CH703968B1 - Method for diagnosing cartilage cell defects and method for producing a cartilage transplant. - Google Patents

Abstract

Disclosed are an in vitro diagnostic method and a method for growing cartilage graft. Common to both methods is that (a) a sample of cartilage is dissolved to recover isolated cartilage cells and (b) the isolated cartilage cells obtained in step (a) are separated and then proliferated for a period of time such that the Proliferation of individual cells by formation of cell populations is recognizable. For diagnosis, the proliferative capacity of the cells is then determined by the size of the cell populations, while in cartilage growth, the cells with the best proliferative capacity are selected and applied to a three-dimensional resorbable scaffold on which the cells are then grown until the graft becomes the desired one Has size and cell density.

Description

Field of invention

The present invention relates to the field of cartilage transplant surgery, including associated diagnostics.

State of the art

According to the methods common today, healthy cartilage tissue is removed from the patient by means of a biopsy. Cartilage cells are isolated from this and placed on a three-dimensional scaffold made of absorbable material and then multiplied in vitro until a transplant of the desired size is built up. After reaching the desired size or cell density, the cartilage transplant is implanted in the damaged area.

With this approach, the life of the transplant has been found to be critical. In a considerable number of patients, the lifespan is only about 1 year.

[0004] The aim of the present invention was therefore to increase the service life and / or the quality of cartilage transplants.

This goal was achieved by two measures, on the one hand by improved diagnosis and on the other hand by an improved transplant.

Presentation of the invention

The object of the invention is therefore an in vitro method for assessing the quality of a cartilage sample or of cartilage mass.

Another object of the invention is a method for producing a cartilage transplant.

The object of the present invention was achieved by providing an in vitro method for assessing the quality of cartilage mass, which is characterized in that in vitro (a) a sample of cartilage mass is dissolved in order to obtain isolated cartilage cells, (b) the isolated cartilage cells obtained in step (a) are isolated and then proliferated for a certain period of time in such a way that the proliferation of the individual cells can be recognized by the formation of cell populations, (c) the ability of the cells to proliferate is determined based on the size of the cell populations.

Step a) is carried out essentially analogously to today's methods for obtaining cartilage cells, in particular with digestion of the cartilage mass, e.g. a biopsy, using hyaluronidase.

The cells obtained in step a) are then singulated and kept for a predetermined time, e.g. cultured for 4 days under conditions that allow proliferation. In order to create conditions that are as close to nature as possible, the culture medium should not be specially enriched with hormones and / or proteins / peptides. Rather, a minimal medium such as Dulbecco’s Modified Eagle’s Medium is advantageous. This contains - inorganic salts as a source for Ca, Fe, Mg, SO4 <2> <–>, PO4 <3> <–> as well as NaCl, KCl for isotonicity; - Amino acids; - vitamins; - Other components, namely the energy sources D-glucose, and sodium pyruvate and possibly phenol red for determining critical changes in the pH value.

Hormones and proteins / peptides are not contained in this medium, since the cartilage quality is to be determined under conditions that are as natural as possible. As already mentioned above, such are preferably not added and if so, it must be ensured that they are always used in the same concentrations and with constant purity / activity, since the ability to proliferate is assessed on the basis of findings obtained with cartilage material of known quality which is why the tests need to be standardized as much as possible. In special cases, an antibody against TNF-alpha can be added, since it has been found that poor cartilage proliferation is associated with high TNF-alpha concentrations in many (up to 70%) of the patients.

In parallel (or in special cases alone) the prolifertion can be tested in culture medium containing autologous blood serum, i. Blood serum of the patient from which the cartilage material originated. Since the autologous blood serum can contain growth-inhibiting factors, proliferation in a medium containing such serum gives information on the expected ability to proliferate in the body, but not necessarily information on the quality of the cartilage material as such. If the parallel experiment results in significantly poorer proliferation, it is advisable to examine the blood for growth-inhibiting factors and to specifically inhibit them.

In the quality check according to the invention, the cells are, for example, greatly diluted, placed in a Petri dish and grown on agar agar / Dulbecco’s Modified Eagle’s medium at 37 ° C.

The number of cell divisions of the individual cells or the most proliferative x% of the cells (for example the best 5% of the cells) after a predetermined time are determined and the mean value is calculated.

For a period of 4 weeks, up to three cell divisions have so far been found to be bad, about 5 divisions as good and 10 divisions as very good in non-enriched minimal medium. If the ability to proliferate is good or even very good, a cartilage implant can be expected to have a good service life; if it is divided into 3 or less, success is fundamentally in doubt. The above-mentioned ability to divide was determined in a process in which the culture medium remained unchanged during the process duration of 4 weeks, i.e. was neither supplemented nor renewed. If the culture medium is supplemented or renewed, these values or the period may change somewhat.

The process result, in particular the cells that proliferate best under the process conditions, can be selected and applied to a three-dimensional framework made of resorbable material and grown thereon. A minimal medium such as Dulbecco’s Modified Eagle’s Medium is used as the breeding medium. Standard values can be selected for the temperature and the relative humidity (r.h.). The temperature is usually around 37 ° C.

For cartilage cultivation, autologous blood serum can also be added, for example if this has led to an increased ability to proliferate in a parallel experiment or if specifically inhibitors against substances impairing cell growth, such as TNF-alpha, are added.

The culture medium is preferably changed every few days. The replacement takes place, for example, every 2 to 8 days, usually every 3 to 6 days, in particular every 4 to 5 days. The usual proliferation time to the finished transplant is 3 to 8 weeks, usually at least 4 weeks, in particular 4 to 6 weeks.

Since the ability of cells to proliferate is genetically determined, in the case of cartilage cells with a very deep ability to proliferate, e.g. through partial return towards the embryonic state before the production of the cartilage transplant an increase in the ability to proliferate can be achieved. Such a return can be achieved, for example, by bombarding with antisense RNA, which is directed against (i) three special loci on chromosomes 1 and 6 and / or (ii) against telomeres. From such “rejuvenated” cells, analogously to the method described above, the cells that proliferate best are selected for the production of the transplant and used as described above.

As mentioned briefly above, it has also been found that, in addition to determining the ability to proliferate, determining the content of surface cytokines, in particular TNF-alpha, is an important indicator of the condition of the cartilage and thus also of the success of the transplant is.

Various factors can lead to an increase in the TNF-alpha content, for example a genetic predisposition, which can also manifest itself in the form of certain diseases, such as psoriasis, or a tick bite often many years ago. A corresponding predisposition can be determined by detecting a special polymorphism on chromosome 6, which is found in particular in connection with increased TNF-alpha content and psoriasis, and / or by detecting tick genes in the patient's body, e.g. by in vitro examination of a blood sample, e.g. by means of PCR using suitable primers that are as selective as possible.

Information on this can be found e.g. in Kerstin Bade, Identification of Psoriasis-Associated Polymorphisms in the MHC (2004), dissertation to obtain a doctorate from the Faculty of Mathematics and Natural Sciences at the Christian-Albrechts-Universität zu Kiel and the literature cited therein.

TNF-alpha (tumor necrosis factor) is a multifunctional proinflammatory cytokine that is involved in the rapid defense against infections. In diseased skin of people suffering from psoriasis vulgaris, it was found that TNF-alpha is overexpressed and that this promoter polymorphism on chromosome 6, position -238, is associated with type I psoriasis and psoriatic arthritis. (Bade, K. [2004], in particular page 112 and the literature cited therein).

With a high or very high content of TNF-alpha, the probability of a rheumatological disease such as osteoarthritis / arthritis is high and the probability of a good long-term success of a cartilage transplant is low. The likelihood of long-term success can be increased if TNF-alpha is blocked, e.g. by TNF-alpha inhibitors such as antibodies or antisense RNA. Such TNF-alpha inhibitors can be administered prophylactically and / or before the transplant and / or with the transplant and / or subsequent to the transplant. A good type of administration is by means of a depot in the transplant itself. Such a depot can be in the form of active substance-containing microglobules with rapid and / or delayed release into the transplant, e.g. into the resorbable matrix, so that the release is controlled by dissolution, diffusion and resorption processes. Alternatively, both antibodies and antisense RNA can be injected into the damaged joint or in its immediate vicinity. As a further variant, cells that are to be used for cartilage formation can be genetically modified in vitro so that at least some of them are capable of producing an antibody or antisense RNA against TNF-alpha themselves.

Description of embodiments of the invention

Both for assessing the quality of cartilage mass and for producing the transplant, a sample of cartilage mass is dissolved in vitro in order to obtain isolated cartilage cells. This is done according to known methods, e.g. using hyaluronidase.

The isolated cartilage cells obtained in the above step are so isolated that there is enough space around each cell so that the cell clusters remain clearly distinguishable even after about ten proliferation. The cells are then proliferated under proliferation conditions for a certain period of time. The cell populations formed are then assessed and, in the case of the diagnosis, compared with comparison data obtained under identical conditions and selected for the cultivation of a transplant.

The proliferation of the individual cells for the quality assessment is preferably carried out in two-dimensional cell cultures so that the populations formed by the individual cells can be easily identified or counted. Usual two-dimensional cell cultures are grown in Petri dishes on agar-agar. What is special about this diagnostic method is that the culture medium required for proliferation is preferably a minimal medium that preferably only has the components necessary for proliferation, such as the most essential trace elements, vitamins, amino acids and energy donors, for example a medium that - inorganic salts as a source for Ca, Fe, Mg, SO4 <2> <–>, PO4 <3> <–> as well as NaCl and KCl; - Amino acids; - vitamins; - D-glucose, and sodium pyruvate and optionally phenol red contains or consists of these substances and water.

A well-suited medium is, for example, a medium which is suitable for the cultivation of lymphocytes. One such medium is, for example, Dulbecco’s Modified Eagle’s Medium, which, in addition to water, contains the substances listed in Table 1 and in particular only contains these substances.

In a particularly preferred embodiment, the minimal medium should be free from hormones and / or proteins / peptides. In particular, it should not contain any growth factors or, if so, in precisely reproducible amounts, purity and activity.

Table 1:

[0030]

* The magnesium concentration can be adapted to cartilage cells, while the other amounts usually remain within the specified range.

The temperature is kept around 37 ° C for both the proliferation and the transplant cultivation, in particular at 37 ± 0.5 ° C.

If a hormone- and protein- / peptide-free medium is used for proliferation or transplant cultivation, a period of at least 3 weeks, preferably approx. 4 weeks, has proven to be favorable for the cultivation to assess the ability to proliferate a period of 3 to 8 weeks, especially from 4 to 6 weeks.

For the determination of the ability to proliferate, all cell populations of a sample or only some of the cell populations can be used. It is essential that the cells that proliferate best are taken into account and that the reference samples are examined identically. For example, the best 5% can be evaluated, recognizable from the largest cell populations or the best 10%.

In the transplant cultivation, preferably only the cells with the best proliferation capacity are selected and applied to a three-dimensional, resorbable framework. This selection can result in a larger or a smaller number of cells depending on the proliferation capacity and quantity. Should e.g. If a large transplant is produced with massively proliferating cells, more cells are preferably applied to the resorbable framework than if the ability to divide is very high. If the proliferation is poor, the cells can also be treated with antisense RNA to increase the cells' ability to proliferate. In this case, antisense RNA is added to the cells in vitro, for example they are bombarded with antisense RNA. This antisense RNA can be directed against telomeres, for example.

[0035] This is followed by a selection of the cells that proliferate best.

The treatment with antisense RNA can be carried out on an unselected cartilage cell sample and selection can then take place, or the cartilage cell sample can be (pre) selected with regard to the optimally proliferating cells and only the best proliferating cells are subjected to treatment with antisense RNA , optionally followed by a second selection after treatment.

The selected cells are then applied to an absorbable framework and grown on this absorbable framework until the transplant has the desired size and cell density. Depending on the conditions, this is usually the case after 3 to 8, in particular 4 to 6 weeks in minimal medium. If hormones and / or proteins and / or peptides, in particular growth factors, are added, then cartilage growth can be accelerated, but with the risk that damaged tissue will develop that tends to form tumors.

Suitable resorbable framework materials are, for example, felt-like fiber fabrics the size of the defect to be treated, for example framework materials made of polylactones (PLA), or frameworks made up of alginate beads.

Another important factor that can be used for the success of the transplant and also for assessing the osteoarthritis / arthritis risk is the determination of the cytokine content on the cartilage tissue, in particular the TNF-alpha content. This level can indicate a predisposition, for example due to a disease such as psoriasis or a tick bite that may have occurred many years ago.

Surprisingly, it was found that in up to 70% of patients with cartilage defects, the TNF-alpha values are too high and that in this group of patients the proportion of people suffering from psoriasis is above average. Various polymorphisms can be assigned to these patients, e.g. one on chromosome 6.

In the course of determining the cause, it was also found, completely surprising, that tick genes can be detected in patients with cartilage defects and an increased concentration of TNF-alpha than in a reference group of people of similar age and gender structure.

In principle, all tick genes can be used, but preferred gene sequences are those with a high selectivity, since this can reduce the background and lower the detection limit.

The usual detection limit by means of PCR is around 10 <-10>, but recent research suggests that much lower detection limits can be achieved when using specially selective primers.

Claims (17)

1. In vitro method for assessing the quality of cartilage, wherein in vitro (a) dissolving a sample of cartilage to recover isolated cartilage cells (b) isolating the isolated cartilage cells obtained in step (a) and then proliferating them for a certain period of time such that the proliferation of the individual cells becomes recognizable by the formation of cell populations, (C) the proliferative capacity of the cells is determined by the size of the cell populations. 2. The method according to claim 1, characterized in that the dissolution of the cartilage mass includes the addition of hyaluronidase. 3. The method according to any one of claims 1 or 2, characterized in that the proliferation takes place in two-dimensional cell cultures, for example on agar-agar plates, with a minimal medium. 4. The method according to claim 3, characterized in that the minimal medium - inorganic salts as a source of Ca, Fe, Mg, SO4 <2> <->, PO4 <3> <-> as well as NaCl and KCl; - Amino acids; - vitamins; - D-glucose, and sodium pyruvate and optionally phenol red contains. 5. The method according to any one of claims 3 or 4, characterized in that the minimal medium is free of hormones and / or proteins / peptides, so that the cartilage quality can be determined under natural conditions as possible. 6. The method according to any one of claims 1 to 5, characterized in that the period in step (b) is about 4 weeks. 7. The method according to any one of claims 1 to 6, characterized in that for determining the proliferation capacity in step (c) only a certain proportion of cell populations is used, in particular a certain percentage of the best proliferating cells or the largest cell populations. 8. The method according to any one of claims 1 to 7, characterized in that the Proliferationsfähigkeit in parallel (i) with pure minimal medium and (ii) with minimal medium enriched with autologous blood serum, and then comparing the (i) proliferative capacity determined in pure minimal medium with the proliferation determined in (ii) minimal medium supplemented with autologous blood serum. 9. A method for producing a cartilage graft, characterized in that (i) dissolving a sample of cartilage in vitro to recover isolated cartilage cells, (ii) isolating the isolated cartilage cells obtained in step (i) and then proliferating them for a certain period of time in such a way that the proliferation of the individual cells becomes recognizable by the formation of cell populations, (iii) the cells with the best proliferative capacity are selected and applied to a three-dimensional resorbable scaffold, (iv) the cells are grown on the resorbable scaffold until the graft has the desired size and cell density. 10. The method according to claim 9, characterized in that the dissolution of the cartilage mass includes the addition of hyaluronidase. 11. The method according to any one of claims 9 or 10, characterized in that the proliferation in step (ii) takes place in two-dimensional cell cultures, for example on agar-agar plates, with a minimal medium. 12. The method according to any one of claims 9 to 11, characterized in that the cultivation is carried out on the resorbable scaffold with a minimal medium. 13. The method according to any one of claims 11 or 12, characterized in that the minimal medium - inorganic salts as a source of Ca, Fe, Mg, SO4 <2> <->, PO4 <3> <-> as well as NaCl and KCl; - Amino acids; - vitamins; - D-glucose, and sodium pyruvate and optionally phenol red contains. 14. The method according to any one of claims 11 to 13, characterized in that the minimal medium is free of hormones and / or proteins / peptides, so that the cartilage quality can be determined under natural conditions as possible. 15. The method according to any one of claims 9 to 14, characterized in that the period in step (ii) is about 4 weeks and / or in step (iv) 4 to 6 weeks. 16. The method according to any one of claims 9 to 15, characterized in that the cells in step (ii) and / or step (iii) by means of antisense RNA which is directed against telomeres, are converted into a better proliferating state. 17. The method according to any one of claims 9 to 16, characterized in that the cells are grown in a culture medium containing an inhibitor of TNF-alpha.