Journal of Biotechnology 148 (2010) 4655

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

A novel concept for scaffold-free vessel tissue engineering: Self-assembly of

microtissue building blocks
Jens M. Kelm a,b, , Volker Lorber a , Jess G. Snedeker f , Drthe Schmidt a,c , Angela Broggini-Tenzer d ,
Martin Weisstanner d , Bernhard Odermatt d , Anita Mol e , Gregor Znd a , Simon P. Hoerstrup a,b,c
a

Regenerative Medicine Program, Department of Surgical Research, University of Zrich, CH-8091 Zrich, Switzerland
Competence Center for Applied Biotechnology and Molecular Medicine, Microscale Tissue Engineering Group, VetSuisse Faculty,
University of Zrich, CH-8057 Zrich, Switzerland
c
Clinic of Cardiovascular Surgery, Department of Surgery, University of Zrich, CH-8091 Zrich, Switzerland
d
Department of Pathology, University Hospital, Zrich, Switzerland
e
Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
f
Department of Orthopedics, University of Zrich, Balgrist, CH-8008 Zrich, Switzerland
b

a r t i c l e

i n f o

Article history:
Received 10 August 2009
Received in revised form 28 February 2010
Accepted 2 March 2010

Keywords:
Vascular grafts
Extracellular matrix
Gene expression analysis
Regenerative medicine
Biomedical engineering
3D cell culture technology
Mechanical strain bioreactor

a b s t r a c t
Current scientic attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show
substantial limitations, thereby preventing routine clinical use. In the present report, we describe a
novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue selfassembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a
vascular shape and apply pulsatile ow and circumferential mechanical stimulation. Microtissues composed of human artery-derived broblasts (HAFs) and endothelial cells (HUVECs) were accumulated and
cultured for 7 and 14 days under pulsatile ow/mechanical stimulation or static culture conditions with
a diameter of 3 mm and a wall thickness of 1 mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, -SMA, Ki67, VEGF).
Self-assembled microtissues composed of broblasts displayed signicantly accelerated ECM formation
compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under
both, static and ow/mechanical stimulation conditions. A layered tissue formation was observed only
in the dynamic group, as indicated by luminal aligned -SMA positive broblasts. We could demonstrate
that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity
makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of
any foreign material.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Due to an ever growing aging western population with cardiovascular disease and therefore increasing numbers of bypass and
reconstructive vascular interventions, there is a substantial clinical need for appropriate vascular graft materials. In the context
of coronary bypass surgery, small diameter vascular grafts, such as
saphenous vein and mammary artery, are regularly in high demand
thus resulting in the unwanted usage of native autologous materi-

Corresponding author at: Center for Applied Biotechnology and Molecular

Medicine and Regenerative Medicine Program, University of Zrich, Winterthurerstrasse 190, CH-8057 Zrich, Switzerland. Tel.: +41 44 6353800;
fax: +41 44 6356840.
E-mail addresses: jens.kelm@cabmm.uzh.ch, jens.kelm@insphero.com
(J.M. Kelm).
0168-1656/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2010.03.002

als. Development of arterial replacements to substitute native vein

and artery segments for peripheral or coronary revascularization is
more than 50 years old, but still native autologous replacements
are considered to be the gold standard (LHeureux et al., 2007).
Synthetic grafts, especially for small diameter (<6 mm) applications, have been associated with various complications including
acute thrombosis, restenosis and compliance mismatch, susceptibility to infections and lack of growth, (Hoerstrup et al., 2006;
Zilla et al., 2007). As such, novel strategies to create allogeneic
and autologous living tissue-engineered blood vessels (TEBVs) in
vitro are considered to be acceptable alternatives and are therefore
actively being pursued by numerous investigators. Since the rst
report of TEBVs by Weinberg and Bell (1986), a wide variety of biomaterials (synthetic and natural biopolymers) and cell sources have
been investigated for their eligibility to produce TEBVs (Couet et al.,
2007; Isenberg et al., 2006). Two scaffold-based conceptual strategies are prevalent: (1) implantation of instructive scaffold materials

J.M. Kelm et al. / Journal of Biotechnology 148 (2010) 4655

promoting in vivo cellular ingrowth and tissue maturation through

recruitment of endogenous cells (Campbell et al., 1999) and (2)
implantation of functional living tissue replacements generated in
vitro produced from cells seeded on tubular scaffolds and matured
in biomimetic bioreactors (Hoerstrup et al., 2001, 2006). However,
when considering the importance of the immunological response
to such scaffolds and the efciency by which they can be produced,
endogenous tissue derived from autologous cells may be deemed to
be the most appropriate extracellular matrix. A successful scaffoldfree concept has been introduced by LHeureux et al. (1998), where
single-cell sheets were wrapped in a multi-step process around a
stiff support tube. The resulting tissue was reminiscent of native
tissue and displayed good functionality in vivo, displaying the high
potential of pure cell-based concepts. However, the whole production process required more than 4 months and necessitated
multiple processing steps (LHeureux et al., 2006).
Current approaches have shown high potential. But long production times and complex processing steps have hampered

47

their routine clinical implementation. In the present report, we

describe a scaffold-free concept to produce small diameter TEBV
using microtissue composed of myobroblasts and endothelial
cells. Microtissues produce higher amounts of extracellular matrix
required to create scaffold-free TEBV.

2. Material and methods

2.1. Isolation of primary cells
In order to obtain primary human artery-derived broblasts
(HAFs), de-endothelialized vessel segments of human arteries were
minced and cultivated in a 37 C humidied 5% CO2 -containing
atmosphere and Dulbeccos modied Eagles medium (DMEM,
Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf
serum (FCS; cat. no. A-15-022, lot no. A01129-242; PAA Laboratories, Linz, Austria) and 1% penicillin/streptomycin solution

Fig. 1. A schematic image of the bioreactor setup, displaying the three components: (i) the pulsatile pump, (ii) the assembly device and (iii) the medium reservoir. (B)
Cross-section through the microtissue assembly device with the inlet silicon tube enabling circumferential mechanical stimulation and a reverse medium outow. (C) The
casting mould to assemble the microtissues consist of an inner silicon tube (inow, mechanical stimulation), two distance spacer of 1 mm thickness determining the vessel
wall thickness and an outer non-adhesive, porous cask to retain the microtissues within the assembly device.

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J.M. Kelm et al. / Journal of Biotechnology 148 (2010) 4655

(Invitrogen). Pure HAFs which had migrated out of the tissue pieces
after 1014 days were serially passaged and expanded for 46
weeks under aforementioned conditions to desired cell numbers
(Hoerstrup et al., 1998).
2.2. Cell culture
Human artery-derived broblasts were expanded in DMEM supplemented with 10% FCS, 1 ng/ml bFGF (PeproTech EC, London, UK)
and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Isolation of human umbilical cord vein endothelial cells (HUVECs)
were processed according to Hoerstrup et al. (1998). HUVECs were
expanded in Endothelial Growth Medium (EGM2 + Single Quot Kit
Components, Lonza Group Ltd., Basel, Switzerland) supplemented
with 10% FCS. All cell types were cultivated at 37 C in a humidied
5% CO2 -containing atmosphere.
2.3. Microtissue production
Monolayer cultures of HAF were trypsinized and single-cell
suspensions seeded with 10,000 cells/drop into 60-well plates
(HLA plate, Greiner-Bio One, Frickenhausen, Germany). In order to
enable gravity-enforced microtissue formation in hanging drops,
the 60-well plates were incubated upside down. Microtissues were
produced and maintained in DMEM medium (Invitrogen) supplemented with 10% FCS and 1% penicillin/streptomycin solution.
HAF-HUVEC microtissue cocultures were produced in two steps:
(i) production of the core feeder spheroid composed of HAF by 2day cultivation in hanging drops followed by (ii) addition of 1000
monodispersed HUVECs per drop and tissue (Kelm et al., 2005).
After additional 5-day cultivation, microtissues were harvested and
loaded into the assembly device.

2.6. Immunohistological and uorescence analysis

Immunohistochemistry was performed using the Ventana
Benchmark automated staining system (Ventana Medical Systems, Tuczon, AZ) and following primary antibodies: collagen IV
(BMA Biomedicals AG, Augst, Switzerland), von Willebrand factor (polyclonal rabbit, DakoCytomation, Glostrup, Denmark) and
anti--smooth muscle actin (-SMA, clone 1A4; Sigma Chemical
Company, St. Louis, MO), anti-vascular endothelial growth factor
(VEGF) (AK77, Lab Visison, Fremont, CA) and anti-Ki67 (Mib-1,
DakoCytometry). Primary antibodies were detected with the Ventana iVIEW DAB detection kit, resulting in a brown reaction product.
Sections were counterstained with hematoxylin and covered with
a glass cover slip.
For immunouorescence analysis, the TEBV was harvested,
washed twice in phosphate-buffered saline (PBS; Sigma Chemicals,
St. Louis, MO, USA), xed for 1 h in PBS containing 4% paraformaldehyde and subsequently washed three times for 5 min in PBS. The
tissue was then permeabilized for 60 min in PBS containing 0.5%
Triton X-100 (Sigma Chemicals). The vessel was sequentially incubated with the primary antibody targeting von Willebrand factor
(vWF; Sigma Chemicals) for 12 h at 4 C and the detection antibody, anti-rabbit Cy2 (Jackson Immunochemicals, West Grove, PA)
for additional 12 h at 4 C. Nuclei were visualized by DAPI (Sigma
Chemicals) and F-Actin by A633-Phalloidin (Molecular Probes Inc.,
Eugene, OR, USA). For microscopy, the vessel has been cut longitudinal to expose the luminal side and analyzed by confocal laser
scanning microscopy (CLSM; Leica TCS SP5, Leica Microsystems,
Glattbrugg, Switzerland).
3. Results
3.1. Bioreactor design

2.4. Expression analysis

For gene expression analysis, cells were either grown as monolayer (2D culture) in DMEM supplemented with 10% FCS or
assembled to microtissues (2500 cells/MT) in the same medium.
After 4 days in culture the RNA was extracted from 2D and microtissue cultures by TRIReagent (MRC, Molecular Research Center)
according to the manufacturers protocol.
Resulting RNA quality was assessed on an Agilent 2100 Bioanalyzer and further processed using the Agilent Microarray Platform.
Samples were one-colour (Cy3) labelled and analyzed on a 4 44k
whole human genome chip according to the manufacturer protocol
(Agilent) and scanned using the Agilent microarray scanner. Further
data analysis was performed in the Rosetta Resolver. Ratios were
built between 3D and 2D culture for each cell line and differentially expressed genes were collected with the following criteria:
p-value < 0.01, at least 2-fold change (0.5 > ratio > 2), absolute intensity > 0.5 for at least one data point. Differentially expressed genes
were analyzed in a gene ontology program (Database for Annotation, Visualization and Integrated Discovery (DAVID)) for their
functional properties.

A novel microtissue assembly device concept was designed to

(i) assemble microtissues to a tubular shape and (ii) enabling circumferential mechanical stimulation in a pulsatile bioreactor. The
system consisted of the assembly device connected to a pulsatile
pressure device with a control unit (Mol et al., 2005) driven by 1 bar

2.5. Bioreactor characterization

Silicone tubing was obtained from Dow Corning (Silastic Rx50,
Dow Corning, MI, USA). The pressure in the bioreactor system was
analyzed using a custom-made pressure chamber system enabling
to adjust ow and mechanical stimulation in the bioreactor system
(Mol et al., 2005). Flow rates were measured using an ultrasonic
ow measurement system (Sono TT, em-tec, Finning, Germany).
The silicon tube was xed within the vascular ow transducer
(Sono TT, 2 mm em-tec) and the ow measured for three different
amplitudes.

Fig. 2. Overview of the technical parameters of the bioreactor system and the resulting mechanical strain applied on the tissue during mechanical stimulation in the
bioreactor.

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49

Table 1
Gene expression ratio of primary human broblasts (primary cell line I, II and II) cultured in 3D versus monolayer sheets of 67 extracellular matrix-related genes analyzed
in a gene ontology program (Database for Annotation, Visualization and Integrated Discovery) for their functional properties. Dark red displays 10 higher expression in
3D, orange displays 210 higher expression in 3D and green displays a higher expression ratio in 2D cultures.

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J.M. Kelm et al. / Journal of Biotechnology 148 (2010) 4655

Table 1 (Continued )

inlet pressure (oxycarbon) and a medium reservoir (Fig. 1A). The

assembly device was placed within a 50 ml falcon tube which was
closed by a custom-made stainless steel cap harboring the connectors for inlet and outlet ow (Fig. 1B). The tissue assembly device
comprised of an inner silicone tubing and a non-adhesive outer casket with a pore size of 150 m. Spacer rings determined the wall
thickness of 1 mm at the bottom and the top of the vessel (Fig. 1C).
The medium ow was directed in a circular system through the
inner silicone tube to the base of the tube and redirected to the
outlet in the cap. This conguration enabled mechanical stimulation through the silicon tube and oxygen and nutrient diffusion
through the outer periphery of the tissue (Fig. 1C). The vessels were
created by microtissue building blocks composed of human arteryderived broblasts and umbilical cord endothelial cells, which were
placed into the assembly device. To gain optimal oxygen and nutrient diffusion the outer casket was removed after 25 days, and
the pressure increased up to 55 mmHg (Fig. 2). The bioreactor
system was placed in a humidied incubator at 37 C containing
5% CO2 .

3.2. Microtissue building blocks

Scaffold-free tissue engineering approaches are based on the
capability of cells to produce their own extracellular matrix (ECM).
To evaluate the capacity of human broblasts to produce ECM in
monolayer and microtissue cultures we performed a differential
expression analysis with broblasts of three different patients (primary cell line I, II and III) on a 44k whole human genome array. We
observed that following the analysis of about a 1000 genes, which
were differentially expressed (data not shown), the most dramatic
differences in expression levels were associated with extracellular
matrix-related genes (see Table 1). Matrix-related gene transcripts
such as collagens, bronectin, laminin, glucosaminoglycans (GAGs)
and matrix metallopeptidases (MMPs) were highly upregulated
in microtissue cultures (Table 1). These results could be further
substantiated by immunohistological analysis of collagen IV, an
integral component of the ECM to match mechanical demands of
a vessel (Poschl et al., 2004). HAF-composed microtissues cultured
without ascorbic acid exhibited a higher collagen IV production

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51

Fig. 3. Immunohistological comparison of collagen IV expression of human artery-derived broblasts (HAFs), cultured for 6 days (A) in monolayer cultures with 150 M
ascorbic acid, (B) monolayer cultures without ascorbic acid and (C) in microtissue cultures.

after 6 days compared to conuent monolayer cultures either

with or without supplementation of 0.25 mg/ml ascorbic acid 2phosphate (Fig. 3).
As previous studies demonstrated highly improved microtissue assembly and cell survival by incorporating endothelial cells
into the building blocks (Kelm et al., 2006), human umbilical vein
endothelial cell coated microtissues were used to assemble the vessels (Fig. 4A and B). However, even if the broblasts were isolated
from the arterial wall, only a low amount of alpha-smooth muscle
actin (-SMA) could be identied, signifying that the cells did not
adopt a myobroblastic or smooth muscle cell phenotype in this
format (Fig. 4D). Vascular endothelial growth factor (VEGF), as a
marker for hypoxia, could be detected only at a very low level. Cell
proliferation could be observed predominantly in the peripheral
cell layer of the microtissue reecting most likely endothelial cell
proliferation (Fig. 4F).
3.3. Vessel assembly and maturation
In order to produce small diameter TEBV, microtissues harvested from the hanging drop cultures were transferred into the
assembly device. Between 4000 and 5000 microtissues are required
to obtain a vessel of 5 mm in length and 1 mm wall thickness.
The resulting tissues were harvested after 7 (Fig. 5AD) and 14
days (Fig. 5EH) under dynamic as well as after 14 days under
static culture conditions (Fig. 5IL). Association of the microtissues occurred already within 7 days in the bioreactor but individual
microtissues were still visible. After an additional 7-day cultivation
either applying pulsatile ow and mechanical stimulation or under
static culture conditions, microtissues fused to a homogenous tissue. With regard to the already high expression of collagen in the
microtissue cultures, the tissue contained a high amount of collagen IV. However, cellular reorganization of the broblasts lining

the luminal part along the axis of the vessel could only be observed
after 14 days of ow and mechanical stimulation (Fig. 5C, G and
K). Under static culture conditions, microtissues assembled as well
into a homogenous tissue, however such a system gave rise to a
hypoxic environment as indicated by increased VEGF production
(Fig. 5D, H and L).
In contrast to the individual building blocks, in which a differentiated phenotype could not be observed, luminal -SMA positive
cells were detected exclusively in tissue-engineered vessels after
14 days with mechanical stimulation (Fig. 6). The -SMA positive
cells corresponded closely to the cells which have been aligned
according to the circumferential mechanical strain. Cell proliferation as detected by staining Ki67 was very low (Fig. 6GI).
3.4. Endothelialization of the lumen
Endothelial cells were distributed throughout the vessel wall
after 7 and 14 days in the bioreactor (Fig. 7AC). However, endothelial cells could not be detected on the luminal surface of the
mechanically stimulated vessel after 14 days (Fig. 7B). The high
background staining observed within the -SMA positive broblasts was most likely related to the binding of vWF to the collagen
brils (Furlan, 1996). However, luminal endothelialization is essential to prevent thrombosis after transplantation. HUVECs placed
into the lumen of the vessel for 24 h adhered to the cellular surface
as shown by the positive staining for vWF (Fig. 8).
4. Discussion
The option of creating living small diameter blood vessels from
autologous cells offers many potential advantages compared to current synthetic vascular grafts, such as the absence of thrombotic
occlusion and the inherent potential of healing and remodeling

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Fig. 4. Characterization of microtissue building blocks composed of human artery-derived broblasts (HAFs) coated after 2 days with human umbilical vein endothelial cells:
(A) macroscopic, (B) hematoxylin and eosin staining, and immunohistological analysis of (C) von Willebrand factor, (D) VEGF, (E) -SMA and (F) Ki67 antigen.

according to the needs of the specic environment. In particular,

the critical shortage of native autologous vascular graft material (saphenous vein, mammary artery etc.) especially in todays
often repeated coronary bypass surgery may be suitably addressed
(Stegemann et al., 2007).
Scaffolds as substrates for cellular adherence are an integral
component for the successful construction of tissue-engineered
blood vessels. A wide variety of natural and polymeric biomaterials such as polyglycolic acid (PGA), polylactic acid (PLA) have been
successfully used for small diameter TEBV (Bordenave et al., 2008;
Gong and Niklason, 2008; Stegemann et al., 2007). However, the
ideal scaffold material is considered to be endogenously produced
extracellular matrix. Autologous ECM provides several advantages
over articial scaffold materials for TEBV such as prevention of
an adverse immunological response, providing an environment
for controlled cell proliferation to develop sufcient tissue mass,
mouldable design to tubular constructs, as well as mechanical stability and stimulating production and organization of matrix brils

such as collagens. The rst complete biological-based engineered

blood vessel was introduced by LHeureux et al. (1998) and has been
successfully tested in a preclinical primate model conrming the
potential of full-biological replacements (LHeureux et al., 2006).
However, extended production times of more than 4 months, most
likely due to a decelerated ECM production in the cell sheets,
impairs routine clinical applicability.
Here, we present a novel concept to produce small diameter
blood vessels devoid of any foreign material using microtissues as
minimal building blocks. In contrast to scaffold-based technologies,
cellular proliferation was restricted solely to the cell expansion
phase. Once embedded within a 3D environment, cellular proliferation was dramatically reduced and a marked upregulation of
a variety of ECM-related genes as compared to their monolayer
counterparts was evident. Most noticeably were the collagens,
in association with high expression of matrix metallopeptidases,
indicative of continual ECM remodeling which is a requirement
of cellular and matrix reorganization under mechanical strain.

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53

Fig. 5. Macroscopic and histological analysis of assembled small diameter tissue-engineered vessels constructed with microtissue building blocks: (AD) 7 days dynamic
culture, (EH) 14 days dynamic culture and (IL) 14 days static culture conditions. Cross-sections of the vessel constructs were stained for (B, F and J) hematoxylin and eosin,
and immunohistological analysis for (C, G and K) collagen IV and (D, H and L) VEGF.

Mechanical stimulation has a well-known high impact on the differentiation of broblasts (Wang et al., 2003, 2006). Fibroblasts
and matrix located close to the luminal surface responded to the
mechanical force by reorganizing and aligning according to the
circumferential strain. This resulted in a layered tissue structure
reminiscent of the tunica media and tunica adventia. Initially, cells
in the building blocks lacked -SMA, however myobroblast differentiation was induced by mechanical stimulation as shown by the
appearance of -SMA positive cells in the luminal organized layer
after a 14-day culture period in the bioreactor. Single differentiated
myobroblasts could also be detected in the static control at the
luminar and vessel surfaces, which may be related to an elongation
of single cells on the surface with the development of associated
stress bers (Wang et al., 2003).
Endothelial cells were directly incorporated within the vessel
wall as this microtissue conguration has previously been shown

to have a positive effect on microtissue assembly (Kelm et al., 2006).

In addition, we also consider the incorporation benecial in its
ability to accelerate the connection to the host capillary system.
Moreover, it is believed that trans-interstitial capillary ingrowth
from the adventia or peri-graft tissue wall, contributes to the
endothelialization along the lumen of the graft (Brewster et al.,
2007).
A robust and time-efcient biotechnological process for the production of tissue-engineered living vascular grafts is a key factor for
successful clinical routine implementation. We have demonstrated
that self-assembled microtissues generated through a robust upscalable production process can be used as minimal building blocks
to produce small diameter TEBV. The accelerated ECM production
of microtissues and their maturation/differentiation capacity in the
vessel format makes this approach highly attractive to generate
fully autologous small diameter blood vessels devoid of any for-

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Fig. 6. Immunohistological analysis of (A, B, D, E, G and H) -SMA and (C, F and I) Ki67 in the assembled, engineered small diameter vessel, after 7 days dynamic cultures,
14 days dynamic cultures and in the 14 days static control culture.

Fig. 7. Endothelial cell distribution was evaluated by immunohistological analysis of von Willebrand factor after (A and B) 7 days ow and mechanical stimulation, (C and
D) 14 days ow and mechanical stimulation and (E and F) 14 days static control culture.

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55

Fig. 8. HUVECs coated onto the luminar surface of the tissue-engineered vessel after 7-day cultivation in the bioreactor. Endothelial cells were visualized en face of the
luminal area by immunouorescence staining of von Willebrand factor (shown in green), nuclei (shown in cyan) and F-Actin stained with A633-coupled Phalloidin (shown
in red). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of the article.)

eign material not only for therapeutic use but also for substance
evaluation.
Acknowledgements
This work was supported by the Swiss National Foundation (SNF
310000-120432). Angela Broggini-Tenzer and Martin Weisstanner
were supported by the UBS acting on behalf of a client, the Swiss
paraplegic research, Nottwil and the cancer league of the canton
Zrich. We also thank Hans Ruedi Sommer for his technical support
and Peter Richards for critical comment on the manuscript.
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