WO2012055851A1

WO2012055851A1 - Chondrons for cartilage therapy

Info

Publication number: WO2012055851A1
Application number: PCT/EP2011/068618
Authority: WO
Inventors: Bernd Rolauffs; Wilhelm Aicher
Original assignee: Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum
Priority date: 2010-10-27
Filing date: 2011-10-25
Publication date: 2012-05-03
Also published as: DE102010050460A1

Abstract

The present invention relates to the use of chondrons to treat damage to or degeneration or diseases of cartilage, bone, menisci or intervertebral discs, in particular osteoarthritis, arthrosis, or osteochondritis dissecans, in particular in humans and horses.

Description

Chondrone for cartilage therapy

The present invention relates to the use of endogenous structures for cartilage, bone, meniscus and disc therapy and a method for obtaining these endogenous structures.

Cartilage damage such as diseases or degenerations can have many causes. Common to all damage, however, is the loss and alteration of the matrix located between and above the chondrocytes. As a result, the cartilaginous surface initially becomes rough, then cartilage substance is lost, it develops an arthrosis, which in turn limits the function of the affected joint.

Chondrosis is generally referred to as the degenerative changes of the cartilage. In intervertebral discs, there is a narrowing of the intervertebral space between one or more vertebral segments compared to the remaining vertebral bodies, whereby the height of the intervertebral spaces decreases. The cause may be either congenital or acquired changes, the latter being degenerative, inflammatory, or operative. In addition, if the underlying bone involved, it is called osteochondrosis. Changes in the cartilaginous and osseous tissues in the large and small joints of the musculoskeletal system lead to osteoarthritis, which can also be caused by congenital or acquired changes. The cause may be degenerative, inflammatory, post-traumatic factors and diseases of the central nervous system in question. The resulting in an arthritis / osteoarthritis from the cartilaginous cartilage pieces, or the resulting state is referred to as osteochondritis dissecans, which also occurs in the context of an accident or today medically identifiable causes and also causes the development of osteoarthritis / osteoarthritis.

In recent decades, surgical procedures for cartilage repair have been established. In articular cartilage repair treatment, the goal is removal of the diseased cartilage and restoration of a cartilage surface. A modern, promising and clinically established therapy for cartilage damage is the implantation of the patient's own, so-called autologous chondrocytes in a matrix. This procedure is referred to as autologous chondrocyte transplantation, abbreviated to ACT (English: ACI). In the medium term, an ACT leads to good clinical success in smaller, well-defined cartilage lesions. However, larger defects or corresponding defects on opposing cartilage surfaces can not be treated with it. In addition, the ACT also leads to the formation of only a hyaline-like cartilage, the quality of which neither clinically nor histologically corresponds to the hyaline cartilage. This is reflected in a reduced biomechanical quality of the hyaline-like (fibrous) cartilage, whereby further degeneration and thus also the development of osteoarthritis can only be deferred, but can not be reversed. It is therefore an object of the present invention to provide a comparison with the prior art improved therapy option with which even larger defects can be treated.

According to the invention this object is achieved by the use of chondrons for the treatment of cartilage, bone, meniscus or disc damage or diseases and the resulting osteoarthritis / osteoarthritis.

Further, we solved the invention by a method for obtaining chondrons, can be isolated with the body's own or allogenic and xenogeneic chondrons.

The object underlying the invention is thereby completely solved. The use of chondrons provides a means by which on the one hand larger cartilage defects can be treated and on the other hand the long-term survival of the chondrocytes contained in the chondrons is improved.

In the present case, a "chondron" is understood as meaning the unit comprising chondrocyte and the surrounding pericellular environment, i. the pericellular glycocalyx and the pericellular capsule, understood. Chondrocytes and their pericellular environment form a structural and functional unit.

In the ACT currently used, healthy cartilage cylinders of a patient, for example, from the knee joint, are removed. From this, chondrocytes are isolated, expanded in the laboratory, applied to a carrier material and implanted in the cartilage defect. The isolation of the chondrocytes destroys the pericellular matrix, and thus the structure of the chondron. As a result, the isolated chondrocytes produce insufficient amounts of glycosaminogly- and collagen type II, whereby the biomechanical properties of this cartilage formed from the ACT are inferior to those of the original cartilaginous tissue.

The present inventors have recognized that the isolation of physiologically complete chondrons from the cartilage tissue and their use to form cartilage replacement tissue offers an improved approach to the therapy of cartilage damage compared to the method known in the art.

It is particularly preferred if the chondrons for the treatment of cartilage damage or diseases of the large joints such. the knee, hip, jump, shoulder or elbow joint or the spine, in particular for the treatment of disc damage, are used.

In the field of treatments for affected joints and spinal columns of a patient, in particular a mammal, eg. Of a human, the need for effective and durable therapy is still great. The cartilage damage or diseases of joints in these areas include, for example, osteochondritis dissecans, osteoarthritis, arthritis and osteoarthritis, ie inflammatory and degenerative joint diseases.

With the use of chondrons according to the invention, such diseases, ie cartilage degeneration and the resulting osteoarthritis / arthrosis can be treated in a targeted manner, as well as a progression of osteoarthritis can be prevented or delayed. Chondrons, as explained above, form a functional unit of chondrocytes with their immediate surrounding pericellular matrix, and can be obtained therefrom mechanically and enzymatically from cartilaginous tissue. Both metabolically and biomechanically, chondrons are of great functional importance because their structure plays a major role in biomechanical, biophysical and biochemical interactions between chondrocyte and ECM. Furthermore, Chondron pellets form significantly more glycosaminogly- kane and collagen type I as chondrocyte pellets, which is why they are particularly suitable for the claimed use in comparison to the chondrocytes hitherto used isolated in the ACT. The inventors have in fact recognized that due to the preserved, original pericellular matrix of the chondrons, the formation of a hyaline-like matrix in cartilage damage can be promoted much more strongly than a pericellular matrix, which must be newly synthesized by expanded chondrocytes.

Furthermore, the use of chondrons in the diseases / injuries listed offers the advantage that they provide as an important aggrecan reservoir the essential markomolecule for the hydrodynamic cartilage function and for cartilage proteoglycans. In addition, the chondrons also provide mechanical protection for chondrocytes from biomechanical overload. Furthermore, unlike chondrocytes, chondrons are non-immunogenic, and therefore much more compatible in their use than these.

The use of the invention thus offers the advantage that not only young patients or those with isolated, smaller cartilage defects can be treated, but now older patients and / or patients with major cartilage damage, currently still by an implantation of artificial joints or only be treated by conservative measures such as painkillers etc.

In one embodiment of the invention, the chondrons are used for the treatment of osteoarthritis / osteoarthitis or osteochondrosis dissecans.

As already mentioned above, "osteoarthritis" or arthrosis is understood to mean a joint disease with destruction of the cartilage surfaces. "Osteochondrosis dissecans" is understood to mean the detachment of a cartilaginous piece (flake) by, for example, an accident or also by other unexplained causes. which can lead to the development of arthritis / osteoarthritis. These diseases occur especially in the knee joint, in the upper ankle joint, in the hip joint, in the shoulder joint and in the elbow joint, not only in humans, but also in animals, such as in dogs, (racing) horses, cats, cows or pigs.

In a further development of the use according to the invention is preferred when chondrons are used, which were obtained from autologous, so the body's own, cartilage material.

This embodiment has the advantage that it can be used on the body's own and thus patient's own material, which possibly minimizes unwanted reactions of the body to the chondrons.

[0021] According to yet another embodiment, chondrons obtained from heterologous, allogenic or xenogenic cartilage material are used. By "allogeneic" material is meant cartilage material from a donor of the same species as the patient to be treated, and "xenogeneic" material from cartilage material from a donor of a species other than the patient into whom the chondrons are to be reincorporated. This embodiment has the advantage that it is also possible to treat patients in whom cartilage material which is insufficiently good or in quantity can be provided for the isolation of chondrons.

In the present case, it is understood that the chondrons can be used after their provision either directly into the area to be treated with cartilage damage or cultivated in vitro, which can be used after cultivation, the chondrons with or without a corresponding carrier material in the joint.

Therefore, the present invention also relates to the use of chondrons derived from cartilage material for the in vitro production of cartilage. replacement tissue. These tissues can then be implanted in the affected area of the patient from whom the original cartilage material has been removed.

In a preferred embodiment of the use according to the invention, the chondrons are obtained as follows: a) in vitro isolation of chondrons from a biological cartilage tissue sample of a patient by means of comminution and / or enzymatic digestion, in particular Dispase II and collagenase P, wherein the Digestion is preferably carried out by incubation with the enzymes for a period of 5 hours and preferably at 37 ° C with stirring, followed by sieving, preferably with a 100 μm sieve, to obtain chondrons; b) in vitro expansion of the chondrons by culturing the chondrons obtained in step a) in monolayer or three-dimensional culture in standard medium;

Step b) can be carried out, for example, the following standard media: F12 + GlutaMAX, Gibco 21885 (250 ml), DMEM + GlutaMAX, Gibco 31765 (250 ml), FBS Superior, Biochrom S0615 (50 ml), PenStrep, Gibco 15140 (10 ml), partricin (fungicides), biochrome A2812 (5 ml), L-ascorbic acid phosphate magnesium salt, Sigma Aldrich A8960 (0.5 ml).

It is particularly preferred if the cartilage replacement tissue is obtained after adaptation under GMP conditions.

According to the invention, the thus isolated chondrons can then be used directly and / with carrier material for the treatment and thus for the generation of cartilage replacement tissue in vivo, or for the in vitro production of cartilage replacement tissue. In the present case, a "biological sample" is understood to mean any material already taken from a patient's body, preferably tissue, more preferably cartilage tissue, the viable cells, in particular chondrons, chondrocytes or their precursors, cell aggregates, and / or other cell-free material contains. In this case, the biological sample may be an endogenous sample, ie a sample of the patient to be treated with the chondrons obtained therefrom, or an allogeneic / xenogeneic sample.

In the present case, it is overall preferred if the patient is a mammal, preferably a human. In addition to the use in humans, of course, analogous applications in other mammals, such as horses, camels, dogs, cats, etc., are also being considered since there is an increasing interest in effective treatment of osteoarthritis / osteochondrosis dissecans / cartilage damage consists of these animals.

According to the invention, the chondrons thus obtained for the treatment of cartilage, meniscal, bone or disc damage, - diseases, or -degenerationen, or for the treatment of osteoarthritis / osteochondrosis dissecans, in particular a human, used become.

The chondrons used according to the present invention may be further administered together with pharmaceutically acceptable carriers and / or excipients, and optionally therapeutically active substances. In this case, "pharmaceutically acceptable excipients or excipients" as used herein means any substance / composition to be administered to a patient in pharmacy which does not or not adversely affect cells, and / or which can support or facilitate the pharmacological use of the chondrons , By "therapeutically active substance" is further understood any substance used for the purpose of treating or ameliorating a clinical picture of a patient. It is understood that the above-mentioned features and those yet to be explained not only in the combination specified, but also in other combinations or alone, without departing from the scope of the present invention.

The invention will be explained in more detail in the following description and the accompanying drawings.

[0034] In the drawings:

Fig. 1 (A) is a schematic representation of the chondron organization and the surrounding collagen fibers with chondrocyte (C), pericellular glycocalyx (PG), pericellular capsule (PC), territorial matrix (M) and interteritorial (IM) matrix ; and (B) a three-dimensional representation of a solitary chondroma; Reference: C. Anthony Poole; 1997;

Fig. 2 Fluorescence microscopy image with collagen type VI staining after isolation from human cartilage tissue A) from chondrocytes and B) from chondrons; in the present case two chondrons: B) on the left a chondron, which contains a cell; B) on the right a chondron, which contains two cells (own data);

Fig. 3 fluorescence microscopy images with A) a collagen type II stain, B) a collagen type VI stain, C) an aggrecan molecule stain;

Fig. 4 A) fluorescence microscopy images of a chondroma consisting of several cells, B) transmitted light microscopy image of the same chondron, C) and D) collagen type II fibers thereof Chondrons as a link to the extracellular cartilage matrix (own data);

5 shows a diagram of the gene expression profiles of chondrons from osteoarthritic joints (OA chondrons) and in comparison of chondrocytes from the same arthritic joints (OA chondrocytes) and chondrocytes from a joint of a young patient, who has an autologous chondrocyte transplantation (ACT) of the above shown chondrocytes (ACT chondrocytes); and

Fig. 6 is a diagram for the relative cell number of chondrons and

Chrondocytes after a certain culture period.

In Figure 1, in Figure A, a chondrocyte is shown in its pericellular environment, the chondron. The chondrocyte and its pericellular environment form a functional unit and have a multifaceted importance for the function of the hyaline cartilage. As can be seen from Fig. 1, a chondron consists of a single or multiple chondrocytes (C) enclosed by the pericellular glycocalyx (PG) and a fibrillar pericellular capsule (PC). The capsule defines the pericellular matrix around the chondrocyte.

To obtain chondrons, osteoarthritic (hereinafter also abbreviated to "OA") chondrocytes were isolated from patients in whom the knee joint was surgically replaced (n = 28, average age: 68 years). After collagenase / dispase digestion at different concentrations and over different time periods, their pericellular matrix was maintained or removed. Non-OA chondrons and non-OA chondrocytes from healthy knee joints were used as controls. The successful isolation was confirmed by immunofluorescence (collagen type 6A3 antibody, Santa Cruz, USA). The cells were incubated at 37 ° C., 5% CO 2 and in a culture medium consisting of F12: DEMEM 1: 1, 10%. FBS, 0.1% ascorbic acid either in three-dimensional peptide hydrogels or cultivated in culture bottles to monolayers. To study gene expression, cDNA was synthesized and quantitative PCR was performed for GAPDH and other genes.

The fluorescence microscopy images in Figure 2 show that living chondrons could be recovered from human tissue with their intact structure; Furthermore, the fluorescence microscopy images in Fig. 3 show that the obtained chondrons produce the fractions, collagen type II, type VI and aggrecan, which are fundamental for cartilage function, in quantities which can be detected by conventional techniques.

Fig. 4 shows that chondrons can be obtained with their fiber network. This may be. relevant for improved / superior function of the chondrone. The isolation of the fiber network and the use of chondrons with and without this fiber network is included in the invention.

In Fig. 5, a gene expression analysis is shown, which clearly shows that important for cartilage function genes collagen type II, Aggrekan and COMP are expressed by chondrons significantly and statistically significantly increased. This significantly increased gene expression of the relevant cartilage components thus shows that chondrons are superior to chondrocytes. In particular, gene expression of chondrons, even from osteoarthrotic diseased joints, is shown to be superior to the chondrocytes of the same joint as well as a young and relatively healthy subject to ACT.

In Fig. 6, the results of the experiment for comparing the relative cell numbers of chondrons and chondrocytes are shown, wherein it was shown that recovered chondrons show a significantly faster proliferation rate than chondrocytes, so that in a laboratory phase of a given length More living chondrons are available for transplantation than chondrocytes. The introduction of chondrons into the human or animal joint as a therapy has not yet been performed, but can be based on the data presented herein with the previously clinically proven, approved and used in clinical routine daily methods. The isolated chondrons can be introduced instead of the usually used chondrocytes in a carrier, which is sewn or glued or inserted after punching a cartilage defect in this defect. Alternatively, z. B. also be used a gel which is injected into the defect. All methods approved for ACT are also eligible.

Claims

claims

1. Use of chondrons for the treatment of cartilage, meniscal, bone or disc damage, degeneration or disease.

2. Use according to claim 1, characterized in that the chondrons are used for the treatment of diseases of the knee, ankle, hip, shoulder, elbow joint and other joints of the musculoskeletal system or the spine.

3. Use according to claim 1 or 2, characterized in that the chondrons for the treatment of osteoarthritis, osteoarthritis, osteochondrosis dissecans, rheumatoid arthritis, are used.

4. Use according to one of the preceding claims, characterized in that chondrons obtained from autologous cartilage material are used.

5. Use according to one of claims 1 to 3, characterized in that chondrons obtained from heterologous, allogenic or xenogenic cartilage material are used.

6. Use of chondrons derived from cartilage material for the in vitro production of cartilage replacement tissue.

Use according to claim 6, characterized in that it comprises the following steps: in vitro isolation of chondrons from a biological sample patient; b) in vitro expansion and cultivation of the chondrons to form a cartilage replacement tissue.

8. Use according to one of claims 1 to 7, characterized in that the chondrons are used for the treatment or for the production of cartilage replacement tissue of a mammal.

Use according to claim 8, characterized in that the mammal is a human or a horse.