process.

ACKNOWLEDGMENTS

We are very grateful to Dr. Tim Ganey (Medical Center,

Atlanta, GA, USA) for discussion and helpful comments

and to Prof. A. Herrmann (Humboldt University of Berlin,

Institute of Biology/Biophysics, Berlin, Germany) for crit-

ical discussion and referring the manuscript.

REFERENCES

1. Buckwalter JA, Lane NE 1997 Athletics and osteoarthritis.Am J Sports Med 25:873 881.

2. Gold GE, Bergman AG, Pauly JM, Lang P, Butts RK, Beau-lieu CF, Hargreaves B, Frank L, Boutin RD, Macovski A,Resnick D 1998 Magnetic resonance imaging of knee cartilagerepair. Top Mag Reson Imag 9:377392.

3. Saxon L, Finch C, Bass S 1999 Sports participation, sportsinjuries and osteoarthritis: Implications for prevention. SportsMed 28:123135.

4. Gillogly SD, Voight M, Blackburn T 1998 Treatment of ar-ticular cartilage defects of the knee with autologous chondro-cyte implantation. J Orthop Sports Phys Therap 28:241251.

5. Mayhew TA, Williams GR, Senica MA, Kuniholm G, DuMoulin GC 1998 Validation of a quality assurance program forautologous cultured chondrocyte implantation. Tissue Eng4:325334.

6. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-JanssonE, Lindahl A 2000 Two- to 9-year outcome after autologouschondrocyte transplantation of the knee. Clin Orthop 374:212234.

7. Temenoff JS, Mikos AG 2000 Review: Tissue engineering forregeneration of articular cartilage. Biomaterials 21:431 440.

FIG. 6. Three-week-old chondrocyte aggregates after different time

points in co-culture with human condyle from an osteoarthritic patient

(HE staining). (A) After 45 minutes, multiple focal adhesion points

were formed, and surface cells of the aggregate have already changed

their morphology from flattened to spherical cells (arrows). (B) After

18 days, the aggregates became flattened; new matrix was synthesized.

This new cell-rich matrix (filled arrowhead) differs from condyle

cartilage because it has lower cell density (open arrowhead). Cells of

the in vitro aggregates integrated well into the in vivo condyle cartilage

(arrows).


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8. Hunziker EB 1999 Biologic repair of articular cartilage. Defectmodels in experimental animals and matrix requirements. ClinOrthop 367(Suppl):S135S146.

9. Uchio Y, Ochi M, Matsuaki M, Kurioka H, Katsube K 2000Human chondrocyte proliferation and matrix synthesis cul-tured in atellocollagen gel. Inc J Biomed Mater Res 50:138 143.

10. Grande DA, Pitman MI, Peterson L, Menche D, Klein M 1989The repair of experimentally produced defects in rabbit artic-ular cartilage by autologous chondrocyte transplantation. J Or-thop Res 7:208 218.

11. Ochi M, Uchio Y, Tobita M, Kuriwaka M 2001 Currentconcepts in tissue engineering technique for repair of cartilagedefect. Artif Organs 25:172179.

12. Sittinger M, Perka C, Schultz O, Haupl T, Burmester GR 1999Joint cartilage regeneration by tissue engineering. Z Rheuma-tol 58:130 135.

13. Frenkel SR, Di Cesare PE 1999 Degradation and repair ofarticular cartilage. Front Biosci 4:D671D685.

14. Sims CD, Butler PE, Cao YL, Casanova R, Randolph MA,Black A, Vacanti CA, Yaremchuk MJ 1998 Tissue engineeredneocartilage using plasma derived polymer substrates andchondrocytes. Plast Reconstr Surg 101:1580 1585.

15. Bassleer C, Gysen P, Foidart JM, Bassleer R, Franchimont P

1986 Human chondrocytes in tridimensional culture. In VitroCell Dev Biol 22:113119.16. Stefansson K, Wollmann RL, Moore BW, Arnason BG 1982

S-100 protein in human chondrocytes. Nature 295:63 64.17. Landry J, Bernier D, Ouellet C, Goyette R, Marceau N 1985

Spheroidal aggregate culture of rat liver cells: Histotypic re-organization, biomatrix deposition, and maintenance of func-tional activities. J Cell Biol 101:914 923.

18. Freed LE, Hollander AP, Martin I, Barry JR, Langer R,Vunjak-Novakovic G 1998 Chondrogenesis in a cell-polymer-bioreactor system. Exp Cell Res 240:58 65.

19. Buckwalter JA, Mankin HJ 1997 Articular cartilage. Part I:Tissue design and chondrocyte-matrix interactions. J BoneJoint Surg Am 79:600 611.

20. Benya PD, Shaffer JD 1982 Dedifferentiated chondrocytesreexpress the differentiated collagen phenotype when cultured

in agarose gels. Cell 30:215224.21. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, BourguignonJ, Lasselin C, Feisinger P 1994 Reexpression of cartilage-specificgenes by dedifferentiated human articular chondrocytes culturedin alginate beads. Exp Cell Res 212:97104.

22. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, BlockJA, Aydelotte MB, Kuettner KE, Thonar EJ 1994 Phenotypicstability of bovine articular chondrocytes after long-term cul-ture in alginate beads. J Cell Sci 107:1727.

23. Kimura T, Yasui N, Ohsawa S, Ono K 1987 Chondrocytesembedded in collagen gels maintain cartilage phenotype dur-ing long-term cultures. Clin Orthop 186:231239.

24. Homminga GN, Buma P, Koot HWJ, van der Kraan PM, vanden Berg WB 1993 Chondrocyte behaviour in fibrin glue invitro. Acta Orthop Scand 64:441 445.

25. Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy

DC, Barlow SK, Langer R 1994 Biodegradable polymer scaf-folds for tissue engineering. Biotechnology 12:689 693.

26. Sittinger M, Reitzel D, Dauner M, Hierlemann H, Hammer C,Kastenbauer E, Planck H, Burmester GR, Bujia J 1996 Resorb-able polyesters in cartilage engineering: Affinity and biocom-patibility of polymer fiber structures to chondrocytes.J Biomed Mater Res 33:57 63.

27. Lindenhayn K, Perka C, Spitzer R, Heilmann H, PommereningK, Mennicke J, Sittinger M 1999 Retention of hyaluronic acidin alginate beads: Aspects for in vitro cartilage engineering.J Biomed Mater Res 44:149 155.

28. Hutmacher DW 2000 Scaffolds in tissue engineering bone andcartilage. Biomaterials 21:2529 2543.

29. Nehrer S, Breinan HA, Ramappa A, Hsu HP, Minas T, Short-kroff S, Sledge CB, Yannas IV, Spector M 1998 Chondrocyte-seeded collagen matrices implanted in a chondral defect in acanine model. Biomaterials 19:23132328.

30. Hering TM, Kollar J, Huynh TD, Varelas JB, Sandell LJ 1994Modulation of extracellular matrix gene expression in bovinehigh-density chondrocyte cultures by ascorbic acid and enzy-matic resuspension. Arch Biochem Biophys 314:90 98.

31. Ronziere MC, Farjanel J, Freyria AM, Hartmann DJ, HerbageD 1997 Analysis of types I, II, III, IX and XI collagenssynthesized by fetal bovine chondrocytes in high-density cul-ture. Osteoarthritis Cartilage 5:205214.

32. Ruggiero F, Petit B, Ronziere MC, Farjanel J, Hartmann DJ,Herbage D 1993 Composition and organization of the collagennetwork produced by fetal bovine chondrocytes cultured athigh density. J Histochem Cytochem 41:867 875.

33. Amadio PC, Ehrlich MG, Mankin HJ 1983 Matrix synthesis inhigh density cultures of bovine epiphyseal plate chondrocytes.Connect Tissue Res 11:1119.

34. Stewart MC, Saunders KM, Burton-Wurster N, Macleod JN2000 Phenotypic stability of articular chondrocytes in vitro:The effects of culture models, bone morphogenetic protein 2,and serum supplementation. J Bone Miner Res 15:166 174.

35. Kameda T, Koike C, Saitoh K, Kuroiwa A, Iba H 2000

Analysis of cartilage maturation using micromass cultures ofprimary chondrocytes. Dev Growth Differ 42:229 236.36. Denker AE, Nicoll SB, Tuan RS 1995 Formation of cartilage-

like spheroids by micromass cultures of murine C3H10T1/2cells upon treatment with transforming growth factor-beta 1.Differentiation 59:2534.

37. Buckwalter JA, Rosenberg LC, Hunziger EB 1990 Articularcartilage: Composition, structure, response to injury, andmethods of facilitating repair. In: Ewing JW (ed.) ArticularCartilage and Knee Joint Function: Basic Science and Arthro-scopy. Raven Press, New York, NY, USA, pp. 19 56.

38. Anderer U 1992 Investigations of Cell Proliferation in Sphe-roids: Analysis of cAMP and Cell Cycle Distribution in Re-lation to Cell Communication. Doctoral thesis. Research Cen-ter Karlsruhe, Karlsruhe, Germany.

39. Grundmann K, Zimmermann B, Barrach HJ, Merker HJ 1980

Behaviour of epiphyseal mouse chondrocyte populations inmonolayer culture. Morphological and immunohistochemicalstudies. Virchows Arch [Pathol Anat] 389:167187.

40. Lefebvre V, Peeters-Joris C, Vaes G 1990 Production of col-lagens, collagenase and collagenase inhibitor during the ded-ifferentiation of articular chondrocytes by serial subcultures.Biochim Biophys Acta 1051:266 275.

41. Blanco FJ, Geng Y, Lotz M 1995 Differentiation-dependenteffects of IL-1 and TGF-beta on human articular chondrocyteproliferation are related to inducible nitric oxide synthaseexpression. J Immunol 154:4018 4026.

42. Guerne PA, Sublet A, Lotz M 1994 Growth factor responsive-ness of human articular chondrocytes: Distinct profiles inprimary chondrocytes, subcultured chondrocytes, and fibro-blasts. J Cell Physiol 158:476 484.

43. Rudert M, Hirschmann F, Schulze M, Wirth CJ 2000 Bioar-

tificial cartilage. Cells Tissues Organs 167:95105.44. Muller Kliesner W 1987 Multicellular spheroids. A review on

cellular aggregates in cancer research. J Cancer Res ClinOncol 113:101122.

45. Sutherland RM 1988 Cell and environment interactions intumor microregions: The multicell spheroid model. Science240:177184.

46. Carlsson J, Acker H 1988 Relations between pH, oxygenpressure and growth in cultured cell spheroids. Int J Cancer42:715720.

47. Serum-free systems, module 2C:0 1998 In: Doyl A, NewellDG (eds.) Cell and Tissue Culture: Laboratory Procedures.John Wiley & Sons, West Sussex, England.


8/10/2019 suruh

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48. Buddecke E 1989 Hemmstoffe der Nukleinsaure- und Protein-biosynthese. In: Buddecke E (ed.) Grundri der Biochemie.Walter de Gruyter, Berlin, Germany, pp. 114 116, 199 201.

49. Sutherland RM, McCredie JA, Inch WR 1971 Growth ofmulticell spheroids in tissue culture as a model of nodularcarcinomas. J Natl Cancer Inst 46:113120.

50. Santini MT, Rainaldi G 1999 Three-dimensional spheroidmodel in tumor biology. Pathobiology 67:148 157.

51. Brauner T, Hulser DF 1990 Tumor cell invasion and gap

junctional communication. II. Normal and malignant cellsconfronted in multicell spheroids. Invasion Metasasis 10:3148.

Address reprint requests to:Dr. Jeanette Libera

co.don AG

Molecular Medicine, Biotechnology,

and Tissue Engineering

Warthestrasse 21

D-14513 Teltow, Germany

Received in original form August 31, 2001; in revised form March11, 2002; accepted April 3, 2002.


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