Prospects of micromass culture technology in tissue engineering.

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ssBioMed CentHead & Face Medicine
Open AcceReview
Prospects of micromass culture technology in tissue engineering
Jörg GK Handschel1, Rita A Depprich*1, Norbert R Kübler1, Hans-
Peter Wiesmann2, Michelle Ommerborn3 and Ulrich Meyer1
Address: 1Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany,
2Department for Cranio- and Maxillofacial Surgery, Westfälische-Wilhelms-Universität Münster, Waldeyerstr. 30, 48149 Münster, Germany and
3Department for Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf,
Germany
Email: Jörg GK Handschel - handschel@med.uni-duesseldorf.de; Rita A Depprich* - depprich@med.uni-duesseldorf.de;
Norbert R Kübler - kuebler@med.uni-duesseldorf.de; Hans-Peter Wiesmann - depprich@med.uni-duesseldorf.de;
Michelle Ommerborn - ommerborn@med.uni-duesseldorf.de; Ulrich Meyer - ulirich.meyer@med.uni-duesseldorf.de
* Corresponding author
Abstract
Tissue engineering of bone and cartilage tissue for subsequent implantation is of growing interest
in cranio- and maxillofacial surgery. Commonly it is performed by using cells coaxed with scaffolds.
Recently, there is a controversy concerning the use of artificial scaffolds compared to the use of a
natural matrix. Therefore, new approaches called micromass technology have been invented to
overcome these problems by avoiding the need for scaffolds. Technically, cells are dissociated and
the dispersed cells are then reaggregated into cellular spheres. The micromass technology
approach enables investigators to follow tissue formation from single cell sources to organised
spheres in a controlled environment. Thus, the inherent fundamentals of tissue engineering are
better revealed. Additionally, as the newly formed tissue is devoid of an artificial material, it
resembles more closely the in vivo situation. The purpose of this review is to provide an insight into
the fundamentals and the technique of micromass cell culture used to study bone tissue
engineering.
Background
The in vitro formation of bone- or cartilaginous-like tissue
for subsequent implantation [1-3] is, as described, com-
monly performed by using scaffolds. Recently, there is a
controversy (e.g. biocompatibility, biodegradability) con-
cerning the use of artificial scaffolds compared to the use
of a natural matrix [4]. Skeletal defect regeneration by
extracorporally created tissues commonly exploits a three-
dimensional cell-containing artificial scaffold. As indi-
cated before, a number of in vitro studies have been per-
most of these materials were generally shown to allow
spacing of skeletal cells in a three-dimensional space, not
all materials promote the ingrowth of cells within the scaf-
folds [8]. Rather, supporting cellular function depends, as
described, on multiple parameters such as the chosen cell
line, the underlying material, the surface properties and
the scaffold structure. Some in vitro studies indicate that a
material itself may impair the outcome of ex vivo tissue
formation, when compared to a natural tissue-containing
matrix. Additionally, in the in vivo situation defect regen-
Published: 09 January 2007
Head & Face Medicine 2007, 3:4 doi:10.1186/1746-160X-3-4
Received: 31 July 2006
Accepted: 09 January 2007
This article is available from: http://www.head-face-med.com/content/3/1/4
© 2007 Handschel et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 4
(page number not for citation purposes)
formed to evaluate the cell behaviour in various three-
dimensional artificial scaffold materials [5-7]. Whereas
eration can be critically impaired by the immunogenity of
the material, the unpredictable degradation time and by

Head & Face Medicine 2007, 3:4 http://www.head-face-med.com/content/3/1/4
side effects caused by degradation products [4]. Based on
these consideration matrices close to the natural extracel-
lular matrix are regarded as most promising in skeletal tis-
sue engineering by some researchers. A recently
elaborated approach in extracorporal tissue engineering is
therefore the avoidance of non-degradable scaffolds, that
are resorbed at a different time rate than the skeletal tissue
regeneration by itself proceeds. Therefore, new
approaches have been invented to overcome these prob-
lems by renouncing scaffolds.
What is the theory of micromass technique?
It is well known that tissue explants can regenerate com-
plete organisms [9]. Basic research has indicated that
regeneration of simple animals and microtissues can be
achieved by re-aggregation approaches using micromass
technique [10]. Investigations on skeletal development
gave first insight into this micromass biology [11-13]. The
micromass technology relies to a great extent on the pres-
ence of the proteinacious extracellular matrix. As
described before, the extracellular matrix may exert both
direct and indirect influences on cells and consequently
modulate their behaviour. At the same time, these cells
alter the composition of the extracellular matrix. This may
be accomplished in a variety of fashions, including differ-
ential expression of particular extracellular matrix compo-
nents and/or proteases such as metalloproteinases by cells
in the local microenvironment. Whereas most investiga-
tions concerning micromass technology were performed
in developmental studies, only limited literature is availa-
ble concerning the use of this technique in tissue engi-
neering [14]. A large body of evidence has confirmed that
a minimal cell number is required in three-dimensional
tissue-like constructs to induce the differentiation of mes-
enchymal precursors along the chondrogenic and osteo-
genic pathways (reviewed in [15]). In contrast,
mesenchymal precursors seeded in low-density micro-
masses adopt features of a fibroblastic phenotype and
abolish cell differentiation, when mimicking a low-den-
sity condensation [16-18]. These findings indicated that a
"critical" cell mass is necessary to proceed with a specific
extracellular matrix formation. A threshold amount of
precursor cells is necessary to form a three dimensional
extracellular matrix structure around these cell masses
promoting their differentiation. The extracellular matrix
in the microenvironment then interacts with cells to fur-
ther develop towards a specific tissue. The absence of the
requisite extracellular matrix components would lead to
decreased recruitment of precursors to the condensations,
causing a subsequent deficiency in chondrocyte or osteob-
last differentiation. In vitro studies with chondrocytes con-
firmed these findings, showing that the ability of
mesenchymal precursors to initiate chondrogenic differ-
condensation process, which varies by the density of the
condensation [19].
Technical aspects of the micromass technology
In the context of tissue engineering, ex vivo tissue genera-
tion may be optimised by the use of cell re-aggregation
technology. The re-aggregate approach is a method to gen-
erate, in an attempt to mimic the in vivo situation, a tissue-
like construct from dispersed cells, under special culture
conditions. Therefore, the self-renewal (cell amplifica-
tion), spatial sorting and self-organisation of multipoten-
tial stem cells in combination with the self-assembly of
determined cells are the basis for such an engineering
design option. Technically, cells are dissociated and the
dispersed cells are then reaggregated into cellular spheres
[14]. In order to technically refine scaffold-free spheres,
cells are kept either in regular culture dishes (as gravitory
cultures), in spinner flasks, or in more sophisticated bio-
reactors. In contrast to conventional monolayer cell cul-
tures, in which cells grow in only two dimensions on the
flat surface of a plastic dish, suspension cultures allow tis-
sue growth in all three dimensions. It was observed that
cells in spheres exert higher proliferation rates than cells
in monolayer cultures, and their differentiation more
closely resembles that seen in situ. This finding may be
based on the spatial configuration in a three-dimensional
matrix network. Different culture parameters (sizes of the
culture plate, movement in a bioreactor, coating of culture
walls) are all crucial to the process. Roller tube culture sys-
tem have been shown to be suitable for cultivation of tis-
sue explants in suspension. The cultivated and fabricated
tissues may be used for studying the primary mixing of
cells, and the patterns of cell differentiation and growth
within growing spheres in order to improve the outcome
of microsphere cultivation. In addition, some culture con-
ditions could aid the development of high-throughput
systems, and allow manipulation of individual spheres. It
seems worthwhile elaborating new bioreactor technolo-
gies and culture techniques to improve the ex vivo growth
of scaffold-free tissues. Technically, short-term re-aggrega-
tion experiments, which last from minutes to a few hours,
can be distinguished from long-term studies. Short-term
re-aggregation has been used widely to evaluate basic
principles of cell-cell interactions and cell-matrix interac-
tions, whereas long-term cultivation (days to several
weeks) is suitable in ex vivo tissue engineering strategies.
Recent studies on the re-aggregation approach aim to
solve two aspects: to fabricate scaffold-free, three-dimen-
sional tissue formation and at the same time to investigate
basic principles of cellular self-assembly [20,21]. As in
monolayer cultures, which facilitates the study of cell-
material interactions, suspension cultures allow the eval-
uation of cell action towards a three-dimensional space.Page 2 of 4
(page number not for citation purposes)
entiation is dependent upon cell configuration within a The re-aggregate approach enables to follow tissue forma-
tion from single cell sources to organised spheres in a con-

Head & Face Medicine 2007, 3:4 http://www.head-face-med.com/content/3/1/4
trolled environment. Thus, the inherent fundamentals of
tissue engineering are better revealed. Additionally, as the
newly formed tissue is avoid of an artificial material, it
more closely resembles the in vivo situation.
Cell sources for micromass technology
Cells from cartilage and/or bone were found to be a suit-
able cell source for such ex vivo re-aggregate approaches.
Anderer and Libera [1] developed an autologous spheroid
system to culture chondrocytes and osteoblasts without
adding xenogenous serum, growth factors, or scaffolds,
considering that several growth factors and scaffolds are
not permitted for use in clinical applications. It was dem-
onstrated by such an approach that autologous chondro-
cytes and osteoblasts cultured in the presence of
autologous serum form a three-dimensional micro-tissue
that had generated its own extracellular matrix. Chondro-
cyte-based micro-tissue had a characteristic extracellular
space that was similar to the natural matrix of hyaline car-
tilage. Osteoblasts were also able to build up a micro-tis-
sue similar to that of bone repair tissue without collagen-
associated mineral formation. The fabrication of a self-
assembled skeletal tissue seems not to be limited towards
certain species, as results from bovine and porcine
chondrocyte and osteoblast cultivation led to the forma-
tion of species-related cartilage-like or bone-like tissue.
However, conditions allowing cartilage formation in one
species are not necessarily transposable to other species.
Therefore, results with animal models should be cau-
tiously applied to humans. In addition, for tissue-engi-
neering purposes, the number of cell duplications must
be, for each species, carefully monitored to remain in the
range of amplification allowing redifferentiation and
chondrogenesis [22].
It was recently observed, that even complex cellular sys-
tems can be generated ex vivo without the use of scaffolds.
Co-cultures of osteoblasts and endothelial cells for exam-
ple resulted in the formation of a bi-cellular micromass
tissue renouncing any other materials. Other organotypic
cultures, used to develop engineered tissues other then of
skeletal origin, confirm that it is feasible to create tissue
substitutes based on re-aggregated spheres technology.
Examples of these strategies include liver reconstruction,
synthesis of an artificial pancreas, restoration of heart
valve tissue and cardiac organogenesis in vitro [23].
Future prospects and challenges
Several investigations have suggested that after in vivo
transfer of such reaggregates, tissue healing is improved in
sense of a repair tissue that mimics the features of the orig-
inal skeletal tissue [1,24]. Especially preclinical and clini-
cal cartilage repair studies demonstrated that tissue
tissues is able to impair the formation of fibro-cartilage by
suppression of type I collagen expression, while promot-
ing the formation of proteoglycan accompanied by a dis-
tinct expression of type II collagen. It can be assumed that
the volume of the observed repair tissue was formed by
the implanted chondrospheres itself as well as by host
cells located in the superficial cartilage defect. The mecha-
nisms by which chondrospheres promote defect healing
are complex and not completely understood. Van der
Kraan et al. [4] reviewed the role of the extracellular
matrix in the regulation of chondrocyte function in the
defect site and the relevance for cartilage tissue engineer-
ing. Numerous other studies have confirmed that extracel-
lular matrix of articular cartilage can be maintained by a
distinct number of chondrocytes and that the extracellular
matrix plays an important role in the regulation of
chondrocyte function. In in vitro-generated cartilage-like
tissue a time-dependent increase in the expression of col-
lagen type II, S-100, and cartilage-specific proteoglycans,
paralleled by a reduced cell-matrix ratio was observed in
the microspheres [24]. The transplanted cell/matrix com-
plex was attributed to be responsible for the observed
chondrocyte proliferation, differentiation and hyaline car-
tilage-like matrix maturation in vivo.
The inductive properties of the implantation site may also
be beneficial when a stem cell-based micro-tissue strategy
is chosen. Stem cell tissue engineering using fetal or adult
stem cells in combination with sphere technologies leads
to implantable stem cell-driven tissues (unpublished
data). Typically, stem cells must be amplified to large
quantities in suspension cultures and have access to
appropriate growth factors to establish specially organised
histotypical spheres. These spheres can then be implanted
into the lesioned skeletal site. Although adult stem cells of
various origins can transdifferentiate into distinct cell
types, the transformation of these cell types into function-
ing tissues and their successful implantation by re-aggre-
gation technology needs further elaboration.
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