Nephrol Dial Transplant (2008) 23: 764–766
doi: 10.1093/ndt/gfm812
Advance Access publication 8 December 2007
Case Report
See http://ndtplus.oxfordjournals.org/
Tissue formation following implantation of cultured elastic
chondrocytes for treatment of vesicoureteral reflux
Nevenka Kregar Velikonja1 , Andrej Coer2 , Miro Gorenšek3 , Miomir Knežević4 and Andrej Kmetec5
1
Educell d.o.o. Letališka 33, 2 Medical Faculty, University of Ljubljana, Vrazov trg 2, 3 Department of Orthopedic Surgery, University
Medical Centre Ljubljana, Zaloska 9, 4 Blood Transfusion centre of Slovenia, Šlajmerjeva 6 and 5 Department of Urology, University
Medical Centre Ljubljana, Zaloska 7, 1000 Ljubljana, Slovenia
Keywords: endoscopic implantation of chondrocytes;
histology; tissue formation; vesicoureteral reflux
vestigate directly the histopathological alterations resulting
from elastic chondrocyte implantation.
Materials and methods
Introduction
Vesicoureteral reflux (VUR) is the retrograde passage of
urine from the bladder into the ureter and kidneys during voiding. Its pathophysiology appears to be related to
the valve mechanism of the ureterovesical junction, high
intravesical pressure, bladder neurogenic disorder or immature bladder. Abnormal location of the ureteral orifice
is implicated in the short ureteral tunnel, which in turn
compromises the flap-valve mechanism. Endoscopic subureteric injection of various materials has become a universally accepted mode of treatment for VUR in recent years.
Different kinds of injection materials have been used for
injection with various success rates [1–3].
Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences to the
development of biological substitutes that restore, maintain
or improve the function of tissue or of a whole organ [4].
Treatment of VUR by endoscopic implantation of cultured
autologous elastic chondrocytes (ACI—autologous chondrocyte implantation) under the ureteral orifice—a method
based on tissue engineering technologies—has proved to
be successful in eliminating or downgrading high-grade
VUR and has enabled surgical treatment to be abandoned
in paediatric patients [5]. We have developed a method
for implantation of cultured elastic chondrocytes, together
with the patient’s autologous plasma, for treating VUR in
patients with chronic renal failure who are candidates for
renal transplantation [6].
The effectiveness of cell implantation for VUR treatment is usually evaluated by X-ray contrast cystography,
but it is normally not possible to investigate the quality of
regenerated tissue. In the present case we were able to inCorrespondence and offprint requests to: Nevenka Kregar Velikonja, Educell d.o.o. Letališka 33, 1000 Ljubljana, Slovenia. Tel: +386-31-648-186;
Fax: +383-1-5438-203; E-mail: [email protected]
Implantation of cultured elastic cartilage cells for treatment
of VUR in patients with chronic renal failure has been approved by the Ethical Committee at the Ministry for Health,
Republic of Slovenia (No. 63/04/02).
A 46-year-old male patient with end-stage renal disease
(ESRD) and grade IV bilateral VUR was treated by endoscopic injection of autologous chondrocyte suspension.
The procedure was evaluated 6 weeks later by X-ray contrast cystography, revealing a decrease of VUR to grade I/II
on the right, but no downgrading of VUR on left side.
Nephroureterectomy had to be performed more than
6 months after chondrocites implantation on the left side
due to pyelonephritis and sepsis, which was probably a
result of persistent infective foci in native kidney and urinary tract abnormality as was double ureter with knicking
in lower part. X-ray cystography and ultrasound examination namely revealed reflux in double ureters which were
dilated, knicking and forming small diverticula. Therefore
we assume that the complication was not related to the
endoscopic cell implantation procedure. In the course of
this surgical intervention, the site of cell implantation was
isolated for histological analysis.
A small piece of elastic cartilage from the patient’s ear
was taken under local anaesthesia as a source of elastic
chondrocytes. Cells were isolated by collagenase (1 mg/ml,
Sigma GmbH, Germany) digestion and cultivated in an
AIMV cell culture medium (Gibco Ltd, UK) supplemented
with 2% patient’s autologous serum. A suspension of
6 × 107 chondrocytes in a 5 ml thrombin solution was implanted endoscopically, simultaneously with an equal volume of the patient’s blood plasma. The total volume of
suspension implanted under the refluxing ureteral orifice
was 10 ml, with a cell concentration of 6 × 106 /ml.
The tissue sample obtained from the vesicoureteral junction was immediately immersion-fixed in neutral buffered
formalin for 24 h before processing and embedding in
C The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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Tissue formation following implantation of cultured elastic chondrocytes
765
Fig. 1. Histology of regenerated tissue, revealing cartilaginous tissue formation under the vesicoureteral junction (A: 20× magnitude, B: 100×
magnitude), haematoxilin-eosin staining.
Fig. 2. Histological and immunohistological analysis of tissue formed after autologous elastic chondrocyte implantation for treating VUR. Weigert van
Giesen staining shows the presence of elastic fibres (A). Immunostaining shows the abundant presence of collagen type II (B) and the local and weak
presence of collagen type I (C).
paraffin wax. From paraffin blocks, consecutive 5-µmthick sections were cut and prepared for histochemical
and immunohistochemical staining. The first section was
stained with haematoxylin and eosin and the second, for
elastic fibre analysis, with Weigert van Gieson (WvG) staining.
Additionally, two consecutive sections were stained immunohistochemically with anti-collagen I and anti-collagen
II antibodies respectively (Chemicon International, CA).
Sections were deparaffinized, hydrated, soaked in 3% hydrogen peroxide for 30 min at room temperature and then
incubated in blocking buffer. Immunohistochemistry was
performed using polyclonal rabbit anti-human collagen
type I or anti-collagen type II antibodies. The samples
were incubated with primary antibody dilutions of 1:200
overnight at 4◦ C. Immunoperoxidase detection was employed using the ABC method (DAKO, Denmark). The
antibody binding sites were visualized by incubation with
a diaminobenzidine–H2 O2 solution and finally contrastained with haematoxylin. Human skin and cartilage from
a nose septum tissue sample were used as positive/negative
controls. The specificity of the reaction was determined using non-immune serum in place of primary antibody as the
negative control.
Results
Histological analysis of the bioptic sample revealed formation of cartilage-like tissue at the site of elastic chondrocyte
implantation (Figure 1A). There was no formation of gran-
uloma tissue or migration of the implanted cells to distant
sites. No allergic or inflammatory response was visible.
Haematoxylin/eosin staining showed the tissue morphology and cell distribution to be similar to the native elastic
cartilage (Figure 1B). The presence of elastic cartilagespecific proteins in the intercellular matrix was detected
by specific histology and immunohistological staining
(Figure 2). WvG staining proved the extensive presence
of elastic fibres (Figure 2A). Further, immunostaining
for cartilage-specific collagen type II (Figure 2B) and
cartilage nonspecific collagen type I (Figure 2C) shows
the predominant presence of the former, whereas collagen
type I is expressed more weakly and fibres are present only
locally.
Discussion
Various implant materials have been used for VUR treatment and their long-term efficacy remains controversial.
Materials for implanting should be easy to inject, without
systemic side effects, and should make a good augmentation
in the subureteral plane of the ureter orifice. Such materials
need to possess two properties: firstly to resist degradation
and reside in the implantation sites for a long period of time,
and secondly, to enhance tissue regeneration and establish
permanent subureteral tissue [3]. Injections of polytetrafluoroethylene (Teflon) or collagen are the materials most used
in the endoscopic treatment of VUR. Although the principle of endoscopic treatment is valid, there are concerns
regarding the long-term safety and effectiveness of these
substances [5].
766
There are several reasons why cultured elastic chondrocytes should be used for augmenting the ureteral orifice in
patients with VUR. In addition to the fact that a biopsy
of ear cartilage tissue is relatively easy to obtain, with
little or no donor site morbidity, the ability of cartilage
cells to produce abundant extracellular matrix is very important. As a result of the latter, implanted cells provide
a constant source of bulking material and should influence the biomechanical and morphological properties of
the ureteral orifice in such a way as to correct the VUR
grade.
In the described case, bilateral VUR grade IV was
downgraded on the right side; however, treatment of the
left side was not successful due to morphological abnormalities of the ureter, observed as double ureter knicking from kidney to ostium where they rejoined, which is
also evident on histological sections (Figure 1A). Consecutive nephroureterectomy provided the opportunity for
histopathological analysis of the implanted site.
Histopathological analysis showed the abundant presence of elastic fibres in the regenerated tissue, with a distribution of cells similar to that in native elastic cartilage.
Immunohistological analysis revealed massive production
of collagen type II. The cartilage deposit was well encapsulated and created a mass effect with the surrounding tissue,
without causing any inflammatory reaction or granuloma
tissue formation. These analyses demonstrate the good
in vivo regeneration capacity of human ear elastic chondrocytes proliferated in vitro, as well as their ability to form
tissue with very similar properties to those of native elastic
cartilage in a non-homologous tissue environment, when
used for treating VUR.
Tissue engineering approaches, in which human cartilage
is used as a cell source, generally require the expansion of
cell populations in monolayer cultures. Change of the cartilage phenotype during multiple passaging in a monolayer
was documented as early as 1977 [7]. The ability of in vitro
cultured articular chondrocytes to regenerate hyaline cartilage, such as tissue in joint cartilage lesions, is supported
by extensive histological evidence in clinical trials on ACI
[8,9]. The ability of in vitro cultured elastic chondrocytes to
regenerate in vivo, however, rests mainly on evidence from
animal studies [10,11].
This case of tissue formation, following elastic chondrocyte implantation at the site of the vesicoureteral junction,
constitutes the first evidence for the in vivo capacity of
N. K. Velikonja et al.
implanted elastic chondrocytes to retain tissue formation
in described non-homologous use in humans, and provides
an important argument for further investigation of the redifferentiation capacity of human elastic chondrocytes and
further development of their potential for different tissue
engineering applications.
Acknowledgments. The work was performed within Slovenian Research
Agency financed project L3-6265.
Conflict of interest statement. Cultured elastic chondrocytes for treatment
of VUR are produced by Educell Ltd as UroArtTM product. Nevenka
Kregar Velikonja (lead author) is the director of the company and Miomir
Kneević is a member of advisory board of the company. UroArtTM product
is in development and is not a commercial product. Other co-authors are
not linked to the company.
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Received for publication: 18.4.07
Accepted in revised form: 17.10.07