Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
1999-03-04
2001-06-05
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S297500, C435S182000, C435S382000, C623S023630
Reexamination Certificate
active
06242247
ABSTRACT:
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.
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.
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.
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.
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.
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.
It is also suggested, e.g. in the publication U.S. Pat. No. 3703575 (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.
Examples of such methods are described in the following publications:
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.
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 bio-compatible, resorbable matrix (32×10
6
to 120×10
6
cells per cm
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.
According to the method described in U.S. Pat. No. 4,963,489 (Naughton), again a three-dimensional, artifical 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.
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 monolayer 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.
According to the method described in U.S. Pat. No. 5,326,357 (Kandel), chondrocytes are applied to a layer of filter material (MILLICELL®-CM having a pore size of 0.4 &mgr;m) in a mono-layer with a cell density of 1.5×10
6
cells per cm
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.
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.
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.
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. According to the methods mentioned above, the problem of de-differentiation of chondrocytes is solved by immobiliz
Bittmann Pedro
Mainil-Varlet Pierre
Muller Werner
Rieser Franz
Saager Christoph P.
Beisner William H.
Birch & Stewart Kolasch & Birch, LLP
Sulzer Orthopedics Ltd.
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