WO1998007452A1

WO1998007452A1 - Method of sterilising material for implantation

Info

Publication number: WO1998007452A1
Application number: PCT/GB1997/002265
Authority: WO
Inventors: Donald Francis Walker
Original assignee: Sulzer Vascutek Limited
Priority date: 1996-08-21
Filing date: 1997-08-21
Publication date: 1998-02-26
Also published as: AU3950297A

Abstract

A method of sterilising material is disclosed, the material being for implantation into a human or animal body. The method includes treating the material with a substance non-reactive with a sterilising agent to be used, the substance being selected so as to maintain certain physical characteristics of the material such as flexibility and/or structure of cells or extra cellular material. Suitable substances include water soluble non-volatile substances for example glycerol. The method can be used on graphs for implantation or on biological material such as vascular tissue etc. and has the advantage that the substance does not react with water and so the material can be treated in solution without drying out or becoming brittle.

Description

METHOD OF STERILISING MATERIAL FOR IMPLANTATION

This invention relates to a method of treating a graft for implantation into a body.

Biologic tissue grafts of human and animal origin such as heart and venous valves and blood vessels are well known. Biologic vascular grafts exhibit good patency (the characteristic of remaining open in the body) but have been shown to lack stability in the body in the longer term, and can elicit immune responses in the host. Current approaches to countering instability and antigenicity in situ include treating the graft with a cross-linking agent such as glutaraldehyde or Denacol, or inducing cross-linking in the graft by other means such as dye-mediated photo-oxidation. Dye-mediated photo-oxidation is preferred since the treated graft has physical characteristics which are closer to the natural tissue and low immunogenicity. There are also some reports of dye-mediated photo-oxidation eliciting grafts with better patency and lower incidence of thrombosis and anastomotic hyperplasia.

Unlike glutaraldehyde treatment, dye-mediated photo- oxidation does not sterilise the graft, and additional sterilisation treatment is necessary before the graft can be implanted. The current preferred sterilisation method is treatment with ethylene oxide (EtO) . However, EtO cannot be directly applied to a graft held in aqueous solution such as saline, since EtO reacts with water; the penetration of EtO into the tissue may also be limited, and the removal of EtO after sterilisation can present problems. Equally, the graft cannot simply be allowed to dry out to allow the application of EtO, since it would become brittle and could not be used without extensive re-hydration, and would be susceptible to damage.

According to the present invention there is provided a method of sterilising material for implantation into a human or animal body, the method comprising treating the material with a substance non-reactive with a sterilising agent to be used, the substance maintaining at least some of the physical characteristics of the material, and sterilising the treated material with said sterilising agent.

The physical characteristics of the material which may be maintained by treatment with the substance include flexibility, and/or structure of cells or extracellular material such as collagen, particularly the microstructure of collagen.

Preferably the sterilising agent and the substance are different. The substance preferably comprises a water- soluble non-volatile substance, and the sterilising agent can comprise, for example, ethylene oxide. A suitable substance might be glycerol. Other possible substances include sugars such as sorbitol .

Viewed from one aspect, the present invention provides a method of treating material prior to implantation in a human or animal body, said method comprising the following steps:

a. contacting the material with a protective substance;

b. optionally, drying the treated material of step a) ;

c. sterilising the material of step b) by contact with a sterilising agent; and

d. optionally, humidifying the sterilising material of step c) .

As stated above, the substance may be water-soluble sugars such as sorbitol or glycerol . Suitable solutions range from 5% to 100%, usually in 50% ethanol or in water. Where water is used as a solvent it should preferably be RO grade. Preferred solution concentrations are 30% to 70%, particularly 40% to 60%. Generally, the material is incubated in the substance for at least 12 hours at above ambient temperature (usually 34-38°C) . Following sterilisation, the treated material may be humidified to reduce the need for or extent of rehydration prior to implantation.

Optionally, the substance may contain additives which may be of biological interest. In this way the substance will provide a vehicle to preload the additives (which may be water or alcohol soluble biologically active agents) into the biological material. Examples of suitable additives include anticoagulants, antibiotics, steroids, hormones and growth factors. Heparin is of particular interest. Thus, in a further aspect, the present invention provides a method of introducing an active agent into material prior to implantation into a human or animal body, said method comprising treating said material with a substance comprising said active agent and non- reactive to the sterilising agent to be used, the substance maintaining at least some of the physical characteristics of the material, and sterilising the treated material with said sterilising agent. Preferably, the substance is glycerol and heparin and the sterilising agent is EtO.

Surprisingly, it has been found that this methodology increases the activity of heparin in the treated material .

Preferably the material is biological material, such as vascular tissue etc.

The pre-sterilising treatment enables the material substantially to retain certain physical characteristics, such as flexibility, and can suitably replace at least some of the water contained in the material.

It is preferred that the substance is non-toxic, and glycerol is extremely advantageous in that regard.

The material can, after being treated, be drained and/or washed to remove excess glycerol or other substance, prior to implantation.

The present invention also provides a method of treatment of material for implantation in a human or animal body, the method comprising treating the material with glycerol. The present invention will now be illustrated by way of the following, non-limiting examples:

Example 1: Plasticization of material with σlvcerol in preparation for EtO sterilisation

Samples are stored in 20% EtOH. A solution of 40% glycerol (AnalaR) in RO grade water is warmed up to 37 °C, and is poured over the material to be treated in sufficient quantity to cover completely the material in its container. The material and glycerol is then incubated preferably at 34-39°C for around 16 hours or more, preferably with agitation (125 MOT/min or 50rpm equivalent) . At the end of the incubation the tissue is removed from the glycerol and the excess glycerol is drained off. The treated material can then be left to dry, eg in class 100 air before being packaged (eg) in blister packs under a clean room environment. The material can then be sterilised by treatment with EtO, before humidification at 100% RH for 24hrs at 37 °C and packaging (eg) in a foil pouch.

EtO Sterilisation (example) A vacuum of 20 inches Hg (which is equivalent to 252mm Hg absolute pressure) is first drawn on a chamber. Steam is then injected into the chamber increasing the relative humidity.

The load is then conditioned for 1 hour, reaching a temperature of 35 to 39°C, and allowing the environment within the product packaging to reach a relative humidity of not less than 55% RH. At the end of conditioning, a vacuum is then pulled on the chamber for a short time to being it back to 20 inches Hg vacuum. EtO gas is then injected into the chamber; pressure within the chamber rises slowly from 20 inches Hg vacuum to 5.5 inches Hg vacuum (620mm Hg absolute pressure). EtO gas concentration reaches lOOOmg/lt. EtO gas exposure is initiated once the pressure reaches 5.5 inches Hg vacuum and lasts for 4 hours.

At the end of 4 hours exposure, EtO is removed from the chamber. Pressure within the chamber is cycled between 20 inches Hg vacuum and 2 inches Hg vacuum (709mm Hg absolute pressure) for 2 hours.

Example 2: Biologic Graft Initial Sterilisation Validation

Bovine Carotid and Thoracic arteries have been plasticised in a variety of solutions to allow sterilisation by Ethylene Oxide. So far, testing has been performed to show that there is no significant level of residual Ethylene Oxide or ethylene chlorohydrin. This example was performed to ensure that the EtO process is effective and penetrates the biologic graft.

Materials and Methods One large bovine carotid was removed from storage in 50% Ethanol, and plasticized in 50% glycerol according to the method set out in Example 1. The carotid artery was chosen, over the thoracic, as a worst case sample since it has a thicker wall. All side branches were closed off with cable ties (RS COMPONENTS). One spore strip (AMSCO SPORDEX 500) was inserted and pushed half way down the artery. The ends were then tied off with cable ties (RS COMPONENTS) . The artery sample was packaged in double blisters and passed through for sterilisation. The sample was then sterilised by EtO treatment, and once sterilised, the sample was removed from its packaging. To avoid excess handling the artery was cut with scissors, the spore strip was removed and aseptically transferred to a Nutrient Broth (BECTON DICKINSON) . This was then placed in an incubator (B AND T UNITEMP) at 32 °C.

Results There was no sign of growth in the nutrient broth after 24 or 48 hours. Positive controls, set up at the same time, showed growth after 24 hours. The EtO sterilant successfully penetrates the carotid artery wall since all other openings, such as side branches, are tied off. This shows that EtO sterilisation is a valid method for the treatment of biological material.

Example 3: Physical Testing of Bovine Artery Samples

15 x 15cm samples of Bovine carotid and thoracic arteries were transferred to 50% ethanol. All samples were plasticized in a solution of 50% glycerol in 50% ethanol. Once plasticized, samples were drained and allowed to dry for 1 hour, to remove excess glycerol.

5 samples were then packaged in double viewpack, the other 10 samples were EtO sterilised by the protocol set out in Example 2. Of these 10, 5 were then humidified for 1 hour, post-sterilisation, to reduce rehydration time.

Suture Pull-Out All samples were rehydrated by incubation in Reverse Osmosis water at 37°C for 10 minutes, with agitation equivalent to 50 rpm. Two sites were chosen for suture pull out, one at the end of the sample and one at the side; both were pulled at 90° to the sample. The suture hole was placed 2mm away from the edge of the sample using a loading jig.

A 4/0 Dacron suture was then threaded through and loaded into a yarn jig, on a Lloyds LRX Tensile tester. The sample was held in place by jaw clamps and a constant gauge length of 20mm was set. The test was performed at lOOmm/min and a data analysis package was used to calculate the load required to pull the suture out.

Maximum Load and Stress All samples were rehydrated, as before, prior to testing. A 5cm sample was loaded onto two eccentric roll grips on a Lloyds LRX tensile Tester. The test was performed with a gauge length 20mm at lOOmm/min, a data analysis package was used to determine the load and maximum stress required to pull the sample apart. Maximum stress is a measure of strength which is calculated from the maximum load and the cross sectional area of the sample. Sample dimensions were measured by vernier calipers.

Results The results from suture pull out, maximum load and maximum stress are shown below. Each sample is compared to an untreated natural sample, which is the partner of the treated sample. The results show that the physical properties of treated bovine arteries are unaffected by the plasticization and sterilization processes.

SD 2.37 SD 2.85 1.533

Carotid Arteries (continued)

Treatment

Samples

11C SIDE PLASTICIZED, STERILISED 11C TOP

AND HUMIDIFIED

12C SIDE 5.06 9.05 12C TOP 8.80 7.27 13C SIDE 8.50 8.41 13C TOP 10.52 9.41 14C SIDE 7.78 4.77 14C TOP 8.97 8.81 15C SIDE 7.88 6.84 15C TOP 6.06 9.23

X = 8.54 X = 8.37 N = 10 SD = 2.12 SD = 1.65

Mean of all natural samples x = 9.16, SD 2.38, 15 treated samples x = 8.46, SD 2.46, 15

Suture Retention Results: Thoracic Arteries

Treatment

Samples

1A SIDE PLASTICIZED 1A TOP 2A SIDE 2A TOP 3A SIDE 3A TOP 4A SIDE 4A TOP 5A SIDE 5A TOP

10

SD 1.25 SD = 1.22

inued

Example 4: Plasticization of Biologic Grafts

Bovine Carotid and Thoracic arteries (fixed by dye- mediated photo-oxidation) were stored in 20% or 50% ethanol at 2-8°C. Artery sections were plasticized in glycerol solutions as previously described, with the objective of producing a graft which could be sterilised by Ethylene Oxide and packaged dry.

Materials and Methods

Plasticization A range of glycerol (PROLABO) solutions from 5 to 50% were made up in 50% ethanol (MERCK) . All alcohol solutions were made up in purified water (RO grade) . Plasticization was carried out overnight (>16h) at 37°C, with gentle agitation (equivalent to 50 r.p. .) in sterile plastic vessels.

Measurements were made of artery lengths and wall thickness using vernier calipers (BATY). Diameter measurements were not made since the artery samples do not hold a regular shape when dried. If wall thickness is recovered then diameter will also be recovered. Initially the samples were dried overnight at room temperature (20°C), before sterilisation, in order to reduce excess glycerol before packaging and produce a dry sample. The same effect can be obtained by draining the sample against the vessel wall to remove excess glycerol and drying for 1 hour at room temperature. At higher concentrations of glycerol e.g. 50%, samples remained slightly tacky to touch.

Sterilisation Once plasticized, samples were packaged in graft blisters (XT-POLYMER AND TYVEK) . Double packaged samples were then sterilised under EtO for 7*5 hours at 35-39°C. Once sterilised, samples were desiccated. One set of samples was also humidified in an attempt to hydrate the samples and reduce the time taken to fully hydrate at point of use.

Rehydratjpri Samples were rehydrated by incubation in RO water at 37°C, with gentle agitation (50 r.p.m.) for about 10 minutes. This was very effective for humidified samples of higher glycerol concentrations. These samples could also be rehydrated over the same time scale at room temperature (20°C) with intermittent manual mixing. Rehydration was measured by comparing dimensions of samples before and after.

Residuals Artery samples plasticized in 30, 40 and 50% glycerol were sterilised, desiccated and humidified. These were then tested for ethylene oxide residuals by Gas Chromatography. Levels of ethylene oxide, ethylene chlorhydrin and ethylene glycol were measured to ensure that EtO sterilisation did not pose any problems in terms of residuals.

Results

The results below show the recovery of a number of dimensions after rehydration.

82 33 48 95 29 62 97 43 97 93 84 97 98 100 96 81 29 29 93 67 85 96 57 79 98 100 84

99 100 99

These results show that, after 10 minutes of rehydration at room temperature the best recoveries are made by humidified 50% glycerol samples. Thoracic arteries recover their dimensions more evenly than Carotid arteries , and are easier to handle . Initially it was thought that 50% glycerol would pose a problem in terms of packaging i.e. some glycerol would leave the graft and stick to the packaging, spoiling its appearance, and also that the graft itself would feel "slimy" to the touch. However, if the graft is sufficiently drained and dried it does not affect the appearance of the blister. The grafts are preferably not handled until rehydrated, after which they behave like a normal untreated artery.

Residuals Sample Artery Treatment Ethylene Oxide Ethylene No. Chlorhydrin 2 Carotid 302 Glycerol <1 ppm 1.5 ppm 3 Carotid 402 Glycerol <1 ppm 2.4 ppm 4 Carotid 502 Glycerol <1 ppm <1.0 ppm 5 Thoracic 302 Glycerol <1 ppm <1.0 ppm 6 Thoracic 402 Glycerol <1 ppm 2.0 ppm 7 Thoracic 502 Glycerol <1 ppm <1.0 ppm

These results show that the ethylene oxide level does not pose a problem. The ethylene chlorhydrin (ECH) levels are higher but again pose no problem, as can be seen from the maximum allowable limits for ECH in ISO 10993. For permanent exposure devices, the levels are as follows: 12 mg / 24h 60 mg / 30 days Not to exceed 2 mg / day 50 g / Lifetime

The 40% glycerol treated Carotid sample contained 2.4 ppm ECH or 2.4 μg/g. This would give 24μg for a 10 g graft. The limit per day is 2000 μg of ECH.

The use of 50% glycerol plasticizing solution, and post sterilisation hu idification, permits rehydration at room temperature for 10 minutes with occasional mixing. The point of use rehydration step could be performed in operating theatres with warm, sterile, normal saline infused through a dedicated port in the packaging to retain sterility. It could also be used to incorporate other solutions, for example, heparin or antibiotics.

Since glycerol keeps the dimensions of the grafts stable there would be little dimensional change during processing, therefore limiting concern over shrinkage or swelling on implantation. Thoracic arteries have a thinner wall and rehydrate more rapidly and evenly; they are easier to handle and to suture especially when sleeved.

Example 5: Plasticization of Bovine Pericardium

The physical characteristics of samples of bovine pericardium were assessed, before and after sterilisation, to assess the affect, if any, of the plasticization - sterilisation process on the pericardium. In order to provide a more hydrated end product, we also looked at varying glycerol concentrations and the use of post sterilisation humidification.

Materials and Methods

Sample preparation 20 pericardium samples were stored in 20% Ethanol. These were cut in half longitudinally and placed in solutions of 50% ethanol (BDH) .

10 specimens were left untreated for use as controls and were rehydrated in R.O. water for 15 minutes, at room temperature, prior to testing.

Plasti jzatJlon The following glycerol (PROLABO) solutions were prepared in 50% ethanol (BDH); 50%, 60%, 70%, 80%, 90% and 100%. Plasticization was performed according to the established procedure i.e. at least 16 hours incubation at 37 °C with agitation. Following this plasticization, samples were drained and dried for one hour at room temperature and then packaged in double blisters.

Sterilisation All test samples were sterilised under EtO. Following sterilisation samples which were not to be humidified were placed in foil pouches. Samples for post sterilisation humidification were placed in 100% humidity chambers, at room temperature, for between 2 and 24 hours. These samples were then foil pouched. An initial investigation into humidification time was performed on the 50% glycerol samples (1-10). The remaining samples of varying glycerol content were humidified for 24 hours.

Tensile Testing The maximum load required to break the sample was measured on a Tensile Tester (LLOYDS INSTRUMENTS). The sample was loaded onto two eccentric roll grips, these were then pulled apart, at a constant rate of lOOmm/min, until the sample broke. Maximum Load was calculated by a Data Analysis package on the LRX tensile tester. This package also calculated maximum stress or tensile strength based on the cross sectional area of each sample. All samples were rehydrated in RO grade water for 15 minutes, at room temperature, prior to testing. Suture Retention The load required to pull a 4-0 polyester suture from each sample was measured on the LRX tensile tester Lloyd instruments. The suture hole was placed 2mm from the edge of each sample using a perspex loading jig (VASCUTEK) . The suture was passed through and both ends loaded into a Bollard grip, the sample was held in an Eccentric Roll grip. The suture was pulled through at a rate of 100 mm/min, the maximum load was calculated by a Data Analysis package on the LRX.

Results The results are summarised in the tables below. A. CONTROL SAMPLES

As expected, for natural tissue, there is a great variation in all results . Sample 9 has noticeably lower value for Maximum Load and Stress, this may have been caused by poor loading of the sample. It may also signify the extent of variation that exists in naturally derived tissue, as the suture retention result for sample 9 is very close to the mean. B . TEST SAMPLES

80.70 12.54 10.51

S.D. 17.80 2.75 2.80

These results also show a wide variation, with mean values for treatment slightly lower than those for control samples . Sample 4 shows a noticeably lower maximum load value, in common with control sample 2. It is unlikely that these samples were both poorly loaded to the same extent .

These tensile tests were performed in order to determine if plasticization and sterilisation have an adverse affect on the physical strength of the pericardium. The results obtained show that, while the mean values for treated samples are lower, there is no significant decrease in physical strength after treatment. This is especially true when the degree of natural variation is taken into account.

State of hvdration The state of hydration was determined visually when the foil pouches and blisters were removed. Carotid artery samples, treated in the same way, were included to provide a more recognisable "marker for wetness".

The 50% glycerol samples with no humidification appeared dry and felt dry to the touch, though they were not rigid or too dehydrated. The higher glycerol concentrations appeared wet and felt slimy to the touch. At very high concentrations 80-100%, the samples felt rigid and stiff.

Increasing periods of rehydration improved the appearance of 50-70% glycerol samples, they also felt softer and more natural. Above 70% the samples were still quite slimy and rigid.

Humidification had much the same effect on pericardium samples, at 50-60% glycerol these felt more like their natural counterparts. Samples treated in 50% glycerol, with post sterilisation humidification of at least 4 hours, felt more natural than any others. It is possible to plasticize and sterilise bovine pericardium in the same way as bovine arteries . This does not adversely affect the physical strength of the tissue. Post sterilisation humidification allows for a more fully hydrated end product. Since it is no longer required that the material be dried, it will be possible to modify the plasticization method to further reduce the scope for dehydration.

Example 6 : Heparin Loading on Bovine Carotid Arteries

The purpose of this experiment was to investigate the possibility of loading heparin into the biologic graft using the plasticisation process. Heparin is a commonly used anticoagulant. It was chosen because of the benefit it might bring in terms of minimising thrombosis. Heparin is an ideal test substance. It is water soluble and there are commercially available assay kits, providing a means of loading and detection.

Materials and Methods

Bovine carotid arteries (Sulzer Carbomedics) were supplied, stored in 20% ethanol, and rinsed in RO grade water prior to use.

40% glycerol (ANAL R grade) was made up in RO grade water.

100 mg sodium heparin from porcine intestinal mucosa (Leo Labs) was added to 30 ml of 40% glycerol. Heparin activity was quoted at 172 IU/mg. Therefore, the activity of the final solution was 17200 IU.

The artery sample was then incubated at 37°C ±2°C with gentle agitation for 16 hours, in the heparin-glycerol solution .

Following incubation, the artery was removed, drained of excess glycerol and dried under Class 100 air for one hour. At this point, 2 x 1cm sections were removed to act as non-sterile test samples. The remainder of the artery was then sterilised by ethylene oxide (see Example 2 ) .

Heparin Assay /Range 0-1.0 IU/ml)

Heparin assay was performed by modifying a commercially available diagnostic kit. The Coacute Heparin Kit (Chromogenix) is a chromogenic assay for determining heparin activity in human plasma. In order to use the kit to analyse biologic samples, control normal plasma is added to the reaction buffer and incubated at 37 °C for one hour. The sample for analysis, in this case a section of biologic graft, is added to the plasma- buffer dilution and incubated for a further one hour at 37 °C. The resulting test dilution is tested as normal.

The following samples were assayed in duplicate: 1. Natural artery 2. Heparinised artery 3. Heparinised sterilised artery

(A405 refers to the absorption coefficient of the test sample at 405mm)

These results show that it is possible to load and recover heparin from biologic grafts, though the recovery is very low.

Increasing the heparin concentration, the temperature or the incubation period might all improve loading. However, this cannot be at the expense of any of the biologic graft's other physical characteristics.

These results also suggest that sterilisation by ethylene oxide increases heparin activity. However, non-uniform loading, irregular size and shape or the presence of side branches could also account for the greater activity.

The carotid artery is a thick artery covered in a layer of "furry" adventitia. Thoracic arteries - these are thinner, have little or no adventitia and should prove easier to load and assay.

Example 7: Heparin Loading on Bovine Thoracic Arteries Materials and Methods

The materials and methods followed were as for

Example 6, except that thoracic artery was used instead of carotid and heparin control samples were made in PBS and plasma.

The Chromogenic Heparin Coacute Kit was used once again.

Test Samples 1. Heparin plasma control 2. Biologic PBS control 3. Heparinised biologic 4. Non-heparinised biologic spiked with 10 μl heparin saline 5. Non-heparinised biologic washed and spiked with 10 μl heparin saline

Sample 1 was tested to investigate the accuracy of the test kit and also ensure a positive control.

Sample 2 was tested to ensure that the biologic graft incubated in PBS did not interfere with the test, or give false positive results.

Sample 3 was tested to investigate heparin loading and recovery from thoracic artery.

Sample 4 was tested to ensure that the biologic graft and glycerol did not interfere with heparin recovery.

Sample 5 was tested to ensure that glycerol did not interfere with heparin recovery. Results

Discussion

Unfortunately, these results highlight the inadequacy of the chosen assay system. The heparin plasma control system was spiked to contain 1.87 IU. Had it been spiked to contain 0.87 IU, some more useful information might have been obtained. Nevertheless, it does show that a heparin plasma control can be prepared and successfully analysed in this way.

The biologic negative control contained a background heparin activity of 0.2 IU. This could have been caused by carry over or contamination. Previous results and those from Sample 5 rule out the possibility of interference.

The heparinised biologic. Sample 3, contained >1.0 IU/cm. The results from Samples 4 and 5 show that the activity was due to loaded heparin and not any interfering factors . Conclusion

These results show that it is possible to load and recover heparin from biologic grafts and that this is easier in thoracic arteries. Neither the biologic graft nor any of the constituents used in its processing interfere with the assay. Data analysis is hindered by the fact that the assay limit is 1.0 IU/ml.

Alternative methods such as radio labelling could also be useful.

The result from Sample 3 suggests that a 1 cm section of thoracic artery, placed in a solution containing >1000 IU heparin, will display >1.0 IU heparin activity.

Heparin is a water soluble anticoagulant. This work suggests that it may be possible to passively load any water or alcohol soluble active substance including, but not limited to, anticoagulants, steroids, hormones and antibiotics.

Modifications and improvements may be incorporated without departing from the scope of the invention.

Claims

1. A method of sterilising material for implantation into a human or animal body, the method comprising treating the material with a substance non- reactive with a sterilising agent to be used, the substance maintaining at least some of the physical characteristics of the material, and sterilising the treated material with said sterilising agent.

2. A method as claimed in Claim 1 wherein the physical characteristics of the material which may be maintained by treatment with the substance include flexibility, and/or structure of cells or extracellular material.

3. A method as claimed in Claim 1 or Claim 2 wherein the sterilising agent and the substance are different.

4. A method as claimed in any one of the preceding Claims wherein the substance comprises a water-soluble non-volatile substance.

5. A method of treating material prior to implantation in a human or animal body, said method comprising the following steps:

(a) contacting the material with a protective substance;

(b) optionally, drying the treated material of step (a);

(c) sterilising the material of step (b) by contact with a sterilising agent; and

(d) optionally, humidifying the sterilising material of step (c).

6. A method as claimed in Claim 5 wherein the substance is a water-soluble sugar such as sorbitol or glycerol.

7. A method as claimed in Claim 5 wherein the substance is in a solution of concentration between 30% and 70%.

8. A method as claimed in either one of Claims 6 or 7 wherein the substance is in a solution of concentration between 40% and 60%.

9. A method as claimed in any one of Claims 5 to 8 wherein the material is incubated in the substance for at least 12 hours at above ambient temperature .

10. A method as claimed in any one of the preceding Claims wherein the substance contains an additive which is of biological interest.

11. A method as claimed in Claim 10 wherein the substance contains heparin.

12. A method of introducing an active agent into material prior to implantation into a human or animal body, said method comprising treating said material with a substance comprising said active agent and non-reactive to the sterilising agent to be used, the substance maintaining at least some of the physical characteristics of the material. and sterilising the treated material with said sterilising agent.

13. A method as claimed in Claim 12 wherein the substance is glycerol and heparin and the sterilising agent is ethylene oxide.

14. A method as claimed in any one of the preceding Claims wherein the material is biological material such as vascular tissue.

15. A method as claimed in any one of the preceding Claims wherein the substance is glycerol.

16. A method substantially as herein before described with reference to the accompanying examples.