GB2075819A - Biosynthetic micrografts from chorionic vessels and process for preparing same - Google Patents

Abstract

Biosynthetic micrografts of 0.5 mm. to 2.0 mm. external diameter from mammalian chorionic vessels, for use in reconstructive surgery, are prepared by harvesting chorionic vessels from the placenta and then treating with reagents to make the vessels non- antigenic and non-thrombogenic. The treated vessels are then suitable for implantation into another mammal without immunological rejection. Preferably blood is removed from the vessel before treating with a tanning agent such as an aldehyde or an alcohol to reduce antigenicity. The vessel may then be washed and treated with anti- thrombogenic agent such as L-sodium glutamate.

Description

SPECIFICATION Biosynthetic micrografts from chorionic vessels and process for preparing same This invention relates to flexible segments of mammalian placental or chorionic vessels which have been prepared for use as biosynthetic micrografts of 0.5 mm. to 2.0 mm. external diameter and process for preparing such blood vessels.

The blood vessels coursing over the foetal surface of the mammalian placenta have been called placental vessels or chorionic vessels by different authorities. In the description of this invention and claims, the term chorionic vessels will be used for the said vessels.

At present, the smallest available artificial blood vessel grafts for use in human beings have external diameters of 4.0 mm. While these grafts function satisfactorily for replacement of diseased blood vessels in the lower limb above the ankle, they are nevertheless too large for reconstruction of vascular defects in blood vessels with external diameters smaller than 2.0 mm. such as in the digits. In the fields of reconstructive microsurgery, blood vessels with external diameter of 0.5 mm. to 2.0 mm. are constantly encountered and the need for an off-theshelf vascular graft is often felt. This invention relates to a process for treating chorionic vessels from mammalian placentae with reagents to render the vessels non-antigenic and non-thrombogenic so as to render them suitable for implantation as vascular grafts without immunological rejection.The use of this invention would be mainly as a vascular graft but this graft can also be used for many other non-vascular purposes in reconstructive surgery as will be enumerated in this application.

A major advance in the field of reconstructive surgery has been the advent of microvascular surgery. It is now possible to join blood vessels with external diameters of 0.5 mm. to 2.0 mm. with reliable patency. A surgeon with special training in micro-surgery, with the aid of optical magnification by an operating microscope using specially designed microinstruments and microstructures finer than the human hair, is able to join tiny blood vessels successfully. It would not be possible to join these tiny blood vessels using only the naked eye.

The ability to anastomose tiny blood vessels enables the reconstructive microsurgeon to transplant tissues from one part of the body to another in one operative procedure so that at the end of the operation the transplanted tissue obtains a new blood supply from the recipient site. It has made possible such operations as the transfer of toes to a hand with amputed fingers in one operative step.

Microsurgery has revolutonised many fields in surgery. In plastic and reconstructive surgery, it has enabled the transplantation of flaps of skin, muscle and bone, singly or in combination to cover large tissue defects which would have been impossible to cover or which would have required multiple operations and long hospital stays to achieve the same result. It has also made possible the successful reattachment of completely amputated digits and limbs. In the field of orthopaedic surgery, bone with an intact blood supply can be transferred to fill a bony defect. In general surgery, a segment of bowel taken from the abdominal cavity can be transplanted to the neck to replace a resected segment of the oesophagus which is diseased.In the field of neurosurgery, a fresh new blood supply can be brought in to increase the nutrition to a part of the brain with a poor blood supply.

While the basic principles of vascular surgery apply in macroas in microvascular surgery in establishing a successful patent anastomosis, the technique and training differ markedly. Tissues are much smaller in microsurgery and much more delicate.

They need to be handled with the utmost care and gentleness. The use of the operating microscope, microinstruments and microsutures need to be patiently mastered. A general macrovascular surgeon cannot expect to master overnight the techniques of microsurgery and expect to transfer his skills and competence in macrovascular surgery to microvascular surgery. He will need special training as microvascular surgery is not a mere extension of macrosvascular surgery. It is a skill to be meticulously learned by operating on laboratory animals before applying the techniques to human beings.

Over the last 3 decades the development of artificial blood vessel grafts, be they purely synthetic or grafts of biological origin, has made tremendous advances in vascular surgery. For large blood vessels, the textile grafts of polyester are available to replace the aorta and its bifurcation. For medium sized and small vessels with external diameters of 4 6 mm., the artificial grafts presently available are the polyester grafts, the glutaraldehyde tanned human umbilical cord vessels and the bovine heterografts.

In the field of microvascular surgery there are at present no artificial grafts for vessels with external diameters of less than 2.0 mm. although there is a pressing need for such artificial grafts. It is an object of the present invention to meet this need.

Sometimes in replantation of amputated digits, the stump vessels are found to damaged for a few centimetres proximally as a result of the avulsion or crushing injury which amputated the digit. This segment of damaged vessel has to be excised till a normal segment of blood vessel can be found proximally. This results in a vascular defector defi ciencywhich requires a graft to bridge the ends.

When the microsurgeon faces a situation which requires a graft, he usually uses a segment of vein graft as veins of the body are more readily dispensible than arteries. This segment of vein graft is usually harvested from an easily accessible region as determined by the part of the body operated upon. The donor site of the vein graft may be from the volar aspect of the wrist, the dorsum of the foot or from the donor or recipient site in a free flap operation.The important factors the surgeon has to consider in harvesting a vein graft and implanting it in the vascular system are as follows:1. the ease of dissection of the graft, 2. the diameter of the vessel graft required, 3. the length of graft required and the length of veins available, 4. the configuration required - with or without branches, the number of branches and whether with a bifurcation at one end, 5. the presence of disease in the donor vessels, 6. the presence of valves and the taper in the wrong direction if implanted in the arterial stream which requires reversal of the vein graft, 7. the long term performance of vein grafts in arteries, i.e. late thrombosis, durability and degeneration of grafts e.g. microaneurysm formation.

The mammallian placenta contains both arteries and veins with external diameters of 0.5 mm. to 2.0 mm.

suitable for use a microvascular grafts and for other reconstructive purposes in the human body and in the lower mammals. The umbilical cord which contains 2 arteries and a vein surrounded by Wharton's jeliy divides into smaller branches at its insertion or attachment to the placenta. These arteries and veins are called the chorionic blood vessels and they form an intricate network of branches and bifurcations which are visible on the foetal surface of the placenta. As they course towards the periphery of the placenta they diminish in size.Vessels near the insertion ofthe umbilical cord measure 1 - 2 mm. in external diameter and those nearerthe periphery measure 0.5-1.0 mm in external diameter These vessels lie embedded in the foetal side of the chorionic plate of the placenta and are covered by the amniotic membrane superficially and by part of the chorion. The arteries as a rule cross superficial to the veins. Both the arteries and the veins are suitable for use as grafts.

In the present invention, a flexible segment of mammalian chorionic artery or vein is prepared for use as a bioprosthesis in mammals. The vessels are dissected from the placenta, trimmed of adventitious tissue, shaped and treated with reagents to reduce the antigenicity and make them non-thrombogenic to the vascular stream. The prepared grafts are suitable for implantation into the blood vessels of a mammal.

The placenta may be dissected immediately after birth or it may be stored in a refrigerator at 4" C. for up to 2 days before dissection. Placentae are not used if there is a maternal history of venereal disease, malignancy, septicaemia or hepatitis.

Placenta are also discarded if there is a risk of infection of the placenta and membranes when there is a prolonged interval between rupture ofthe membranes and birth.

The amniotic membrane is first peeled off to reveal the chorionic vessels lying on the chorionic plate. Part of the chorionic membrane covers the chorionicvessels under the ammion. A reasonably long segment of the artery or vein of about 2 - 3 cm.

long without too many side branches is chosen. The chorionic membrane superficial to the blood vessel is gently dissected off the adventitia of the vessel.

The ends of the vessel are then ligated with fine silk and the small side branches are either ligated or coagulated with a disposable or bipolar cautery. The proximal end of the vessel is divided transversely and the vessel is sharply dissected off the rest of the chorionic plate. The distal end of the vessel is then divided. Excess chorionic tissue is trimmed off.

Through one end of the vessel a fine polythene cannula is passed and the lumen is irrigated with heparinised saline and then dilute hydrogen peroxide solution and left in a tank of hydrogen peroxide for half an hour. The polythene cannula should preferably have a closed blunt tip with perforations in the side. This ensures atraumatic irrigation of the lumen. The cannula is left in the lumen of the vessel to act as a mandrel. Polythene is the preferred material although stainless steel, glass, silicone or other inert plastics material may be used. The mandrel should have a lumen in it although it may be solid. It may be uniform in diameter or tapered, curved or straight.

After half an hour in hydrogen peroxide the lumen is irrigated with a solution of an aldehyde to stabilise, sterilise and render the vessels nonantigenic. Glutaraldehyde is the preferred agent with dialdehyde starch the next best. Other agents which may be used are alcohol, formaldehyde and glyoxal.

The concentration ofthe glutaraldehyde is preferably from 0.1 % to 2.0 % by weight, buffered with 1 % sodium bicarbonate or a combination of potassium dihydrogen phosphate and sodium bicarbonate at a pH from 6 to 8.5. At lower concentrations of glutaraldehyde the reduction of antigenicity and sterilisation is incomplete. At too high a concentration, the reaction is too rapid and the vessel wall becomes too brittle and stiff, losing its elasticity and compliance; as a result sutures tend to cut out easily.

The tanning agent causes the vessel to shrink and conform to the shape of the mandrel. The concentration of dialdehyde starch is preferably from 0.5% to 2.0 by weight The vessels are left in a tank of tanning agent for a period from 15 minutes to 7 days and then removed.

The lumen of the vessels are irrigated with normal saline to remove residual traces of glutaraldehyde which may be toxic to tissues and cause an aseptic necrosis of the tissues which it subsequently contacts. A further step to remove residual traces of glutaraldehyde which may be toxic and render the vessels non-thrombogenic is by treatment with agents such as amino acids, alkali salts of amino acids and oxidising agents such as peroxides, peracids and hypochlorites. The sodium salts of amino acids are preferred in particular L-sodium glutamate, L-cysteine, L-sodium alanine and L-sodium phenylalanine. The best reagent is L-sodium glutamate.

These agents impart a negative charge to the luminal surface of the graft. It is believed that the amine group of the amino acid combines with the carbonyl group ofthe aldehyde leaving the carboxyl group of the combination free to ionise and leave a negative charge on the vessel surface. Negatively charged luminal surfaces are known to be antithrombogenic.

The blood vessels are stored in a sterile sealed container with an appropriate solution such as 40 50 % aqueous alcohol, 1 % sodium bicarbonate, 4 % formaldehyde or 0.5 % glutaraldehyde. The preferred solution is 40 - 50 % aqueous alcohol. Before use the graft should be washed in copius amounts of normal saline to remove residual traces of storage solution.

It can be seen that many variations in the procedure are possible to meet specific and varied requirements. In the selection of segments of vessels, variation is possible. Arteries or veins may be selected according to the thickness of the vessel wall required. Generally it is more advantageous and desirable to replace like with like and replace arteries with arteries and veins with veins. Various diameters of vessels ranging from 0.5 mm. to 2.0 mm. may be selected with the smaller vessels selected from the periphery of the placenta and larger vessels from the centre near the insertion of the umbilical cord.

Straight or curved segments may be chosen and variation is possible with bifurcated or unbranched grafts. The number of side branches can also be varied.

Mandrels used for shaping and irrigation of the vessels may be made of polythene, glass, silicone, stainless steel, plastic or other inert material. The mandrels may be cylindrical or tapered, straight or curved, solid or hollow with a lumen and when hollow with or without side perforations. One end of the mandrel may open or it may be closed with a blunt tip. The vessels after irrigation and removal of blood may be tannd without a mandrel as the tanning agent is able to permeate the wall of the vessel. The use of the mandrel is preferred for vessels with an external diameter of 1.0 mm. or more. Tanning without the mandrel is preferred with vessels with external diameters of less than 1.0 mm.

as it is very difficult to insert a mandrel into a very small vessel without trauma and injury to the vessel wall.

Various cleansing and irrigating solutions to remove blood and residual aldehyde may be used such as water, normal saline, heparinised saline, concentrated heparin, Ringer's lactate, hydrogen peroxide, sodium bicarbonate, alcohol, transplantation perfusate solutions. Anti-thrombogenic agents which may be used are L-sodium glutamate, Lcysteine, L-lysine, L-alanine and L-phenylalanine, amines, hydroxylamines, peracids, peroxides and hypochlorites.

Aldehydes suitable for tanning are glutaraldehyde, dialdehyde starch, formaldehyde and glyoxal; other agents are anhydrous alcohol. Storage solutions may be 40 - 50 % alcohol, 4 % formaldehyde, normal saline or 0.5 % glutaraldehyde.

It may be appreciated that each process may be achieved by a few treatment methods. For example the step of shaping the vessel is achieved by inserting a mandrel in the vessel, excising excess tissue and by the tanning agent which shrinks the blood vessel so that it conforms to the shape and size of the mandrel. It will also be appreciated that one reagent may used for a few different reasons.

For example glutaraldehyde shrinks, shapes, stabilises, sterilises and reduces the antigenicity of the blood vessel and glutamate removes residual aldehyde and renders the vessel non-thrombogenic as well.

It will be realised that the use of the bioprosthesis need not be confined to use as a microsized vascular conduit, although this would be its main use. It could have many non-vascular applications in mammals.

The function of a naturally occurring duct of 0.5 mm.

to 2.0 mm external diameter may be compromised by congenital anomalies or by acquired disease. The naturally occurring ducts which may be repaired, augmented or replaced are: a) the lacrimal drainage system especially the lacrimal canaliculi and the naso-lacrimal duct b) the ducts of the parotid and submandibular salivary glands, c) the bile ducts both intra-hepatic and extrahepatic, d) the pancreatic duct both intra- and extraglandular, e) the ureter, f) the urethra, g) the vas deferens, h) the fallopian tube, i) the semicircular canals and the duct of the cochlea of the ear.

The bioprosthesis may also be used to create a fistulous conduit to connect the middle ear to the external ear through the tympanic membrane to drain the middle ear of secretions.

The bioprosthesis may be used for repairing, augmenting or replacing a nerve of a mammal. It may be used to provide a conduit along which a divided or diseased proximal nerve bundle orfasci- cle may grow towards its distal end and thus bridge a nervous tissue defect. It may be used to cap a nerve end to prevent the formation of a tender neuroma.

In reconstructive surgery of the lymphatic system, the bioprosthesis may be used to create new lymphatics orto repair, augment or replace diseased lymphatic vessels which may be absent, diseased, damaged or ablated.

For defects and deficiencies of the musculoskeletal system, the bioprosthesis may be used as a tubular structure or be slit lengthwise and used as a flat sheet to reconstruct a tendon, ligament or joint capsule. It may also be used as a tendon sheet to reconstruct the gliding mechanism of a tendon or as a pulley over a bony prominence or over a joint.

It will be understood that the descriptions of the processes for repairing the bioprosthesis mentioned above are merely exemplary and that persons skilled in the art may make many variations and modifications without departing from the spirit and scope of this invention.

The present invention will now be illustrated more fully by the following example: Example A placenta was secured immediately after birth. A maternal history of venereal disease, malignancy, septicaemia and hepatitis has been excluded. Under the magnification of an operating microscope, the chorionic artery was harvested from the placenta.

The amniotic membrane was first peeled back to reveal the chorionic artery embedded in the foetal side of the chorionic plate. A segment of artery 2 cm.

long with an external diameter of 1.0 mm. was chosen with a few side branches. Side branches were cauterised with a disposable microcautery set.

The ends of the vessel were ligated with fine silk.

Dissection commenced by excision ofthe chorionic membrane superficial to the adventitia of the blood vessel. The artery was divided at one end and then sharply dissected off the chorionic plate with fine microscissors working from one end of the vessel to the other. At no time was the vessel crushed as handling of the vessel was strictly limited to picking up the vessel by its adventitia only with fine forceps.

The other end of the vessel was then divided and the vessel placed in some normal saline. One end of the vessel was gently dilated and a polythene cannula with an external diameter of 0.6 mm. was inserted into the lumen. The polythene cannula was of the type used for epidural anaesthesia. It had a lumen, a closed end which was blunt and side perforations.

The cannula was passed throughout the length of the vessel. Heparinised saline at a concentration of 100 units per ml was used to irrigate the lumen.

Excess chorionic plate tissue still remaining on the adventitia of the vessel was trimmed off.

Next, hydrogen peroxide 10 volumes % was irrigated through the lumen and the vessel in its mandrel was placed in the solution of hydrogen peroxide for half an hour. The vessel was then placed in a solution of 0.5 % glutaraldehyde which was buffered to a pH of 7.4 by potassium dihydrogen phosphate and sodium bicarbonate. The vessel was left in the glutaraldehyde solution for 72 hours to tan at room temperature. The vessel was washed with 500 c.c. of normal saline for 2 minutes with 2 further changes of saline. After washing the vessel its lumen was irrigated with 2 % L-sodium glutamate and it was left in the glutamate solution to soak for 2 hours.

The vessel was removed from the glutamate solution and placed in a sterile sealed container containing 40 - 50 % ethyl alcohol and stored at room temperature.

Just before the vessel was washed in 3 changes of 500 c.c. of normal saline with gentle stirring. The mandrel was removed and the vessel was implanted into the femoral artery of a rat weighing 420 grams using standard microvascular anastomotic techniques. The rat was anaesthetised with intraperitoneal nembutal. The external diameter of the graft matched very closely the external diameter of the femoral artery of the rat which measured 0.8 mm. When the vascular clamps were removed there was a good flow across the anastomoses and the graft. The patency test showed flow to be full. The operative site was stitched up and the animal recovered from the anaesthesia.

Two weeks later the groin was explored and there was found to be very little reaction around the graft.

The graft was found to be fully patent and blood flow across the anastomoses was good. A transfemoral arteriogram performed through the opposite groin showed both anastomoses to be fully open and patent with good outlines of the anastomoses.

Claims (29)

1. A tubular bioprosthesis for use in reconstructive surgery in mammals, the bioprosthesis having an external diameter of 0.5 mm. to 2.0 mm. and having been obtained from mammalian chorionic arteries or veins and having been treated to reduce its antigenicity.

2. A bioprosthesis in non-tubular form for use as a sheet or a patch in reconstructive surgery in mammals obtained by dividing longitudinally a tubular bioprosthesis as claimed in claim 1, or dividing longitudinally a chorionic artery or vein and then subjecting such an artery or vein to a treatment for reducing its antigenicity.

5. A bioprosthesis according to claim 1 or 2, wherein the chorionic artery or vein is from a human being.

4. A bioprosthesis according to claim 1 substantially as hereinbefore described.

5. A bioprosthesis according to claim 1 substantially as described in the foregoing example.

6. A process for producing a tubular bioprosthesis according to any one of the preceding claims which process comprises the steps of a) removing and separating a chorionic blood vessel from the adjacent and surrounding tissue of a mammalian placenta, b) removing blood from the vessel, c) treating the blood vessel with a tanning or hardening agent to stabilise, sterilise and reduce the antigenicity of the blood vessel and thereby allow transplantation of the treated blood vessel from the placenta of one mammal to another individual or mammal of the same or different species without immunological rejection, and d) treating the blood vessels to remove the tanning or hardening agent and render it non-thrombogenic.

7. A process according to claim 6, wherein in step a) the chorionic vessel is dissected off the chorionic plate for use as a bioprosthesis by an atraumatic technique using fine micro-surgical instruments and viewing through an operating microscope.

8. A process according to claim 6 or 7, wherein in step a) the chorionic vessel segment removed is uniform or tapered, straight or curved, with or without bifurcations where the vessel divides into two equal branches, or, with or without side branches where smaller branches are given off from the side of the main vessel.

9. A process according to claim 6,7 or 8, wherein in step b) a cleansing solution selected from water, normal saline, Ringer's lactate, hydrogen peroxide and transplantation perfusate is used to irrigate the lumen ofthevessel and its exterior.

10. A process according to claims 6 to 9, wherein prior to step c), the cleaned blood vessel is subjected to a shaping treatment.

11. A process according to claim 10, wherein the blood vessel is shaped by inserting in to the lumen of the vessel a mandrel which functions to impart a given configuration to the vessel when the vessel is subjected to step c).

12. A process according to claim 11, wherein the mandrel is of polythene, glass, stainless steel, silicone or an inert plastics material.

13. A process according to claim 11 or 12, wherein the mandrel is straight or curved, of uniform diameter or tapered, with or without a lumen, with or without side perforations, with the end open or closed with a blunt tip.

14. A process according to claim 11, 12 or 13, wherein the mandrel inserted into the lumen of the blood vessel is rotated about its longitudinal axis, to facilitate the removal of excess chorionic plate tissue and shape the external surface of the blood vessel.

15. A process according to any of claims 11 to 14, wherein the shaping is carried out by tanning or hardening the blood vessel by exposure to a tanning or hardening agent while the blood vessel is mounted on the mandrel.

16. A process according to any one of claims 6 to 15, wherein in step c) the tanning or hardening agent used is selected from glutaraldehyde, formaldehyde, dialdehyde starch, glyoxal and alcohol.

17. A process according to claim 16, wherein the tanning or hardening agent is an aldehyde and is used in an amount of0.1%to5%byweight.

18. A process according to claim 16, wherein the tanning or hardening agent is ethyl alcohol and is used in an amount of 50 % to 95 % by volume.

19. A process according to claim 16 or 17, wherein the blood vessel is tanned or hardened by bringing it in contact with a solution of glutaraldehdye of concentration 0.1 % to 2.0 % by weight buffered to a pH of 6.0 to 8.5.

20. A process according to claim 16 or 17, wherein the blood vessel is hardened by bringing it into contact with a solution of dialdehyde starch at a concentration from 0.5 % to 2.0 % by weight.

21. A process according to any one of claims 6 to 20, wherein after step c) the treated vessels are washed to eliminate residual tanning or hardening agent and render the vessels non-thrombogenic with a suitable reagent chosen from L-glutamate, L-alanine, L-phenylalanine, L-cysteine, L-lysine, alkali salts of amino acids, amines, hydroxylamines, peracids, peroxides and alkali hypochlorite.

22. A process according to claim 21, wherein the reagent is the sodium salt of L-glutamic acid.

23. A process according to any one of the claims 6 to 22, further comprising the step of soaking the blood vessel in 70 to 80 % ethanol prior to hardening the vessel in order to produce a stiffer product.

24. A process according to any one of claims 6 to 23, wherein after step d) the blood vessels are stored in a sealed sterile container in preparation for a surgical procedure in a solution selected from alcohol, 4.0 % formaldehyde, 0.5 % glutaraldehyde and normal saline.

25. A process according to claim 24, where the solution is 40 to 50 % aqueous alcohol by volume.

26. A process according to any one of claims 6to 25, wherein the vessels are rinsed with normal saline, heparinised saline, Ringer's lactate or transplantation perfusate solution immediately before implantation in a mammal.

27. A process according to claim 6 substantially as hereinbefore described.

28. A process according to claim 6 substantially as described in the foregoing example.

29. A tubular bioprosthesis according to any one of claims 1 to 5 when prepared by a process as claimed in any one of claims 6 to 28.