WO1995021632A1

WO1995021632A1 - Process for inactivating thermolabile viruses in biological material while preserving collagen properties

Info

Publication number: WO1995021632A1
Application number: PCT/EP1995/000424
Authority: WO
Inventors: Ludwig Baumgartner
Original assignee: Ludwig Baumgartner
Priority date: 1994-02-14
Filing date: 1995-02-07
Publication date: 1995-08-17
Also published as: DE4404625C1; DE4404625C2

Abstract

A process is disclosed for thermally inactivating thermolabile viruses in structured collagen or collagen-containing tissues by means of non-aqueous liquids, vapours or gases at temperatures above the inactivation temperature of the virus while preserving transplant properties.

Description

Process for the inactivation of thermolabile viruses in biological material while maintaining the collagen properties.

The invention relates to a method according to claims 1 to 6 and the use of thermally treated fabrics according to claim 7.

In the manufacture of canned transplants of human origin for use in medicine, e.g. HIV issues are particularly important.

Serological tests for HIV antibodies do not give absolute certainty that the donor is HIV-free; therefore additional process steps are required in the production of canned grafts for virus inactivation.

A chemical treatment using hydrogen peroxide is known for this. However, this method is preferably only suitable for thin, flat material, but is not very suitable for compact materials (bones, tendons) due to insufficient penetration of the agent.

The disadvantage of insufficient penetration and thus insufficient inactivation of viruses in bone materials should therefore be able to be eliminated by thermal methods, since it is known that HIV deactivation takes place at a temperature of 56 ° C. A method for the thermal inactivation of HIV in human bones in an aqueous medium at a temperature of 80 ° C. and a treatment time of about 10 minutes is known (accident surgery 18/92, 1-6 No. 1). However, this method “has the disadvantage that the collagen matrix denatures at the temperature used, so that the osteoinductive properties which are essential for a bone substitute material are lost or at least greatly reduced. HIV can also be inactivated when treated at 80 C, but not the hepatitis viruses B and C that are thermolabile only at 105 C

A method is also known from EP 0141004 B1, in which bone substitute material is produced by thermal sintering of native bones. Here, all organic material is burned, including ev. existing viruses, but also the collagen matrix. A bone replacement material produced in this way no longer has any osteoinductive properties and is therefore unsuitable for transplantation purposes.

It is also known an Inaktivierungsver method which treats bone material with steam. This procedure works, for example, at 121 C for 10 minutes and inactivates both HIV and hepatitis viruses, but at the same time also damages the collagen matrix and the mineral bone structure itself. A bone graft autoclaved in this way therefore has no osteoinductive properties and is therefore completely unsuitable as a bone replacement material.

From US 4,490,361 a virus inactivation by heat treatment by means of organic liquids is known, but this relates exclusively to blood plasma fractions, the pulverized lyophilisate being heat-treated in the form of a suspension.

EP 0 212 040 A1 describes a method for heat inactivation of viruses, but only for factor VIII preparation, the heat treatment being carried out on a lyophilisate in the dry state in the presence of a protective gas or under vacuum.

The aim of the invention is therefore a process which avoids the disadvantages of the chemical or thermal processes listed.

Surprisingly, it was found according to the invention that the gentle production of a biological replacement material for transplantation purposes is possible, whereby both inactivating thermolabile viruses (such as HIV and hepatitis) takes place and the required properties of the collagen or mineral-collagen-containing tissues are largely retained. Thus, the method according to the invention provides collagen or collagen-containing tissues in which thermolabile viruses are thermo-inactivated.

According to this process, the biological material (e.g. dura mater, faszia lata, faszia temporalis, tendons, gums, amnion, skin, cartilage, bones and other collagenous tissues) is subjected to a thermal treatment above the inactivation temperature of viruses, the heat transfer being carried out by means of anhydrous or almost water-free (up to about 10% water content) liquids, vapors or gases. The heat transfer process can take place at normal pressure or at reduced pressure or at increased pressure or alternately. To achieve the desired heat transfer, mixtures of suitable liquids, e.g. also azeotropic mixtures can be used.

Suitable liquids are:

Methanol, ethanol, propanol, isopropanol, isobutanol, acetone, methyl ethyl ketone, glycol, glycerin, propanediol, heptane, isoheptane. Dimethyl sulfoxide, polyethylene glycol silicone oil, 2,3-butanediol.

G neei ■ gne rt.e gases: »C ^o fl ^u 2n» Edelg ^s ase.

The following experiment demonstrates the effectiveness of the method using the example of the shrinkage behavior of a collagen

Fabric during thermal treatment in an anhydrous

Liquid (e.g. .IPA) compared to the same treatment in an aqueous liquid (e.g. water).

Dura mater strips 4 mm wide and 60 mm long are treated in an IPA bath for 20 minutes at 80 ° C:

There is no shrinkage of the tissue.

The same strip treated under the same conditions in a water bath is shortened within vo

Seconds to about 50% of its original length.

The structures of the collagen molecule and the fibrils are obviously irreversibly damaged by the thermal treatment in an aqueous medium, whereas there is no impairment in a non-aqueous medium.

The intactness of the collagen tissue is, however, a prerequisite for the suitability as a surgical transplant material. It is obvious that e.g. a Dura tissue, which has been irreversibly damaged due to shrinkage due to thermal treatment in an aqueous medium, no longer has any native properties.

The intactness of a collagen fiber can be determined by measuring the so-called shrinkage temperature (TS). The TS is defined as the temperature at which the collagen fiber exhibits a maximum of one shrinking interval per temperature interval when heated in an aqueous medium. For example, a strip of a native dura mater with a width of 4 mm and a length of 60 mm shows successive heating in aqueous medium a TS of 69 C (diagram 1). However, this makes it clear that thermal inactivation of the dura or tendons or bones at 69 ° C or above in an aqueous medium is of course prohibited.

With successive heating of native dura to 80 C in an anhydrous medium, e.g. in IPA, however, there is no shrinkage (diagram 1).

Surprisingly, it has also been shown that even temperatures of e.g. Let 105 C end without shrinkage (diagram 1).

Diagram 1:

Shrinkage distance Shrinkage behavior (scale parts) of native dura mater with increasing temperature in

Different properties of fabrics thermally treated in aqueous or non-aqueous medium are also evident in the characteristics of strength and elongation. Native 4 mm wide dura strips of comparable thickness have the following relative tensile strength values:

not thermally treated 100%

20 minutes at 80 ° C in an IPA bath treated 99%

20 minutes at 105 ° C in a glycerin bath treated 96%

20 minutes in a water bath at 80 ° C treated 49%

In the same way, the elongation behavior deteriorates.

The elongation at break of the native, thermally untreated and thermally treated Dura strips in a 80 C warm IPA bath is about 40%, whereas it is 150% for the thermally treated Dura strips for 20 minutes at 80 C in water. Properties of graft tissues which have deteriorated in strength and extensibility in this way are, however, not acceptable for surgical purposes, or only to a very limited extent.

The same applies to bones thermally treated in an aqueous medium:

The pressure load after ten minutes of autoclaving e.g. at 121 C in tensioned water, to values of about 10 - 20% of the initial values.

A thermal treatment in IPA (80 C) or glycerol (105 C) for 20 minutes only results in a reduction of the values to approx. 97%.

The process according to the invention is shown in the examples below. 1. Dura mater is cleaned in the usual process steps and finally freeze-dried for the purpose of preservation. The water content in the dura is now about 5%. For the thermal activation, a 4 x 5 cm preserved piece of Durasta is placed in an IPA bath from

Given 75 C and left in for 20 min.

Existing HIV is inactivated in the dura treated in this way and the mechanical properties of the dura show no deterioration compared to a non-thermally treated dura (tensile strength: minus 1%).

2. A native cancellous bone bone measuring 20x20x20 mm is cleaned in conventional process steps and finally preserved by freeze-drying.

The water content of the bone is now about 5%. For thermal inactivation, the preserved piece of bone is treated in a pressure vessel with IPA for 30 minutes at 3 bar (approx. 110 ° C).

HIV in the bone treated in this way is inactivated and the mechanical properties of the bone show practically no loss of strength compared to a bone that has not been thermally treated.

3. A native hip bone is treated for the purpose of thermo-inactivation in a glycerol bath at 110 ° C. for 60 minutes.

In the bone treated in this way, existing HIV or hepatitis viruses are inactivated and the mechanical properties of the bone show no significant loss of strength when subjected to pressure compared to a bone that has not been thermally treated. The glycerine-treated hip bone can then be stored by freezing, for example at -40 ° C.

The method according to the invention can be applied to biological tissue in all stages of manufacture, e.g. on femoral heads in their native state or on bone material (in the form of cubes or chips), e.g. during the manufacturing process or before, during or after preservation. The same applies to other biological material from bone or collagen tissue,

Due to different material types and dimensions, commercially usable biological replacement material for surgery can be obtained in the context of the method according to the invention by modifications in duration, temperature and pressure.

Claims

claims

1. A method for thermally inactivating thermolabile viruses in collagenous or collagen-containing tissues, characterized in that the tissues in non-aqueous liquids, vapors or gases are subjected to a treatment at elevated temperature which is above the respective inactivation temperature of the viruses, while maintaining their graft properties .

2. The method according to claim 1, that the non-aqueous liquids, vapors or gases can contain a water content of up to 10%.

3. The method according to claim 1 and 2, that non-aqueous liquids or their mixtures, vapors or gases are used.

4. Process according to 1 to 3, that the process temperature is between 50 C and 130 C.

5. The method according to 1 to 4, that the temperature treatment can be carried out under negative, normal or positive pressure or alternately.

6. collagens or collagen-containing tissues in which thermolabile viruses are thermally inactivated, characterized in that they are obtainable by the process according to one or more of claims 1-5.

7. Use of the heat-treated tissue according to claim 1 to 5 and the tissue according to claim 6 as grafts.