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Monolithic Polymers for Cell Cultivation Differentiation and Tissue Engineering.

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DOI: 10.1002/anie.200801872
Tissue Engineering
Monolithic Polymers for Cell Cultivation, Differentiation, and Tissue
Andrea Lber, Andreas Verch, Bettina Schlemmer, Sandra Hfer, Bernhard Frerich,* and
Michael R. Buchmeiser*
Tissue engineering,[1–6] that is, the in vitro seeding of scaffold
materials with living cells and their subsequent implantation
into a defect site, has been proposed in the rehabilitation of
numerous instances of tissue loss or deficiency. Various
strategies have been developed to regenerate bone and
other tissues. These include techniques focusing on molecular,
cellular, and material developments. At a cellular level,
mesenchymal stem cells (MSCs) from various sources have
proven to be appropriate for regeneration of bone and other
tissues.[7–9] Bioreactor systems have been applied to aid the
seeding process. An essential prerequisite, however, is the
availability of suitable scaffold materials that demonstrate
biocompatibility, appropriate biodegradability, mechanical
strength, and allow for neovascularization. Failures in first
clinical studies are presumably related to insufficiencies of
current available scaffold materials, among other things.[8, 10]
Scaffolds in tissue engineering function as matrixes for
delivery of the cells to the defect site. Open structures with
large pores are typically used if the new tissue is expected to
integrate within the host tissue.[7] The morphology of the
scaffold is intended to guide the structure of the engineered
tissue[11] with regard to size, shape, and vascularization.[12] The
use of biodegradable polymers in tissue engineering is
especially attractive.[13] Thus, bioresorption or degradation
of these materials minimizes the foreign-body response and
leads to the formation of a completely natural tissue. The
design and tailor-made synthesis of biodegradability polymers
with suitable mechanical properties is an attractive challenge
[*] Prof. Dr. B. Frerich
Department of Oral and Maxillofacial Surgery
Plastic Facial Surgery, University of Leipzig
and NovaTissue GmbH, Leipzig (Germany)
Homepage: ~ mkg/
A. L9ber, A. Verch, B. Schlemmer, Prof. Dr. M. R. Buchmeiser
Leibniz-Institut f<r Oberfl=chenmodifizierung e.V. (IOM)
Permoserstrasse 15, 04318 Leipzig (Germany)
Fax: (+ 49) 341-235-2584
Prof. Dr. M. R. Buchmeiser
Institut f<r Technische Chemie, Universit=t Leipzig
LinnCstrasse 3, 04103 Leipzig (Germany)
S. H9fer
NovaTissue GmbH, Leipzig (Germany)
[**] This work was supported by the Federal Government of Germany
and the Freistaat Sachsen.
Supporting information for this article is available on the WWW
in tissue engineering. Our objective was to address it in an
interdisciplinary approach with a focus on bone and adipose
tissue replacement.
In search of suitable scaffolds for tissue engineering, we
focused on monolithic supports. Monolithic media represent
uniformly structured matrixes with large interpenetrating
pores, usually in the low micrometer range. During the last 15
years, polymer-based monolithic supports have been introduced into separation science as well as into organic catalysis
and biocatalysis. Major contributions have been made by
Fr6chet, Ŝvec, and co-workers, who initially developed
radical-polymerization-based systems for these purposes.[14]
Our group contributed to this area by developing an approach
based on ring-opening metathesis polymerization (ROMP),
which allowed for a one-pot synthesis of functionalized
monolithic systems. Using this synthetic protocol, a large
variety of monolithic systems have been realized.[15–18]
Recently, we extended this chemistry to the electron-beam
(EB)-triggered synthesis of highly porous monolithic materials.[19]
The use of monolithic supports for tissue engineering has
not been reported to date; instead, sol-gel-derived glasses,
foamed or electrospinning-derived polymers, polymer templating, or plotting techniques have been applied.[20–23] However, a major advantage of monolithic supports is that they
may be created in virtually any form by a simple molding
process. This allows for creating entire parts of the body.
Generally, to be feasible for applications cell cultivation,
differentiation, or tissue engineering, a support has to fulfill
certain criteria. First, it needs to possess a highly porous
structure with pore diameters around 150 mm. Only pores of
such diameters allow for both the substantial ingrowth of the
cells and for vascularization at a comparably late stage of cell
proliferation. Second, the matrix itself needs to be biocompatible. On the one hand, this requires a surface chemistry
that favors cell contact and adhesion. On the other hand, the
polymer must not contain or form, in the course of the
degradation process, any compounds that exhibit cell toxicity.
Furthermore, the matrix should display reasonable mechanical strength and, finally, be biodegradable. The fragments
that form in the course of this degradation process should
have molecular weights smaller than 40 000 g mol 1 in order
be released from the body via the kidneys.
With these requirements in mind, we synthesized porous
monolithic supports by a process based on electron-beaminitiated free-radical polymerization. 2-Methylidene-4phenyl-1,3-dioxolane (M1) was used as monomer, and
trimethylolpropane triacrylate (CL1) was chosen as a crosslinker. A mixture of toluene (microporogen), 2-propanol, and
2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 9138 –9141
In an effort to increase cell adhesion and to provide a
material that allows for the ingrowth of cells, we used more
hydrophilic monomers and turned to a ROMP-based protocol. Thus, by using a mixture of norborn-2-ene (M2) and
pentaglycerol bis(7-oxanorborn-5-ene-2-ylcarboxylate) monoacrylate (CL2) in varying amounts of a microporogen
(toluene) and a macroporogen (2-propanol), different monolithic structures were realized using [RuCl2(Py2)(IMesH2)(CHPh)] (1, Py = pyridine,
IMesH2 = 1,3-dimesitylimidazolin-2-ylidene) as the initiator
and various amounts of pyridine as regulator (Table S2 in
the Supporting Information).
As has been shown for related
systems,[26] the presence of pyridine in amounts as low as
70 ppm allows for the tuning
of the monolithDs structure. It
should be mentioned that the
acrylate group in CL2 acts as a
chain-transfer agent, thus significantly reducing the average
chain lengths of the norborn-2ene derived polymer blocks.
Scheme 1 (right) illustrates the
process of monolith synthesis; a
typical structure with pores
Scheme 1. Monolithic polyester matrix derived from EB-initiated free-radical polymerization (left); ROMParound 200 mm is shown in
derived monolithic matrix (right).
Figure 1. These large pores
dodecan-1-ol (macroporogens) was chosen as solvent. The
optimal mixture shown in Table S1, entry 11 (see the Supporting Information) was applied.[19, 24] The resulting crosslinked copolymer displayed a porous structure with pores up
to 50 mm. Its formation and chemical structure is shown in
Scheme 1 (left). Since the crosslinker CL1 is an ester and the
dioxolane derivative M1 is transformed into an ester,[25] the
copolymer is a polyester that is expected to be biodegradable.
A representative scanning electron microscopy image is
shown in Figure 1 (top).
The specific surface of the monolithic material as determined by N2 adsorption was 12 m2 g 1, revealing a macroporous structure. Storage of the support in a phosphate buffer
(pH 7.2, 200 ppm NaN3) for 1 month at 37 8C revealed a
volume increase from swelling (+ 5 %) and a decrease in mass
( 5 %). Both findings were indicative of a slow disintegration
of the support. It should also be mentioned that the EB-based
process simultaneously sterilizes the matrix.
For biocompatibility studies and tests of in vitro growth
and differentiation on the scaffold, human adipose tissue
derived stem cells (ATSCs) were used. ATSCs have advantages from a clinical point of view owing to their abundant
availability as compared to bone marrow derived stem cells
and are likewise suited for bone and soft tissue (adipose
tissue) engineering. Both tissues are important targets for
regenerative strategies in the craniofacial region. Cultivation
of ATSCs on sterilized polyester-based monolithic supports
revealed rapid cell growth on the surface of the materials,
indicating biocompatibility. However, almost no ingrowth of
the cells into the monolithic structure was observed. Instead,
after 20 days the cells lifted off from the support after initial
growth, suggesting insufficient adhesion. These findings were
attributed to both the hydrophobic character and the surface
structure of the support. This conclusion was supported by the
high water contact angle (1428) observed with these materials.
Angew. Chem. Int. Ed. 2008, 47, 9138 –9141
Figure 1. Porous monolithic scaffolds derived from EB-initiated freeradical polymerization (top) and ROMP (bottom). The scale bars
represent 100 (top), 400 (bottom), and 4 mm (inset, bottom).
2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
cells grew into the pores and partially showed even full
translate into an interparticle-derived porosity of 77 %, as
ingrowth and penetration of the scaffold (Figure 2 A). Finally,
determined by inverse size exclusion chromatography.[27] The
after growth, these cells were then successfully subjected to
protocol used was identified as the one out of 13 (Table S2,
both osteogenic and adipocytic differentiation (Figure 2).
Supplementary Information) that yields monoliths with the
Differentiation of ATSCs into either adipocytes or osteolargest pore diameter. A selection of alternative protocols is
blasts was initiated by addition of isobutylmethylxanthine or
also given in Table S2. To avoid any potential toxic effects of
b-glycerolphosphate and dexamethasone, respectively. Figcatalyst residues, a careful washing of the monolithic structure
ure 2 B shows adipocytes, as identified by the formation of
with a mixture of dimethylsulfoxide, ethyl vinyl ether, and
large univacuolar cells. Figure 2 D, E shows mineralizations by
THF was carried out, which allowed for the virtually
the cells grown within the monolithic material after six weeks.
quantitative removal of the transition-metal-based initiator.
As can be seen, biomineralization was effective with these
Final ruthenium concentrations were below 0.1 ppm (i.e.
materials and is believed to be supported by the free
below the detection limit of inductively coupled plasma
carboxylic acid groups formed from the large number of
optical emission spectroscopy, ICP-OES).
ester moieties. Direct evidence of lineage-specific differN2 adsorption measurements revealed a total surface area
entiation of ATSCs grown on monolith scaffolds was further
of 4 m2 g 1 and thus support the highly macroporous nature of
provided by an immunohistochemical demonstration of
the monolith. Upon storage in a phosphate buffer system, the
osteocalcin expression in osteogenic differentiation (Figinitial water contact angle of 1428 was strongly reduced within
ure 2 F) and S-100 positivity in adipogenic differentiation
13 weeks. In fact, no reliable values could be determined
(Figure 2 C). It is known that the S-100 positivity in the
owing to fast moistening (spreading). This phenomenon is
cytoplasm is typical for adipocytes.
attributed to both the highly hydrophilic character of CL2 and
the high propensity towards
oxidation (in the allylic position) of the poly(norborn-2ene) blocks. On average (n =
4), the YoungDs modulus of
these materials was 1 MPa,
the compressive strength was
7.4 MPa, and the Martens
hardness was 0.3 N mm 2.
These values were found suitable for soft (adipose) tissue
engineering. Polymer degradation was monitored over
240 days. Monoliths became
crumbly, and polymer fragments in the range of 3000–
28 000 g mol 1 were observed.
Importantly, no free acrylic
acid was observed.
In a next step, the ROMPderived scaffolds were cultivated with ATSCs. Within
12 days, the number of cells
on the surface quadrupled. No
additional adhesion factors
such as fibronectine, RGDFigure 2. Histomicrographs of monoliths seeded with adipose tissue derived stem cells after adipogenic
peptides, or matrix factors
(upper row) and osteogenic differentiation (bottom row). Images are vertical cross sections that show the
(aside from differentiation facdegree of cell ingrowth inside the scaffold. A) Low-power magnification with cell ingrowth between the
tors or supplements) comgrayish scaffold material after adipogenic differentiation. Cells with nuclei (colored dark purple) are also
monly used to facilitate or
visible in the center of the scaffold (HE staining). B) High-power magnification of a region with
trigger cell growth were necdifferentiated, mature univacuolated adipocytes in HE staining and C) a similar region labeled with S-100
essary. To study cell ingrowth
antibody (DAB, brown, visible in part in the cytoplasmic border of the vacuolated adipocytes). Asterisks in
(B) and (C) denote mature, univacuolar adipocytes. D) Von Kossa stain shows massive mineralizations
and differentiation on the scaf(arrow, black-brown) after osteogenic differentiation within the monolithic scaffold (counterstaining of cell
folds in a three-dimensional
nuclei with HE). The scaffold material is visible as light-grayish granules on the left-hand side, invaded by
setting, cultivation was percellular ingrowth. E) Region with minor ingrowth and mineralization (counterstain with nuclear fast red).
formed under dynamic condiMineralizations are brown (arrows), cell nuclei are rare in this region (red), and the scaffold material is
tions in rotating culture conrepresented by the light-grayish, birefringent granules. F) Labeling with osteocalcin antibody (visualization
tainers. Culture media were
with BCIP/NBT, blue, counterstain with nuclear fast red); arrows show mineralized nodules. All scale bars
changed weekly. Importantly,
are 100 mm.
2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 9138 –9141
In summary, we have developed novel polymeric monolith-based scaffolds for use in both cell cultivation and tissue
engineering. Current work focuses on enhancing the mechanical strength of the supports and on long-term investigations
of biodegradability as well as on in vitro and in vivo
Received: April 22, 2008
Revised: July 26, 2008
Published online: October 16, 2008
Keywords: biocompatible polymers · cell growth ·
polymerization · tissue engineering
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polymer, engineering, differentiation, cultivation, monolith, tissue, cells
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