GB1565340A

GB1565340A - Fibrous tussue preparations

Info

Publication number: GB1565340A
Application number: GB1636078A
Authority: GB
Original assignee: Oliver R F
Current assignee: Oliver R F
Priority date: 1978-04-25
Filing date: 1978-04-25
Publication date: 1980-04-16

Description

(54) FIBROUS TISSUE PREPARATIONS (71) We, ROY FREDERICK OLI VER, of 33 Kilmany Road, Wormit, Fife, Scotland and ROY ARTHUR GRANT, of 41a Springfield Road, Poole, Dorset, both British subjects, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be perfonned, to be particularly described in and by the following statement: The present invention relates to a fibrous tissue preparation which is suitable for heterotransplantation as a temporary dressing or as a permanent repair for cutaneous wounds and soft tissue injuries, and for application in powdered form as a wound cleansing agent, and to a process for providing such a preparation.

In the past the clinical use of tissue preparations such as dermis for example has been proposed for the repair of soft tissue injuries such as torn tendons, hernias and retroversion of the uterus. In dogs plaited strips of dermis have been used to replace achilles tendons and it was claimed that the tissue assumed the function and to a major degree the histologic characteristics of tendon in about 10 weeks.

Three successful cases of hand tendon reconstruction with dermis have been reported and many other possible clinical applications discussed.

The use of buried dermis grafts to correct defective facial contours has also been reported by Eimer (1920) who obtained good corrections in two cases of sunken cheeks and found the results more permanent than using fat or fascia.

In 1929 Loewe treated 100 cases with dermis implants which included the repair of hernias, dura mater, arthro-plasties of the knee, hip and elbow and recurrent dislocation of the shoulder. Also, Straatsma reported the use of this material for correcting saddle nose deformity and the use of dermis was extended to the treatment of a number of other conditions such as reinforcement for the ligaments of the knee and dislocation of the patella and temporo-mandibular joint.

However, in spite of the foregoing claims the use of buried dermis grafts appeared to gain little in popularity because of the possibility of the formation of epithelial cysts originating from the sweat glands, hair follicles and sebaceous glands present in the transplanted dermis. As well as cyst formation implantation of whole dermis often resulted in foreign body reactions and the appearance of giant cells in the implanted tissue.

In all the cases mentioned so far the implanted dermis was taken from another site in the same individual; in other words autogenous tissue was used.

Homotranspiantation from one individual to another or heterotransplants between individuals of different species was not attempted since whole tissue transplants except in the case of identical twins invariably result in immunological rejection reactions and destruction of the transplanted tissue.

We have now found that the dermal fibrous tissue which is the essential component of dermis for space filling and providing mechanical strength can be purified so that all of cellular elements such as sweat glands, sebaceous glands and vascular tissue are removed thus eliminating cyst formation and foreign body reactions when subsequently implanted into animals. The purification procedure does not signficantly affect the mechanical strength of the fibrous tissue and hence the utility of the purified fibrous tissue for the repair of injuries is not impaired.

We have found unexpectedly that the resulting fibrous protein or fibroelastic tissue preparations have low or absent antigenicity and when homografted between individuals of the same species or heterografted between individuals of a different species do not elicit immunological rejection reactions or foreign body reactions. This is an important finding in practical terms since it means that fibrous tissue preparations taken from animals may be used to repair cutaneous or soft tissue injuries in humans.

According to - one aspect of the present invention there is provided a fibrous tissue preparation of human or animal origin which is suitable for heterotransplantation as a temporary dressing for cutaneous wounds and soft tissue injuries, which preparation is substantially free of antigenic non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins.

According to a further aspect of the present invention there is provided a substantially non-antigenic fibrous tissue preparation of human or animal origin which is suitable for heterotransplantation as a permanent repair for cutaneous wounds and soft tissue injuries, which preparation is substantially free of nonfibrous tissue proteins, substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins, and substantially free of cellular elements. This fibrous tissue preparation is capable of being infiltrated and colonised by the host cells of another individual of the same or different species, revascularized and in some cases reepiderinalized to form a permanent repair for cutaneous wounds and soft tissue injuries.

There is also provided a novel process comprising a number of steps for treatment of suitable human or animal tissues such as dermis, ligament, tendon, areolar tissue, basement membrane or dura mater to render them suitable for homo or heterofiansplant- ation.

According to a further aspect of the present invention there is provided a process for treating fibrous tissue to provide a fibrous tissue preparation which is suitable for heterotransplantation, which process comprises treating the fibrous tissue with two enzymes, one of said enzymes being a proteolytic enzyme which will under the conditions of the process remove non-fibrous tissue proteins, and the other of said enzymes being a carbohydratesplitting enzyme which will under conditions of the process remove antigenic polysaccharides, mucopolysaccharides and glycoproteins from the tissue.

The order in which the enzymes are used in the process is not critical. However, if the enzyme treatment is carried out in two steps and the first of these steps is the treatment of the tissue with the proteolytic enzyme, then it is necessary, because the carbohydrate-splitting enzyme is a protein, to remove the proteolytic enzyme, usually by washing, from the tissue before proceeding to the carbohydrate removing step. Alternatively, the treatment may be carried out in the presence of both the enzymes. If it is, then the process conditions must be so chosen that when one of the enzymes is active the other is inactive. As is well known in the art this is normally achieved by regulating the pH.

Whilst any proteolytic enzyme which under the conditions of the process will remove nonfibrous tissue proteins can be used, the preferred proteolytic enzyme is pure trypsin. We have found that above 20"C the treatment results in an alteration of the fibre structure leading to a lower physical strength. Moreover, low temperatures discourage the growth of microorganisms in the preparation. It is therefore preferred to carry out the treatment at a temperature below 20"C. Moreover, trypsin is more stable below 200C and lower amounts of it are required. Generally for the removal of antigenic protein material it is necessary to treat the dermis or other fibrous tissue for a period of 48 hours or longer, preferably in the presence of an antiseptic and a buffer solution to maintain the correct pH.

In the case of transplants between individuals of the same species fibrous tissue prepared from certain structures such as arterial walls may contain large amounts of mucopolysaccharides such as hyaluronic acid and also polysaccharides. While within a species such carbohydrate material may not be significantly antigenic, nevertheless is does not contribute to the strength of the tissue and may severely obstruct the subsequent recolonization of the graft by host cells such as fibroblasts and interfere with the formation of new capillaries within the graft. Also in the case of zenografts or transplants between individuals of different species residual carbohydrate may give rise to foreign body reactions and infiltration of the graft by lymphocytes. In order to eliminate the possibility of these adverse effects the tissue may be treated with carbohydrate-splitting enzymes.Whilst any carbohydrate-splitting enzyme which under the conditions of the process will remove antigenic polysaccharides, mucopolysaccharides and glycoproteins from the tissue can be used, the preferred carbohydrate-splitting enzymes are amylase, hyaluronidase, and neuraminidase.

We have also found that any residual antigenicity in the tissue can be removed therefrom by reacting it with a cross-linking agent which will under the conditions of the process form a link between the terminal amino groups of lysine groups in the tissue. The preferred crosslinking agents are glutaraldehyde, formaldehyde and nitrous acid.

This further treatment acts specifically to block the cellular immune response in heterografted fibrous tissues i.e. grafts between individuals of different species. This blocking action is well demonstrated by the finding that purified human fibrous tissue implanted in rats elicited an immune response and that this response could be abolished by treatment of the human fibrous tissue with a dilute solution of glutaraldehyde before implantation. This effect is of the greatest practical importance since readily obtainable sources of suitable fibrous tissues are pig skin or bovine skin which if rendered immunologically compatible with the human body would be of the utmost utility in the treatment of large burns when homografting with human skin is at best a temporary expedient and does not lead to a long term repair of the injury.Optionally, prior to the first enzyme treatment the tissue is soaked in cold 0.5-1.9 M sodium chloride solution to remove soluble proteins therefrom and to reduce the amount of proteolytic enzyme required.

The fibrous tissue preparation can be used as a temporary dressing. For example, it could be used as a temporary dressing for a burn for which sufficient donor skin was available for autografting but where the operation had to be postponed for various reasons. In these cit=;tances the material would have the advantage over conventional dressings of inhibiting the onset of wound contraction and granulation tissue formation and over homograft skin in preventing lymphocytic infiltration into the wound.

It was noted in animal experiments that implanted purified dermal fibrous tissue was gradually dissolved at the site of implantation.

This is a major disadvantage when the object of the operation is to obtain a permanent repair rather than a temporary dressing. This disintegration of the implanted material may be prevented by treatment with a stabilizer prior to implantation to stabilize the tissue against the endogenous proteolytic enzymes.

The preferred stabilizers are acrolein, diepoxides, dialdehyde starch, bifunctional sulphones, cyanuric chloride, glutaraldehyde and formaldehyde.

Stabilized purified fibrous tissue implants have been examined at various times up to 280 days after implantation with no obvious changes in structures. However it is important that the strength of any glutaraldehyde solution used to stabilize the tissue should not exceed certain limits. In general the strength of the glutaraldehyde solution should not exceed 0.5%. Treatment with solutions stronger than this has been found to result in hardening and loss of flexibility of the preparation associated with a persistent inflammatory reaction.

In order to make the process economic it is desirable to recover and recycle the enzymes, particularly trypsin when used, since these are very expensive. Recovery is as follows. After treatment of the tissue the solution containing the enzyme is clarified by filtration and passed through a column of a suitable cellulosic ion exchange resin which removes the enzyme by adsorption. The column is then washed with water and the enzyme subsequently displaced with strong (IM) buffer solution at pH 9 in a relatively small volume. The concentrated recovered enzyme solution is then diluted and used for treating a further batch of tissue. Alternatively the filtered enzyme solution may be concentrated by ultra filtration before reuse.

In order to verify the lack of antigenicity in the fibrous tissue this was implanted subcutaneously in rats and biopsied at various times after implantation. The implants which were all recovered intact became recellularized and revascularized with no overt signs of lymphocytic infiltration. The non-antigenicity was further confirmed by sensitizing rats to whole dermis plus epidermis by making implants two and four weeks before implantation of purified fibrous tissue from the same donor.

The implants which were recovered at intervals up to forty weeks after operation showed no differences to those implanted in non sensitized animals with no evidence of lymphocytic infiltration.

The purified fibrous tissue appears to be quite stable and can be stored either deep frozen or freeze dried and can be sterilized with chemicals or gamma ravs.

When implanted the fibrous tissue becomes rapidly incorporated in the tissues and becomes vascularized and recolonized by host cells such as fibroblasts. In cutaneous wounds the implanted purified fibrous tissue becomes overgrown with epidermis provided it is covered by a suitable dressing and eventually assumes the appearance of normal skin. We have also found unexpectedly that implanted purified fibrous tissue will inhibit the contraction of full thickness skin wounds which otherwise causes areas of distortion around the wound. The implantation of purified fibrous tissue into deep skin wounds resulted in a large reduction in the amount of granulation tissue formed in the wounds.Since granulation tissue is a precursor of scar tissue one very important aspect of the use of this material is the prevention of scar formation and consequent disfigurement This is particularly important in the case of cutaneous injuries to the head and hands.

In order that the invention may be more fully understood, the following Example is given by way of illustration only.

EXAMPLE.

Human dermis was treated with a solution of crystalline trypsin (Koch-Light) at a concentration of 2 mg/ml in 0.1M phosphate buffer at pH 9.0 with 0.5 mg/ml sodium azide as a bactericide at 1SOC for 28 days.

The partially purified fibrous tissue was thoroughly washed in saline to remove trypsin and placed in a solution of bacterial < r-amylase for one hour at 1S"C followed by thorough rinsing in saline. The material was then placed in a solution of 0.01% formaldehyde in Tris buffer pH 7.2 for 16 hours at 150C followed by a further washing with saline. The resulting preparation was implanted subcutaneously in hooded Porton rats (strain PVG/C). The implanted preparation became infiltrated with host fibroblasts and revascularized. There was no evidence of lymphocytic infiltration foreign body reactions or giant cell formation in the implanted material.

The preparations of the present invention will have practical applications in various branches of surgery and veterinary medicine for the treatment of hernias, skin wounds, tendon injuries, correction of facial contours, joint defects and generally in plastic and reconstructive surgery and for the repair of soft tissues.

In powdered, lyophilised form the tissue preparation can be used as a wound cleansing agent in all forms of cutaneous injury. It readily absorbs wound exudate, bacteria and wound debris which is then washed from the wound.

According to a further aspect of the present invention, therefore, there is provided a powdered, lyophilised tissue preparation of human or animal origin which is suitable as a cleansing agent for cutaneous wounds and soft tissue injuries, which preparation is substantially free of non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins.

WHAT WE CLAIM IS: 1. A fibrous tissue preparation of human or animal origin which is suitable for heterotransplantation as a temporary dressing for cutaneous wounds and soft tissue injuries, which preparation is substantially free of antigenic non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins.

2. A substantially non-antigenic fibrous tissue preparation of human or animal origin which is suitable for heterotransplantation as a permanent repair for cutaneous wounds and soft tissue injuries, which preparation is substantially free of non-fibrous tissue proteins, substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins, and substantially free of cellular elements.

.A process for treating fibrous tissue of human or animal origin to provide a fibrous tissue preparation which is suitable for heterotransplantation, which process comprisestreating the fibrous tissue with two enzymes, one of said enzymes being a proteolytic enzyme which will under the conditions of the process remove non-fibrous tissue proteins, and the other of said enzymes being a carbohydratesplitting enzyme which will under the conditions of the process remove antigenic polysaccharides, mucopolysaccharides and glycoproteins from the tissue.

4. A process as claimed in claim 3 in which the fibrous tissue is treated with said proteo lytic enzyme, and, before the fibrous tissue is treated with said carbohydrate-splitting enzyme, all or substantially all of said proteolyric enzyme is removed therefrom.

5. A process as claimed in claim 3 in which the treatment is carried out in the presence of both the enzymes, the process conditions being so chosen that when one of said enzymes is active the other is inactive.

6. A process as claimed in any one of claims 3 to 5 in which the proteolytic enzyme is pure trypsin.

7. A process as claimed in any one of claims 3 to 6 in which the proteolytic enzyme treatment is carried out at a temperature below 20"C.

8. A process as claimed in any one of claims 3 to 7 in which the carbohydrate- splitting enzyme is amylase, hyaluronidase or neuraminidase.

9. A process as claimed in any one of claims 3 to 8 in which, prior to the first enzyme treatment, the fibrous tissue is contacted with cold aqueous sodium chloride solution to remove soluble proteins therefrom.

10. A fibrous tissue preparation obtained by a process as claimed in any one of claims 3 to 9.

11. A preparation as claimed in claim 10 which has been sterilized and is ready for use as a temporary dressing for a cutaneous wound or soft tissue injury.

12. A process as claimed in any one of claims 3 to 9 which further comprises the step of treating the fibrous tissue with a reagent which under the conditions of the process remove any residual antigenicity from the tissue.

13. A process as claimed in claim 12 in which the reagent is a crosslinking agent which forms a link between the terminal amino groups of lysine groups in the tissue.

14. A process as claimed in claim 13 in which the crosslinking agent is glutaraldhyde, formaldehyde or nitrous acid.

15. A fibrous tissue preparation obtained by a process as claimed in any one of claims 12 to 14.

16. A preparation as claimed in claim 15 which has been sterilised and is ready for use as a temporary dressing for a cutaneous wound or soft tissue injury.

17. A process as claimed in any one of claims 12 to 14 which further comprises the step of treating the fibrous tissue with a stabilizer to stabilize the tissue against endogenous proteolytic enzymes.

18. A process as claimed in claim 17 in which the stabilizer is acrolein, a diepoxide, dialdehyde starch, a bifunctional sulphone, cyanuric chloride, glutaraldehyde or formaldehyde.

19. A fibrous tissue preparation obtained by a process as claimed in claim 17 or claim 18.

20. A preparation as claimed in claim 19 which has been sterilized and is ready for use as a permanent repair for a cutaneous wound or soft tissue injury.

21. A process for treating fibrous tissue of human or animal origin to provide a fibrous tissue preparation which is suitable for heterotransplantation substantially as hereinbefore described in the Example.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. will have practical applications in various branches of surgery and veterinary medicine for the treatment of hernias, skin wounds, tendon injuries, correction of facial contours, joint defects and generally in plastic and reconstructive surgery and for the repair of soft tissues. In powdered, lyophilised form the tissue preparation can be used as a wound cleansing agent in all forms of cutaneous injury. It readily absorbs wound exudate, bacteria and wound debris which is then washed from the wound. According to a further aspect of the present invention, therefore, there is provided a powdered, lyophilised tissue preparation of human or animal origin which is suitable as a cleansing agent for cutaneous wounds and soft tissue injuries, which preparation is substantially free of non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins. WHAT WE CLAIM IS:

1. A fibrous tissue preparation of human or animal origin which is suitable for heterotransplantation as a temporary dressing for cutaneous wounds and soft tissue injuries, which preparation is substantially free of antigenic non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins.

22. A fibrous tissue preparation obtained by

a process as claimed in claim 21.

23. A powdered, lyophilised tissue preparation of human or animal origin which is suitable as a cleansing agent for cutaneous wounds and soft tissue injuries, which preparation is substantially free of non-fibrous tissue proteins and is substantially free of antigenic polysaccharides, mucopolysaccharides and glycoproteins.

24. A preparation as claimed in claim 15 which has been sterilized, lyophilized and powdered, and is ready for use as a cleansing agent for a cutaneous wound or soft tissue injury.