Bioseed - Biotissue Technologies Gmbh

Table of contents
Introduction 5
Characteristics 6
Clinical effectiveness 9
Safety 15
Indications 17
Surgical technique 18
Clinical cases 24
Forms 28
Bibliography 30
3
BioSeed ® -C
For Articular Cartilage
Regeneration

Three-dimensional polymer carrier
Fleece with CE marking
Approx. 20x10 6 cells per cm 3
Innovative, exclusive, secure fixation
Optimum primary stability of the implant
Cultivation with autologous serum in GMP laboratory
Can be implanted up to 21 days after removal
Any transplantation dates possible thanks to cryoconservation
Can be delivered all over Europe
More than 300 patients successfully treated
Chondrocytes
(Illustration)
5
BioSeed ® -C
Tissue Regeneration Technique
Thanks to autologous chondrocyte transplantation
with 3D matrix, knee injuries (femoral
condyle, tibia plateau, patella), ankle injuries
(talus) or hip injuries (acetabulum) can be
treated with the patient’s own cells. For this
procedure, cells are multiplied in the laboratory,
and then re-transplanted into the damaged
area.
Hyaline articular cartilage cannot regenerate
itself. However, chondrocytes (cartilage cells)
can be multiplied in the laboratory with
corresponding stimulation; they are re-used
in the body and develop into hyaline articular
cartilage.
Before chondrocyte transplantation, a small
amount of cartilage tissue is arthroscopically
removed from a healthy area that is under
less strain from the knee. The cells are then
cultivated for three weeks in vitro and in this
time they are absorbed into a three-dimensional
matrix. The matrix used consists of two
components. These are a carrier fleece which
forms the framework structure and a biological
“glue” which fixes the homogenously
distributed cells in the carrier fleece. This
combination of the two matrix components
provides the chondrocytes with an ideal threedimensional
environment which stimulates
them to redifferentiate and thus assume their
original morphology and function. As a result,
the graft itself is mechanically stable,
supple, flexible and can be cut to fit the size
of defect. Due to these special properties,
a new fixation technique (transosseous fixation)
and arthroscopic transplantation are
possible, and the indication can be extended.
BioSeed ® -C is intended for the treatment of
extensive cartilage defects.
Chondrocyte transplantation is mainly carried
out on patients under 60 who suffer from
constant pain and impaired mobility because
of individual or multiple cartilage damage,
measuring over 1.5 cm 2 , caused by traumas or
focal degenerative changes.

Photo 01
Photo 02
BioSeed ® -C
Characteristics
Autologous chondrocyte transplantation is
nothing new. In studies published by Brittberg
et al. (17) in 1994, clinical application of
autologous chondrocytes for treating cartilage
defects was proven in over 5000 cases. The
results were presented in various publications
(15, 16). The findings of medium- and longterm
studies are particularly promising for
patients who have undergone unsuccessful
cartilage treatment in the past.
A feature of the original technique was the
injection of chondrocyte suspension into the
interior of a “bioactive chamber” under a
periosteal “flap”. Now, technical innovations in
cell culture mean that chondrocytes can be
expanded in a three-dimensional resorbable
carrier fleece, which has resulted in standardised
cell distribution and a simplified implantation
technique (1, 2, 3, 4, 5). The quality of
newly formed hyaline cartilage was researched
in animal experiments (6, 8, 9, 10, 11, 12, 13).
Preclinical tests in horses, for example, show
the characteristic structure of hyaline articular
cartilage 24 months after transplantation of
BioSeed ® -C (7/Photo 04).
6
Photo 03
Photo 04
7
A unit of BioSeed ® -C consists of two rectangular
three-dimensional carrier fleeces,
consisting of resorbable suture materials and
measuring 30 mm long, 20 mm wide and
2 mm high. The carrier fleece contains interconnecting
cavities (over 90 % of the total
volume) where the cultivated cells are evenly
distributed using fibrin glue. The cell density
is 20 x 10 6 /cm 3 .
Due to the properties of the carrier fleece,
BioSeed ® -C has excellent primary mechanical
stability as well as perfect biocompatibility.
In addition, thanks to this matrix, the cells are
distributed homogenously in a three-dimensional
manner, with the chondrocyte phenotype
remaining differentiated.
Autologous chondrocytes are obtained by
arthroscopic removal of cartilage then cultivated
for about 21 days according to GMP
(Good Manufacturing Practice) guidelines.
Only autologous blood serum is used during
the process.
The graft can be cut to any shape required
for transplantation using anatomical or surgical
scissors.
BioSeed ® -C is the result of years of research
carried out by Berlin Charité University of
Medicine and by Freiburg University Hospital.

9
BioSeed ® -C
Clinical effectiveness
Results 6, 12 and 24 months after transplantation
of BioSeed ® -C to treat cartilage
defects of the knee.
This prospective study carried out by Dr. C.
Erggelet from the Department of Orthopaedic
Surgery, Freiburg University Hospital, Germany
began in November 2001. Clinical evaluation
and MRIs were carried out at the following
intervals:
• when BioSeed ® -C was transplanted
• at the 3 month follow-up
• at the 6 month follow-up
• at the 12 month follow-up
• at the 24 month follow-up

Photo 05
Example of
a MRI scan
6 months
after transplantation
of
BioSeed ® -C
into the
femoral
condyle
Photo 06
Second look
arthroscopy
9 months
after transplantation
Transplant
Method
By November 2004, a total of 79 patients
(30 women, 49 men) had been treated with
BioSeed ® -C. The average age of patients was
36 (age range 17 - 64). 42 patients had cartilage
defects in the left knee, 37 patients in
the right knee. All defects were grade IV in
Outerbridge's classification (= cartilage defect
down to the bone). Most of the defects were
situated on the medial femoral condyle. In
17 cases BioSeed ® -C was implanted in the
retropatellar region, in six cases in the trochlea
and in three cases on the lateral femoral
condyle. In one case the transplantation site
was the medial tibia plateau. 21 patients
suffered from two defects: five lesions were
situated on the medial femoral condyle, three
on the lateral femoral condyle, eight on the
trochlea, and five on the patella.
Concomitant surgery was performed in
34 cases, as the following table shows:
Concomitant surgery Number
Micro-fractures 5
ACL reconstruction 4
Repositioning osteotomy 21
Medial capsular shift
Lateral release (transection
2
of the lateral retinaculum) 2
In 71 cases, the transplantation of BioSeed ® -C
was performed by arthrotomy, and in eight
cases by arthroscopy. The results were assessed
according to the IKDC (International Knee
Documentation Committee), KOOS (Knee
Osteoarthritis Outcome Score) and modified
Cincinnati Knee Score.
10
Photo 07
Histology 9
months after
transplantation
Photo 08
Histology
magnified
100 times
11
Results
The MRI scans of all patients at 6 months
after surgery show good integration of the
graft and satisfactory cover of the defect.
The graft has bonded extremely well to the
surrounding articular cartilage and to the
sub-chondral bone. There is still a visible contrast
in colour to the surrounding cartilage,
and the transosseous drill holes can be seen.
There are no signs of articular constriction
or loosening or separating of the graft
(Photo 05).
Histology
This biopsy taken 9 months after implantation
of BioSeed ® -C showed that, histologically,
the graft had integrated well with a firm bond
to the surrounding bone. The characteristic
structure of the hyaline cartilage can be observed
(Photo 07, 08).

Diagram A
Diagram B
Clinical findings
By November 2004 3-month results were
available for 60 patients (3 mo), 6-month
results for 66 patients (6 mo), 12-month results
for 60 patients (12 mo) and 24-month
results for 40 patients (24 mo).
Modified Cincinnati Knee Score
Diagram A shows mean value (mean) and
standard deviation (std) of the score assessed
by doctor and patient. A score of 10
represents the best state of health.
IKDC Score (joint effusion)
Diagram B shows the percentage of patients
with knee joint effusion.
12
Diagram C
Diagram D
13
IKDC Score (subjective knee
evaluation form)
The data in Diagram C are obtained by adding
together the scores for various items. The
score ranges from 0 to 100. The results are
interpreted as a function measurement of the
knee. 100 points on the scale represent unlimited
activity in day-to-day life and a complete
lack of symptoms.
KOOS Score
Diagram D shows changes in each item of
the KOOS evaluation 24 months after surgery.
The score ranges from 0 to 100. 100 is the
best possible state of health. All evaluated
patients are included in the analysis.

Official approval to manufacture drugs using material of human origin.
Photo 09
Production
in the GMPclean
room
laboratories
Photo 10
15
BioSeed ® -C
Safety
BioSeed ® -C is produced from the cartilage
biopsy and blood serum taken from the patient.
At the start of cultivation, the patient’s
blood is analysed in the laboratory for virus
serology.
The carrier fleece needed to cultivate
BioSeed ® -C is completely biocompatible and
consists of surgical, resorbable suture
material.
The fibrin glue, an authorised drug, used in
BioSeed ® -C, is optimised in the laboratory to
match the requirements of the cells. GMP
guidelines (Good Manufacturing Practice) are
followed in the cultivation of BioSeed ® -C.
The 3D chondrocyte grafts are produced
according to European legislation in a GMP
laboratory, specially developed for cell
cultivation and managed under strict criteria.
A licence to manufacture human drugs is
necessary to produce grafts.

Classification according to Outerbridge
Photo 11
17
I II III IV
BioSeed ® -C
Indications
The indications for BioSeed ® -C are: Chondral
traumatic and focally degenerative defects
(grade III and IV in Outerbridge's classification),
measuring over 1.5 cm 2 .
Treatment with BioSeed ® -C is recommended
for application in the femoral condyle, patella,
trochlea, tibia plateau, talus and acetabulum.
BioSeed ® -C can be transplanted while performing
other types of surgery, such as microfractures,
crucial ligament reconstruction,
osteotomy, medial capsular shift and lateral
release.
BioSeed ® -C is indicated for patients under
60 years of age, although there is no age
limit for cultivation.
There should be no previous or acute infections
of the joint.
The patient should not be suffering from an
acute infectious disease, metabolic systemic
diseases or diseases of the immune system.
The same applies to neoplasia.

Phase 01
Phase 02
BioSeed ® -C
Surgical technique
The application of BioSeed ® -C takes place
in two stages: biopsy of cartilage and
transplantation of BioSeed ® -C.
First stage: Arthroscopic biopsy of cartilage
The cartilage biopsy is removed using
arthroscopy. Approx. 250 mg of cartilage
tissue (the equivalent of about six grains of
rice) is removed from a healthy area subject
to less stress (Phase 01).
The cartilage biopsy is placed in a special
biopsy tube (which is then packed in a sterile
larger tube) that contains the liquid for
transportation (Phase 02).
18
Phase 03
Phase 04
19
Approx. 100 ml of blood is collected in order
to cultivate the chondrocytes in the laboratory
(Phase 03).
The biopsy tube and monovettes with the
patient’s chondrocytes and blood are placed
in an insulated container for transportation
(Phase 04).
Together with the completed forms, the patient's
own biopsy and blood are sent to the
laboratory within 72 hours of removal to
begin the cultivation of chondrocytes.

Phase 01
Phase 02
Second stage: transplantation of
BioSeed ® -C
Preparation
The defective zone is prepared using a curette.
In this procedure, the area is cleaned down
to the sub-chondral bone using debridement.
Depending on the defect, a geometrical (rectangular,
triangular or trapezoidal) form is
prepared, which ensures that the graft has
optimum primary stability (Phase 01). It is
advisable to remove all the damaged cartilage
tissue. If the area to be treated is very deep,
two grafts can be placed on top of each other.
For even deeper defects, the sub-chondral
bone can be built up with spongiosa, and the
chondrocyte graft applied.
The prepared area is measured and
BioSeed ® -C is cut to the right size (Phase
02/03).
Implantation
BioSeed ® -C can be fixed in different ways:
For treating large defects or defects without
marginal cartilage, a graft should be fixed
in a stable and secure manner. The anchoring
technique using knots is recommended for
this procedure.
20
Phase 03
Phase 04
21
Anchoring technique using knots (transosseous
fixation or Erggelet technique)
This technique comprises transosseous drill
holes and anchorage by knots. The transosseous
drill holes are produced inserting four
Kirschner wires (Ø 1.7 mm) through the four
corners of the defect site (Phase 04).

Phase 05
Phase 06
Phase 07
Knot technique
Using resorbable surgical thread (Vicryl 2 - 0),
first pull the thread diagonally through one
corner of BioSeed ® -C. The first knot, the anchoring
knot, is positioned about 0.5 – 1 cm
from the graft and the Vicryl thread is
blocked with a clamp. Then one knot, made
of three knots arranged on top of each other,
is formed. Each of these knots consists of
four alternating loops. This three-fold knot
(Ø 1.7 mm) secures fixation in the drilling
channel.
With another clamp, a distance of 0.5 – 1 cm
is set again and the Vicryl thread blocked.
Then a knot, consisting of two knots each
with three alternating loops, is set (Phase 05).
The second knot forms a pulley loop, through
which a pulley thread (Vicryl thread) is later
fed, which in turn is used for pulling in the
anchoring knot (Phase 06). The same procedure
is repeated at all four corners of the graft.
22
Phase 08
Phase 09
Photo 12
23
The pulley threads are pulled through the
prepared loop, then both ends of the pulley
threads are pulled through the loops of the
four Kirschner wires. The graft is then pulled
in through the condyles (Phase 07).
The graft is securely anchored in the defect
area (Phase 08). The pulley threads are pulled
out at the subcutaneous exit.
The same technique can be carried out by
arthroscopy (Phase 09).
Instruments specially designed for this purpose
are available for arthroscopic transplantation.
Sewing in the graft (Fichtl's method)
BioSeed ® -C can also be sewn into the defect.
It is sewn in securely by fixation using the
single knot method at the corners and two
other places on the graft. The suture material
used for this procedure is PDS 6-0.
Fixation with fibrin glue
For this method, after preparation, all that
is needed is to apply fibrin glue to the side
of the defect, position the graft and press
down lightly with the fingers for about three
minutes.

Photo 13
Photo 15
Photo 17
BioSeed ® -C
Clinical cases
Photo 14
Photo 16
Photo 18
Femoral condyle
41-year-old patient with post-traumatic
lesion of the medial femoral condyle
Photo 13 Cartilage defect
Photo 14 BioSeed ® -C implant armed
with 4 vicryl 2-0- threads
Photo 15 Transosseous fixation of
the graft
Photo 16 Transplanted BioSeed ® -C
By kind permission of Dr. P. Bachelin,
Genolier
34-year-old patient with traumatic
lesion of the medial femoral condyle
Photo 17 Measuring the size of
the defect by taking an impression
using film
Photo 18 Transplanted BioSeed ® -C
By kind permission of Dr. F. Zeifang,
Heidelberg
24
25
Photo 19
Photo 21
Photo 23
Photo 20
Photo 22
Photo 24
Arthroscopic transplantation in
femoral condyle
24-year-old patient with traumatic
cartilage defect
Photo 19 Arthroscopic view of the
debrided defect
Photo 20 Pulling in the graft
Photo 21 Anchoring the knots
Photo 22 Transplanted BioSeed ® -C
Arthroscopic transplantation in tibia
41-year-old patient with degenerative
cartilage defect
Photo 23 Debrided defect
Photo 24 BioSeed ® -C transplanted
arthroscopically
By kind permission of Dr. C. Erggelet,
Freiburg

Photo 25 Photo 26
Photo 27
Photo 29
Photo 28
Photo 30
Arthroscopic transplantation in
acetabulum
42-year-old patient with traumatic
defect of acetabulum
Photo 25 Defective area in acetabulum
Photo 26 Graft fixed by adhesion
By kind permission of Dr. A. Fontana,
Monza
Patella
26-year-old patient with traumatic defect
due to partial dislocation of the patella
Photo 27 Retropatellar lesion
Photo 28 Measuring the defect
Photo 29 Cutting the graft to size
Photo 30 Transplanted BioSeed ® -C
By kind permission of Prof. J.-L. Rhenter,
Genolier
26
27
Photo 31
Photo 33
Photo 35
Photo 32
Photo 34
Photo 36
Trochlea
41-year-old patient with traumatic
defect of the trochlea
Photo 31 Area of defective cartilage
Photo 32 Transplanted BioSeed ® -C
By kind permission of Dr. F. Zeifang,
Heidelberg
43-year-old patient with advanced
osteoarthritis of the knee
Photo 33 Defect in the trochlea before
transplantation
Photo 34 24 months after transplantation
By kind permission of Dr. J. Holz,
Hamburg
Talus
29-year-old patient with traumatic lesion
of the ankle joint
Photo 35 Cartilage defect
Photo 36 Sewing BioSeed ® -C into
the defect
By kind permission of Dr. W. Fichtl,
Ulm

BioSeed ® -C
Forms
These forms ensure that each biopsy
and blood sample are handled with
the utmost safety. The form needs to
be completed before treatment with
BioSeed ® -C. When doing so, please
comply with the guidelines enclosed
with the biopsy removal kit.
Production order form
Gives details of the patient, the surgeon
and the amount of graft required. Use
the labels enclosed to identify patients.
Form for cryoconservation /
thawing order
Used for requests to conserve tissue
material for a subsequent operation,
sent by fax.
28 29
Patient consent form
Used to obtain the patient's consent for
the blood analysis required for cultivation
and for consent to use personal data.

BioSeed ® -C
Bibliography
1) Erggelet C., Die Behandlung von Gelenkknorpeldefekten,
Steinkopff Verlag Darmstadt;
2004
2) Erggelet C., et al. Implantation of autologous
chondrocytes in a 3D resorbable
polymer fleece for the treatment of cartilage
defects in the knee joint, ICRS Gent; 2004
3) Burmester G., Tissue Engineering gegen
Arthrose; DZKF 9/10; 2003
4) Preis S., et al. Therapy of cartilage defects
by autologous chondrocyte implantation. DZ
Sportmed. 54: 225-228; 2003
5) Erggelet C., et al. The arthroscopic implantation
of autologous chondrocytes for the
treatment of full thickness cartilage defects
of the knee joint, Arthroscopy Vol 19, No 1
W.B; No. 1 (January) 2003. pp 108-110
6) Sittinger M., et al. Current strategies for
cell delivery in cartilage and bone regeneration;
Current Opinion in Biotechnology 2004,
115: 411-418
7) Kaps C., et al. Molekulare Charakterisierung
von gezüchteten humanen dreidimensionalen
Chondrozytentransplantaten; Der Orthopäde
Springer Verlag 2003, DOI 10.1007/s00132-
32-003-0505-3
8) Barnewitz D., et. al. Tissue Engineering:
Neue Behandlungsansätze bei Knorpelveränderungen
bei degenerativen Gelenkerkrankungen
des Pferdes – erste Ergebnisse einer
Langzeitstudie, Berliner Münchner Tierärztliche
Wochenschrift 116, 157-161 (2003),
Blackwell Verlag, Berlin
9) Risbud M., et al. Tissue Engineering:
advances in vitro cartilage generation; Trends
in Biotechnology Vol.20, No. 8 August 2002
10) Perka C., et. al. Chondrozytentransplantationen
in PGLA/Polydioxanon-Vliesen Orthopäde
29, 112-119 (2000), Springer Verlag
11) Perka C., et al. Tissue engineered cartilage
repair using cryopreserved and noncryopreseved
chondrocytes; Clinical Orthopeadics &
related research 2000; 378: 245-254
12) Duda G., et al. Mechanical quality of
tissue engineered cartilage: results after 6
and 12 weeks in vivo. Biomed Mater Res.
53: 673-677 (2000) John Willey & Sons
13) Sittinger M., et al. Resorbable polyesters
in cartilage engineering: affinity and biocompatibility
of polymer fiber structures to chondrocytes.
J Biomed Mater Res 33: 57-63, 1996.
14) Stellungnahme der Arbeitsgemeinschaft
Autologe Chondrozyten Transplantation (ACT)
und Tissue Engineering – unter der Schirmherrschaft
der DGU und der DGOOC; 2002
15) Peterson L., et al. Autologous chondrocyte
transplantation – biomechanics and long
term durability; American J. Sports; Vol. 30
No.1; 2002
16) Peterson L., et al. Two to 9-year outcome
after autologous chondrocyte transplantation
of the knee. Clin Orthop 212-234, 2000
17) Brittberg M., et al. Treatment of deep
cartilage defects in the knee with autologous
chondrocyte transplantation. New England J.
Med. 331:889-895, 1994
Please see the enclosed CD for the bibliography
and the surgical method.
30
BioSeed ® -C
CD

ioSeed®-C
BioTissue Technologies GmbH
Engesserstr. 4b
D -79108 Freiburg
Tel. +49(0)761 7676-555
Fax +49 (0) 7 61 76 76 - 430
customerservice@biotissue.de
www.biotissue.de
Autologous three-dimensional
chondrocyte graft