U.S. patent application number 09/836218 was filed with the patent office on 2001-08-16 for method for producing cartilagetissue and implants for repairing encholndral and osteochondral defects as well as arrangement for carrying out the method.
This patent application is currently assigned to Sulzer Orthopedics Ltd.. Invention is credited to Bittmann, Pedro, Mainil-Varlet, Pierre, Muller, Werner, Rieser, Franz, Saager, Christoph P..
Application Number | 20010014473 09/836218 |
Document ID | / |
Family ID | 4209753 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014473 |
Kind Code |
A1 |
Rieser, Franz ; et
al. |
August 16, 2001 |
Method for producing cartilagetissue and implants for repairing
encholndral and osteochondral defects as well as arrangement for
carrying out the method
Abstract
Cartilage tissue and implants comprising tissue are produced in
vitro starting from cells having the ability to form an
extracellular cartilage matrix. Such cells are brought into a cell
space (1) and are left in this cell space for producing an
extracellular cartilage matrix. The cells are brought into the cell
space to have a cell density of ca. 5.times.10.sup.7 to 10.sup.9
cells per cm.sup.3 of cell space. The cell space (1) is at least
partly separated from a culture medium space (2) surrounding the
cell space by means of a semi-permeable wall (3) or by an open-pore
wall acting as convection barrier. The open-pore wall can be
designed as a plate (7) made of a bone substitute material and
constituting the bottom of the cell space (1). The cells settle on
such a plate (7) and the cartilage tissue growing in the cell space
(1). The cells settle on such a plate (7) and the cartilage tissue
growing in the cell space (1) grows into pores or surface roughness
of the plate, whereby an implant forms which consists of a bone
substitute plate (7) and a cartilage layer covering the plate and
whereby the two implant parts are connected to each other in
positively engaged manner by being grown together.
Inventors: |
Rieser, Franz;
(Wiesendangen, CH) ; Muller, Werner;
(Wiesendangen, CH) ; Bittmann, Pedro; (Zurich,
CH) ; Mainil-Varlet, Pierre; (Bern, CH) ;
Saager, Christoph P.; (Frieswil, CH) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Sulzer Orthopedics Ltd.
|
Family ID: |
4209753 |
Appl. No.: |
09/836218 |
Filed: |
April 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09836218 |
Apr 18, 2001 |
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09194867 |
Mar 4, 1999 |
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6242247 |
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09194867 |
Mar 4, 1999 |
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PCT/CH97/00220 |
Jun 2, 1997 |
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Current U.S.
Class: |
435/297.1 ;
435/297.5; 435/399; 435/401; 623/23.72 |
Current CPC
Class: |
A61F 2002/30064
20130101; A61B 17/68 20130101; A61F 2002/30449 20130101; Y10S
623/919 20130101; A61F 2002/30762 20130101; A61F 2220/005 20130101;
A61F 2/30756 20130101; A61L 2430/06 20130101; A61L 27/3852
20130101; A61F 2002/30971 20130101; A61F 2002/30004 20130101; A61K
35/12 20130101; A61L 27/3612 20130101; C12N 5/0655 20130101; A61L
27/3654 20130101; A61F 2240/001 20130101; A61F 2250/0014 20130101;
A61L 27/3817 20130101; A61F 2/3094 20130101; A61L 27/3645
20130101 |
Class at
Publication: |
435/297.1 ;
435/297.5; 435/399; 435/401; 623/23.72 |
International
Class: |
C12M 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 4, 1996 |
CH |
1408/96 |
Claims
1. An implant produced by a method for producing an implant
comprising cartilage tissue, comprising the steps of: (a) providing
a cell space of a predefined form, said cell space being limited by
cell space walls having an inside and an outside surface, wherein
said cell space walls are at least in part semi-permeable or have
an open porosity suitable as a convection barrier and wherein the
inside surface of said cell space walls does not discourage cell
attachment; (b) providing cells with a chondrogenic potential of
5.times.10.sup.7 to 10.sup.9 per cm.sup.3 of said cell space; (c)
introducing a mixture consisting of said cells and a suitable
culturing medium, or consisting of tissue particles and a suitable
culturing medium into said cell space so as to form a culture
medium space; (d) positioning said cell space in culturing medium,
such that at least the semi-permeable or porous part of the cell
space walls are immersed in said culturing medium; (e) maintaining
a suitable culturing condition for a time period of sufficient
duration to allow said cartilage tissue and an implant comprising
cartilage tissue to grow in said cell space, wherein the implant
comprises a plate made of an open-pore bone substitute material,
the surface of which is at least partly covered with a cartilage
layer cultivated in vitro, whereby the cartilage layer is anchored
in the bone substitute material by growing into the pores and
surface unevenness of the bone substitute material.
2. The implant according to claim 1, wherein the plate made of the
bone substitute material comprises, at least in the region facing
the cartilage layer, pores of a size of 1 to 20 .mu.m and has a
thickness of 0.5 to 3 mm.
3. The implant according to claim 1, wherein the plate made of bone
substitute material is connected to a further part made of bone
substitute material by means of a cement layer.
4. The implant according to claim 1, wherein the implant is rigid
or plastically deformable.
5. A method for repairing enchondral or osteochondral joint
defects, comprising the steps of: producing an implant comprising
cartilage tissue according to claim 1; and implanting said implant
at a site wherein said enchondral or osteochondral defects are
located.
6. A method for producing auditory bones, nose cartilage, orbital
floors, ear conchs or parts thereof, comprising the steps of:
producing an implant comprising cartilage tissue according to claim
1, wherein said predefined form is in a shape of an auditory bone,
nose cartilage, orbital floor, ear conch, or parts thereof.
7. A method for repairing enchondral or osteochondral joint
defects, comprising the steps of: producing an implant comprising
cartilage tissue according to claim 1; and implanting said implant
at a site wherein said enchondral or osteochondral defects are
located, wherein said implant repairs said enchondral or
osteochondral joint defects.
Description
[0001] The invention is in the field of medicinal engineering and
concerns a method according to the generic part of the first
independent claim, i.e. a method for producing cartilage tissue and
implants for the repair of enchondral and osteochondral defects.
Furthermore, the invention concerns an arrangement for carrying out
the method and implants produced according to the method.
[0002] Cartilage tissue substantially consists of chondrocytes and
extracellular matrix. The extracellular matrix mainly consists of
collagen type II and proteoglycanes the components of which are
exuded into the intercellular space where they are assembled to
form macro molecules. The chondrocytes make up about 5% of the
volume of the cartilage tissue of a grown-up individual.
[0003] Articular cartilage coating the ends of flexibly joined
bones takes over the function of the load distribution in the
loaded joint. For this function the cartilage tissue is capable to
take up water and to release it again under pressure. Furthermore,
the cartilage surfaces serve as sliding surfaces in the joints.
[0004] Cartilage is not vascularized and therefore its ability to
regenerate is very poor, in particular in grown-up individuals and
if the piece of cartilage to be regenerated exceeds but a small
volume. However, articular cartilage often shows degenerations due
to wear or age or injuries due to accidents with a far larger
volume than might be naturally regenerated. This kind of defect of
the cartilage layer makes movement and strain of the affected joint
painful and can lead to further complications such as e.g.
inflammation caused by synovial liquid which comes into contact
with the bone tissue due to the defect in the cartilage layer
covering the bone.
[0005] For these reasons efforts have been made for quite some time
to replace or repair missing or damaged cartilage, especially
articular cartilage by corresponding surgery.
[0006] It is known to repair defects concerning articular cartilage
or articular cartilage and the bone tissue beneath it by milling
the defect location to form a bore of an as precise geometry as
possible, by extracting a column of cartilage and bone of the same
geometry from a less strained location of e.g. the same joint by
means of boring or punching and by inserting this column into the
bore. In the same manner, larger defects with several bores are
repaired (mosaic plasty). These methods are successful but the
actual problem is substantially shifted from a strained part of a
joint to a less strained part of the joint and therefore, is not
really solved.
[0007] It is also suggested, e.g. in the publication U.S. Pat. No.
3,703,575 (Thiele), to repair defects of cartilage with purely
artificial implants (e.g. gels containing proteins and
polysaccharides). It shows, however, that only restricted success
can such be achieved and therefore in recent development solutions
to the problem have been thought in various directions, in
particular based on vital autologous or homologue cells. Vital
chondrocytes or cells able to take over a chondrocyte function are
e.g. cultivated in vitro and then implanted; or vital chondrocytes
are introduced in artificial implants; or vital cartilage tissue is
cultivated at least partly in vitro and is then implanted. This
means that in these recent developments the aim is to produce vital
cartilage in vitro and to implant such cartilage or to populate a
defect site with cartilage forming cells which cells are then to
build tissue at least similar to cartilage.
[0008] Examples of such methods are described in the following
publications:
[0009] According to the method described in U.S. Pat. No. 4,846,835
(Grande), chondrocytes taken from the patient are multiplied in a
mono-layer culture and, for further reproduction, are then
introduced into a three-dimensional collagen matrix in form of a
gel or a sponge in which matrix they settle and become immobile.
After ca. three weeks of cell reproduction, the defect cartilage
location is filled with the material consisting of the collagen
matrix and the cells. In order to hold the implant in the defect
location, a piece of periosteum is sutured over it. The cartilage
regeneration in the region of this kind of transplant is
considerably better than without the transplant.
[0010] According to the method described in U.S. Pat. No. 5,053,050
(Itay), chondrocytes or cells able to take over a chondrocyte
function are introduced into a biocompatible, resorbable matrix
(32.times.10.sup.6 to 120.times.10.sup.6 cells per cm.sup.3) in
which matrix the cells are immobilized. This matrix is implanted,
whereby a cartilage-like tissue forms in vivo. The chondrocytes
used for the implant are previously cultivated, first in a
mono-layer culture and then suspended, whereby they assemble to
form aggregates of 30 to 60 cells.
[0011] According to the method described in U.S. Pat. No. 4,963,489
(Naughton), again a three-dimensional, artificial matrix is used as
carrier material for the implant. This matrix is used for the cell
culture preceding the implantation and is covered with a layer of
connective tissue for better adhesion and better supply of the
cells to be cultivated. After in vitro cell reproduction on the
three-dimensional matrix, the matrix is implanted. The implanted
cells form the cartilage tissue in vivo.
[0012] According to the method described in PCT-WO90/12603 (Vacanti
et al.), again a three-dimensional matrix is used which matrix
consists of degradable polymer fiber materials and on which matrix
the cells settle. The cells cultivated on the matrix or in
mono-layer cell cultures and then introduced into the matrix are
implanted adhering to the matrix and therefore, in an immobilized
state. The matrix is degraded in vivo and is gradually replaced by
extracellular matrix built by the cells.
[0013] According to the method described in U.S. Pat. No. 5,326,357
(Kandel), chondrocytes are applied to a layer of filter material
(MILLICELL.RTM.-CM having a pore size of 0.4 .mu.m) in a mono-layer
with a cell density of 1.5.times.10.sup.6 cells per cm.sup.2. In
vitro culturing of the monolayer produces a thin cartilage layer in
two to four weeks which, in its structure obviously corresponds to
the natural articular cartilage and can be implanted as such.
[0014] It is also known that cartilage can be cultivated in so
called high density cell cultures. Cells are applied to a carrier
and are cultured in a higher density than used for mono-layer
culturing. The culture medium is added only one to two hours after
bringing the cells onto the carrier. After one to three culture
days, the cell layer on the carrier contracts and so-called
microspheres with diameters in the range of 1 mm form. On further
culturing, a cartilage-like tissue forms inside these microspheres
while fibrous cartilage (perichondrium) forms on their surface. For
implants, this kind of inhomogeneous tissue is not suitable.
[0015] Sittinger et al. (Biomaterials Vol. 17, No. 10, May 1996,
Guilford GB) suggest to introduce vital cells into a
three-dimensional matrix for growing cartilage in vitro and to then
enclose the loaded matrix into a semi-permeable membrane. During
the cartilage growth, this membrane is to prevent the culture
medium to wash away compounds produced by the cells and being used
for constructing the extracellular matrix. Implantation of cell
cultures enclosed in this kind of membranes is also known for
preventing immune reactions.
[0016] All methods named above attempt to produce cartilage at
least partly in vitro, i.e. to produce cartilage using vital
natural cells under artificial conditions. The problem encountered
in these attempts is the fact that chondrocytes in these in vitro
conditions have the tendency to de-differentiate into fibroblasts
relatively rapidly, or the fact that it is possible to
differentiate fibroblasts to a chondrocyte function under very
specific culture conditions only. By the de-differentiation the
chondrocytes among other things loose the ability to produce type
II collagen which is one of the most important compounds of
cartilage tissue.
[0017] According to the methods mentioned above, the problem of
de-differentiation of chondrocytes is solved by immobilizing the
chondrocytes in correspondingly dense cultures in a monolayer or in
a three-dimensional matrix. It shows that in this manner
chondrocytes reproduce themselves without substantial
de-differentiation and form an extracellular matrix which is at
least similar to the extracellular matrix of natural cartilage. The
three-dimensional matrix is mostly not only used for immobilizing
the cells but also for mechanical stability after implantation
which is needed because none of the cartilage tissues produced in
the named manner has a stability which could withstand even a
greatly reduced strain.
[0018] The object of the invention is to create a device with which
suitable cartilage tissue or implants which at least partly consist
of such cartilage tissue can be produced in vitro, the cartilage or
implant serving for implantation, especially implantation in
articular cartilage defects. For achieving this object it is
necessary to create an in vitro environment, in particular a
three-dimensional such environment, in which environment
chondrocytes or other cells capable of a chondrocyte function do
not de-differentiate over a longer culture period and perform their
function actively or in which environment cells differentiate to
become active chondrocytes respectively. By solving the problem of
this environment, the main part of the object is achieved as not
de-differentiating, vital chondrocytes according to their natural
function produce the extracellular matrix characteristic for
cartilage and together with this form the cartilage tissue to be
created.
[0019] The implants produced according to the invention consist at
least partly of cartilage tissue produced in vitro and are
especially suited for the repair of enchondral or osteochondral
joint defects. They are to be producible for any possible depth of
such a defect and a defect repaired with the inventive implant is
to be able to carry a normal load as soon as possible after
implantation, i.e. a load created either by pressing or by shearing
forces.
[0020] This object is achieved by the method as defined in the
claims.
[0021] The inventive method is based on the finding that
chondrocytes can build satisfactory cartilage tissue if it is made
possible that a sufficiently high concentration of compounds
produced by the cells and segregated into extracellular spaces is
achieved in a short initial phase and is maintained during the
whole culture period. Under these conditions the differentiated
function of the chondrocytes is fully maintained (they do not
de-differentiate into fibroblasts) and/or it is possible to
differentiate corresponding cells, especially mesenchymal stem
cells or other mesenchymal cells or even fibroblasts to a
corresponding function.
[0022] The first condition is fulfilled by creating a cell
community in which there is, at least at the beginning of the
culture period, a density of cells such that the cells are capable
to produce the amount of compounds necessary for the mentioned
concentrations in the spaces between the cells. The second
condition is fulfilled by accommodating the cell community in a
restricted cell space in which washing out of the named compounds
from the extracellular spaces is prevented.
[0023] The named compounds are especially autocrine factors and
substances serving as components for building the extracellular
structure. These components are especially aggrecanes,
link-proteins and hyalorunates for building
proteoglycane-aggregates and preliminary stages of collagens to
eventually form collagen-fibrils of type II.
[0024] According to the inventive method, the cells are not
immobilized for the in vitro culture but have space at their
disposition, space without a three-dimensional, artificial matrix
in which space the two conditions mentioned above are fulfilled, in
which space it is however largely left to the cells how they are to
settle relative to each other. It shows that in this kind of free
cell space, cells fully practice their chondrocyte-function and a
cartilage tissue having sufficient stability for implantation and
being able to carry at least part of the normal load after
implantation can be cultured.
[0025] According to the inventive method, cells which are capable
of a chondrocyte-function are introduced into an empty cell space,
i.e. into a space containing culture medium only, such that there
is a cell density of ca. 5.times.10.sup.7 to 10.sup.9 cells per
cm.sup.3 in the cell space. This density amounts to a space
occupation of ca. 5% to 100% at an approximate cell volume of
10.sup.3 .mu.m.sup.3.
[0026] The cell space has at least partly permeable walls and is
introduced into a space filled with culture medium for the length
of the culture period which medium is periodically renewed in known
manner. During the culture period, the cell space is arranged to be
stationary in the culture medium or it is moved in it (relative
movement between cell space and culture medium surrounding the cell
space).
[0027] The permeability of the permeable parts of the cell space
wall and the relative movement are to be matched to the relative
dimension of the cell space (depending on the cartilage to be
produced) such that the condition of the washing-out-prevention is
fulfilled.
[0028] For all cases, semi-permeable wall regions (semi-permeable
membranes) with a permeability of 10.000 to 100.000 Dalton (10 to
100 kDa) are suitable, especially for agitated cultures and for
cell spaces of large dimensions (three-dimensional forms). The
named autocrine factors and components for building the
macromolecules of the extracellular cartilage matrix have molecular
weights which are such that they cannot pass through a membrane
with the named permeability.
[0029] It shows that for stationary cultures and cell spaces with
at least one small dimension (thin layers) open-pore walls with
considerably larger pores (up to the region of 10 to 20 .mu.m)
which cannot be effective as semi-permeable walls but merely as
convection barriers are sufficient and that possibly the cell space
can even be open on one side.
[0030] Especially in cell spaces not being moved and containing
cells at a density in the lower region of the given density range,
the cells settle in the direction of gravity and form cartilage
tissues in the form of layers. For producing more three-dimensional
cartilage forms, agitated cell spaces prove to be advantageous.
[0031] For specific cultures and especially for specific forms and
sizes of cell spaces the optimal arrangement (with or without
movement of the cell space in the culture medium) and the optimal
condition of the wall of the cell space or the permeable parts of
this wall respectively must be determined by experiment.
[0032] The cell space has substantially three functions:
[0033] The cell space keeps the community of the (not immobilize)
cells together at a sufficient density such that they do not loose
their specific functionality;
[0034] the cell space restricts the growth of cartilage such that
by choice of the form of the cell space the form of the cartilage
being formed is controlled;
[0035] the cell space wall allows the supply of the cells with
culture medium but prevents the washing out of the substances
produced by the cells and necessary for the growth of
cartilage.
[0036] For producing implants which only partly consist of
cartilage tissue, a part of the permeable wall of the cell space
can additionally have the function of an implant part as will be
described in more detail further below.
[0037] The cells to be brought into the cell space are
chondrocytes, mesenchymal stem cells or other mesenchymal cells.
These cell types are isolated in known manner from cartilage
tissue, from bone or bone marrow or from connective tissue or fatty
tissue. Fibroblasts are also suited, e.g. if factors are added to
the culture medium or the cell space which factors effect
differentiation of the fibroblasts to chondrocytes or if the cells
are treated with this kind of factor before they are brought into
the cell space. The cells can also be multiplied in vitro before
being brought into the cell space.
[0038] It is not necessary to isolate specific cell types from
donor tissue, i.e. mixtures of different cells as usually contained
in such tissues can be brought into the cell space as such. It also
shows that a complete separation of the cells from the
intracellular matrix of the donor tissue is not necessary and thus
possibly tissue particles or mixtures of isolated cells and tissue
particles can be brought into the cell space instead of cells only.
However, care has to be taken that the necessary cell density is
achieved in the cell space possibly by partly separating the cells
from their extracellular matrix, e.g. by means of a short enzymatic
digestion.
[0039] It shows that with the known culture media such as e.g.
HAM-F12 to which 5 to 15% serum is advantageously added, good
results can be achieved. Furthermore, known growth factors and
other components of culture media which support the reproduction of
the cells and the forming of the cartilage matrix can be added to
the culture medium.
[0040] It shows that in the culture conditions created according to
the inventive method the chondrocytes remain active and do not
de-differentiate such that the space is filled with cartilage
tissue in a culture period in the range of ca. three weeks. The
cartilage tissue being formed can, as soon as it has a sufficient
mechanical strength, be removed from the cell space and e.g. be
further cultivated floating freely in the culture medium or it can
remain in the cell space up to directly before being used.
[0041] Cartilage tissue produced according to the inventive method
is used as implant as implant part or when containing autologous
cells as cell-autotransplant or it can be used for scientific in
vitro purposes.
[0042] The following Figures illustrate the inventive method, the
arrangement for carrying out the inventive method as well as
examples of implants produced by means of the inventive method. The
shown examples are implants for repair of enchondral and
osteochondral defects in joints. However, using the inventive
method it is also possible to produce other implants such as e.g.
auditory bones or cartilage for plastic surgery e.g. nose
cartilage, orbital floors, ear conchs or parts thereof.
[0043] FIGS. 1 to 4 show four exemplified arrangements (in section)
for carrying out the inventive method for in vitro production of
cartilage tissue;
[0044] FIGS. 5 to 7 show chondroitin and collagen contents of
different experimental cartilage cultures (of cultures according to
the inventive method and of reference cultures according to known
methods) in comparison with natural articular cartilage;
[0045] FIGS. 8 and 9, for illustrating the mechanical properties of
cartilage produced according to the inventive method, show forces
straining such cartilage (FIG. 8) and straining native cartilage
(FIG. 9);
[0046] FIGS. 10 to 13 show light-microscopical and
electron-microscopical micrographs of cartilage produced according
to the inventive method and of native cartilage.
[0047] FIG. 14 shows a section through an implant produced
according to the inventive method as illustrated in FIG. 2 or 3,
the implant comprising a carrier layer and a cartilage layer
(boundary region between the two layers in section perpendicular to
the layers);
[0048] FIGS. 15 and 16 show exemplified embodiments of the
inventive implants according to FIG. 14 in section perpendicular to
the cartilage layer;
[0049] FIGS. 17 to 20 show examples of applications of implants
produced according to the inventive method for the repair of
enchondral and osteochondral joint defects (sections perpendicular
to the cartilage layer).
[0050] FIG. 1 shows in section an exemplified arrangement for the
inventive in vitro production of cartilage tissue. This arrangement
substantially consists of a defined cell space 1 into which the
cells are introduced and which is arranged in a culture medium
space 2. At least part of the boundary between the cell space 1 and
the culture medium space 2 is formed by a permeable wall, e.g. a
semi-permeable membrane 3. The remaining parts of the boundary
separating the cell space 1 from the culture medium space 2 are not
permeable and consist e.g. of plastic components which give the
cell space 1 the predetermined form and hold the semi-permeable
membrane in place.
[0051] In the shown example, an inner ring 4 and two outer
snap-rings 5 and 6 together with two e.g. substantially circular
pieces of semi-permeable membrane enclose a cell space 1 in the
form of a circular disc.
[0052] The semi-permeable membrane 3 has a permeability of 10.000
to 100.000 Dalton. It e.g. consists of the same material as a
corresponding dialyse tube. It is obvious that in the same manner
as shown in FIG. 1, cell spaces of the most various forms can be
created, into which spaces cells are introduced and in which spaces
these cells build cartilage tissue, the cartilage tissue
substantially assuming the form of the cell space 1 or the form of
the one part of cell space 1 which during the culture period faces
downward in the direction of gravity.
[0053] The culture medium space 2 is a freely selectable space in
which the culture medium is periodically exchanged in known manner.
If the cell space 1 is to be moved in the culture medium space 2
the culture medium space is e.g. a spinner bottle.
[0054] Using an arrangement according to FIG. 1, the inventive
method is e.g. carried out as follows:
[0055] Cells, tissue particles or mixtures of cells and/or tissue
particles as described further above are suspended e.g. in culture
medium and are introduced into the free cell space such that the
cell density is in the range between 5.times.10.sup.7 and 10.sup.9
cells per cm.sup.3.
[0056] The cell space is closed and introduced into the culture
medium space and is left there for a period of time in the range of
approximately three weeks.
[0057] The cartilage tissue formed in the cell space is removed
from the cell space and is either cultivated further swimming
freely on a culture medium or is directly used as implant or as
transplant or for scientific investigations respectively.
[0058] In the a cell space according to FIG. 1, implants consisting
of cartilage tissue, e.g. auditory bones, nose cartilage, orbital
floors or parts thereof are produced.
[0059] FIG. 2 shows a further embodiment of a cell space for
carrying out the inventive method. The cell space 1 is flat and its
one side is limited by an open pore, rigid or plastically
deformable plate 7 made of a possibly biologically degradable bone
substitute material, the other side by a permeable wall, e.g. a
semi-permeable membrane 3 or a more coarsely porous wall. The cell
space has a height of e.g. ca. 3 to 5 mm and any flat form and
extension.
[0060] A cell space as shown in FIG. 2 is especially suited for the
production of an implant for repair of a osteochondral defect. The
implant comprises not only the cartilage tissue grown in the flat
cell space but also the bone substitute plate 7. This bone
substitute plate 7 thus has substantially two functions: during the
growth of the cartilage, it serves as permeable wall for the cell
space 1 and in the finished implant, it serves as anchoring
substrate for the cartilage layer, whereby after implantation this
bone substitute material is colonized in known manner by cells
immigrating from the adjacent vital bone tissue.
[0061] In order for the bone substitute plate 7 to be able to
fulfill the second function named above care must be taken that by
a corresponding arrangement of the cell space in the culture medium
space the cells settle over the whole inner surface of the bone
substitute plate 7 at least for a case in which the initial cell
density is such that the cells have a settling tendency. Therefore,
the cell space 1 is e.g., as shown in FIG. 2, arranged to be
stationary with the bone substitute plate 7 facing downward such
that the cells settle on the bone substitute plate 7 due to the
effect of gravity.
[0062] Furthermore, the bone substitute plate 7 must be formed such
that the cartilage tissue growing in the cell space 1 is able to
grow together with the bone substitute plate 7 in an intermediate
region, thus forming a two part implant which can resist shearing
forces. This kind of growing together is achieved by choosing the
porosity of at least the one surface of the bone substitute plate
on which the cartilage is cultivated such that the collagen fibrils
built in the extracellular cartilage matrix can grow into the pores
and can such anchor the new cartilage in the bone substitute plate.
It shows that for this kind of anchoring of the collagen fibers,
pores of ca. 1 to 20 .mu.m are suitable.
[0063] Furthermore, it is advantageous if a part of the cells which
are brought onto the surface of the bone substitute plate for
cultivating the cartilage settle in uneven places or in pores such
that the growing cartilage tissue growth is connected to the bone
substitute material in these uneven places and pores in a kind of
positive engagement. It shows that cells easily settle in uneven
places or pores if these have a size of at least 20 .mu.m, ideally
between 20 and 50.mu..
[0064] Therefore, the bone substitute plate 7 is to fulfill the
following conditions:
[0065] In order for the cells to be able to be nourished from the
culture medium through the bone substitute plate it must comprise
pores which form continuous canals (open porosity).
[0066] In order for the bone substitute plate to serve at least as
a convection barrier against the washing away of larger molecules
the pores must not be too large and the thickness of the plate must
not be too small.
[0067] In order for the collagen fibrils being built in the growing
cartilage tissue to be anchored in the pores of the plate the pores
must not be larger than 20 .mu.m.
[0068] In order for the cells to be able to settle in uneven places
or pores in the surface of the bone substitute plate such uneven
places or surface pores (surface roughness) having sizes of at
least ca. 20 .mu.m must be provided at least on the one part of the
surface facing the growing cartilage layer.
[0069] It shows that with bone substitute plates having a
correspondingly rough surface, having an open porosity with pore
sizes in the range of 2 to 20 .mu.m and having a thickness of 0,5
to 3 mm, advantageously 0,5 to 1,5 mm satisfactory results can be
achieved. With a plate thickness larger than ca 0,5 to 1 mm, the
region facing away from the bone substitute plate can also have a
coarser porosity, e.g. pores with sizes up to 300 to 700 .mu.m such
as known from bone substitute materials. This kind of porosity
favors the in vivo vascularization of the bone substitute
material.
[0070] As bone substitute material, known osteo-inductive and/or
osteo-conductive materials are suitable, advantageously
biologically degradable such materials which have the mentioned
open porosity and which can be processed to rigid or plastically
deformable plates. Plastically deformable plates can e.g. be
produced from collagen I, from collagen II and hydroxyapatite or
from polylactic acid. Rigid plates can be formed from
tricalcium-phosphate, from hydroxyapatite or from other inorganic
bone substitute materials.
[0071] Treatment of the bone substitute plate 7 with an attachment
factor is unnecessary.
[0072] In a cell space according to FIG. 2 comprising a
correspondingly open-pored bone substitute plate 7, not only
cartilage tissue is grown in vitro but also an implant is produced
which comprises a pre-formed, grown together
cartilage/bone-intermediate region.
[0073] FIG. 3 shows a further, exemplified arrangement for carrying
out the inventive method. In principle this is a combination of the
methods as carried out in arrangements according to FIGS. 1 and 2.
The bone substitute plate 7 is not arranged as part of the
permeable wall of the cell space 1 but lies within the cell space
which cell space is e.g. limited by semi-permeable membranes 3. It
is obvious that in this kind of arrangement the bone substitute
plate 7 does not have to take over the function of a convection
barrier and that due to this the porosity and thickness of the
plate can be chosen at greater liberty.
[0074] The arrangement shown in FIG. 3 is especially suitable for
thin, plastically deformable bone substitute plates 7 which are
complicated to handle as cell space walls.
[0075] FIG. 4 shows schematically a further embodiment of a cell
space 1 which space is especially suitable for cultivating very
thin cartilage layers which are grown together with a bone
substitute plate. In opposition to the cell space according to
FIGS. 1 to 3, the cell space in FIG. 4 is open towards the top such
that at least in the first one to two weeks cultivation must take
place under stationary culture conditions. In opposition to
similar, known arrangements (e.g. U.S. Pat. No. 5,326,357, Kandel)
a bone substitute plate 7 is provided as carrier for the cells and
the cells are not applied to the carrier as mono-layer and are not
immobilized with the help of an attachment factor.
[0076] FIGS. 5 to 7 show results from experiments with the
inventive method (experimental arrangement substantially as
outlined in FIG. 1). Dialyse tubes were used as semi-permeable
membranes.
[0077] Chondrocytes were isolated from bovine shoulders using known
isolation methods. The cells were introduced into the dialyse tubes
and these were moved in a spinner bottle during the culture period,
whereby the cells settled on the bottommost end of the tubes.
HAM-F12 with 5 to 15% serum was used as culture medium. The culture
medium was changed every two days.
[0078] FIG. 5 shows the contents of chondroitin sulfate and
collagen of the cartilage tissue produced according to the
inventive method (in .mu.g per ml of cartilage tissue) as a
function of the duration of the culture period (20, 29 and 49 days)
in comparison with corresponding values of cartilage from bovine
shoulders (age: eighteen months). The results show that the content
of chondroitin sulfate can be even higher in the cartilage produced
in vitro than in natural cartilage, that the content of collagen
however is considerably lower.
[0079] FIGS. 6 and 7 also show, as a function of the duration of
the culture period (0, 7, 20 and 40 days), chondroitin sulfate
(FIG. 6) and collagen contents (FIG. 7) in reference cultures A and
B and of cartilage tissue grown according to the inventive method
after 40 days of culture time (C: cartilage growing in the cell
space in the region of the settled cells, D: culture medium in the
cell space above the settled cells). For references, chondrocytes
were embedded in alginate spheres and cultivated in a stationary
culture (reference A) and in a spinner bottle (reference B).
[0080] FIGS. 6 and 7 make it clear that the cartilage structure in
the experimental arrangement according to the invention is
considerably more successful than in the reference experiments.
[0081] FIGS. 8 and 9 show results of stress experiments on
cartilage produced according to the invention (FIG. 8) and on
native cartilage (FIG. 9) in order to illustrate the mechanical
properties of the cartilage produced according to the inventive
method (for culture conditions see above) compared with the
mechanical properties of native bovine cartilage. The experiment
consists in pressing a punch into the cartilage with a constant
speed (1 micrometer per second) and to stop the punch at a
penetration depth of 200 .mu.m while registering the punch force.
This force first rises approximately proportionally to the
penetration depth and after stopping the punch decreases
(visco-elastic force reduction due to loss of liquid of the
cartilage tissue).
[0082] The two FIGS. 8 and 9 show the punch force in Newton (N) as
a function of the time in seconds (s). In FIG. 8 the registering of
the depth of penetration (travel) is shown in .mu.m.
[0083] The stress experiment with the cartilage produced according
to the invention (FIG. 8) was carried out with a punch having a
diameter of 20 mm and shows a maximal pressure of ca.
0.8N/cm.sup.2. The experiment with the native cartilage was carried
out with a punch having a diameter of 5 mm and shows a maximal
pressure of ca. 30N/cm.sup.2.
[0084] The considerably smaller maximal pressure of the cartilage
produced according to the inventive method can be explained in
connection with FIGS. 10 to 13. These Figures show
light-microscopical micrographs (FIGS. 10 and 11) and
electron-microscopical micrographs (FIGS. 12 and 13) of cartilage
produced according to the inventive method (FIGS. 10 and 11) and of
human cartilage from the medium zone (FIGS. 12 and 13).
[0085] FIGS. 10 and 11 clearly show that the cartilage produced
according to the inventive method contains considerably more
chondrocytes than the native cartilage which can be interpreted as
growth stage for the cartilage cultivated in vitro. The same
interpretation is suggested by FIGS. 12 and 13 in which organelles
are easily visible in the chondrocytes (marked with arrows). In the
native cartilage, the organelles are narrow suggesting little
synthesis activity; in the cartilage produced according to the
invention they are distinctly enlarged which suggests an intense
synthesis activity, i.e. a growth stage.
[0086] Further electron-microscopical investigations of inventively
produced cartilage tissue show that the collagen fibrils form a
dense net therein but that they are thinner than in native
cartilage (after completed growth) and that they are arranged
having random directions. Therefore, the cartilage tissue produced
according to the invention must be looked at as a kind of embryonic
cartilage tissue which, however has the ability to develop in vivo
(after implantation) into `grown-up` cartilage.
[0087] FIG. 14 schematically shows a histological section
(magnification ca. 100-fold) through the intermediate region
between cartilage tissue and bone substitute plate of an implant
which was produced in an arrangement according to one of the FIGS.
2 to 4. The cartilage tissue 10 and the bone substitute plate 7 are
connected to each other in an intermediate region in the manner of
positive engaging means due to the cartilage tissue having grown
into uneven surface places of the bone substitute plate 7.
[0088] FIGS. 15 and 16 show exemplified embodiments of implants
which are produced according to methods as described in connection
with FIGS. 2 to 4 (section through cartilage layer 10). The
implants comprise an intermediate region in which the cartilage
tissue is grown together with the bone substitute plate 7, as shown
in FIG. 14.
[0089] After a culture period of a few weeks, the bone substitute
plate 7 with the cartilage layer 10 having grown on it is separated
from the other wall components of the cell space. Before
implantation, the implant consisting of the cartilage layer 10 and
the bone substitute plate 7 is reduced to the demanded size and
form, if required, and/or is fixed to a further piece 12 of bone
substitute material.
[0090] For processing the implant taken from the cell space, common
surgical methods are used, such as e.g. punching, laser cutting or
milling. For enlarging the bone substitute plate, a further piece
12 of a similar or different bone substitute material is attached
on the one side of the bone substitute plate 7 opposite to the
cartilage layer 10, using a known advantageously biologically
degradable cement.
[0091] After implantation, bone forming cells from the native
environment migrate into the open-pore bone substitute material of
the bone substitute plate 7 and of the attached piece 12 and
micro-vessels grow into the pores of the material. As a result of
this a natural bone tissue develops which gradually replaces the
bone substitute material (7, 12, 13) being gradually degraded.
Hereby it is to be expected that the cartilage cultivated in vitro
is mineralized in the intermediate region 11.
[0092] FIGS. 17 to 20 show enchondral and osteochondral defects
which are repaired with exemplified embodiments of inventive
implants.
[0093] FIG. 17 shows an enchondral defect with a defined form made
by drilling or milling, i.e. a defect which lies in the native
cartilage layer 20 and does not affect the bone tissue 21
underneath the cartilage layer 20. This kind of defect has, in the
human case, a depth of maximally ca. 3 mm and can affect any extent
of the cartilage surface. A piece of cartilage tissue 10' is
inserted into the prepared defect which cartilage tissue was e.g.
cultivated in an arrangement according to FIG. 1 and which
cartilage tissue after cultivation was made to fit the form of the
defect by cutting or punching, if required.
[0094] For a satisfactory fixation of the implant in the defect it
is sufficient at least for small implants to slightly deform the
implant elastically on implantation (press fit). With larger
defects the implant is fixed with known means: e.g. with a piece of
periosteum which is sutured or glued over the implant, with a glue
being introduced between native cartilage and implant (e.g. fibrin
glue) or by suturing the implant to the surrounding native
cartilage.
[0095] FIG. 18 shows a small osteochondral defect which has been
prepared for implantation by drilling an opening having a defined
form (surface extension up to ca. 10 mm, depth up to ca. 3 mm in
the bone tissue), i.e. a defect which does not only affect the
native cartilage tissue 20 but also the bone tissue 21 beneath it.
This defect is repaired with an implant according to FIG. 15, which
implant is fixed with one or several pins. Two pins are shown. The
one pin 22.1 is driven through the implant from its surface by the
surgeon, the other pin 22.2 is previously arranged in the bone
substitute plate 7 of the implant and is driven into the bone by
pressing on the surface of the implant.
[0096] Obviously, it is also possible to fix the implant in the
defect according to FIG. 18 with other fixing means than pins.
[0097] From FIG. 18 it can be seen that in the repaired region the
considerable shearing forces which act upon the intermediate region
between bone 21 and cartilage 20 when straining the joint are taken
over by the intermediate region 11 where the cartilage layer grown
in vitro is grown together with the bone substitute plate 7.
[0098] Instead of an implant consisting of a cartilage layer 10
cultivated in vitro and a bone substitute plate 7, as shown in FIG.
18, the same defect can also be filled with a filling substance and
further repaired with a piece of cartilage tissue cultivated in
vitro (without bone substitute plate), in the same way as this is
shown in FIG. 17. For taking up the shearing forces in such a case
e.g. pins 22.1 reaching right through the implant are to be
provided.
[0099] FIG. 19 shows a prepared osteochondral defect having a depth
of 20 to 30 mm and repaired by implantation of an inventive implant
according to FIG. 16. Shearing forces are again taken up by the
region where the cartilage layer 10 and the bone substitute plate 7
are grown together. As the cement-connection 13 is positioned
within the native bone 21 it is not strained by shearing and thus
need not be reinforced by means of a pin.
[0100] The repair shown in FIG. 18 can also be carried out by
filling the lower part of the bore with bone substitute material
and by implanting an implant according to FIG. 15, possibly by
means of a cement layer 13.
[0101] For larger osteochondral defects a plurality of implants
according to FIG. 19 can be provided (mosaic plasty).
[0102] FIG. 20 shows a large and deep osteochondral defect. It is
so large that the cartilage area to be repaired can no longer be
approximated with an even area. This kind of defect can, as
indicated further above, be repaired with mosaic plasty. However,
as the inventive implants are not limited regarding surface size
and as the bone substitute plates 7 can be made of a plastically
deformable material, the defect is more easily repaired according
to FIG. 20 with an implant according to FIG. 15. For this purpose,
the defect is cut out to a depth of a few millimeters into bone 21
and to a defined form, deeper regions are filled with a plastic
bone substitute material and the implant is positioned in the
defect and fixed with suitable means.
* * * * *