WO2009044407A1

WO2009044407A1 - Small diameter vascular graft from processed cadaver saphenous vein

Info

Publication number: WO2009044407A1
Application number: PCT/IN2008/000404
Authority: WO
Inventors: Guhathakurta Soma; Mammen Cherian Kotturathu
Original assignee: International Centre For Cardio Thoracic And Vascular Diseases (A Unit Of Frontier Lifeline Pvt. Ltd); Department Of Biotechnology
Priority date: 2007-10-03
Filing date: 2008-06-25
Publication date: 2009-04-09

Abstract

The invention in general relates to a process for diminishing the antigenicity and calcification of derived vascular graft when intended to be used as a implant, for rendering such grafts substantially resistant to infection, and for rendering such grafts liable to be stored for a prolonged period of time before implantation.

Description

TITLE :"SMALL DIAMETER VASCULAR GRAFT FROM PROCESSED CADAVER SAPHENOUS VEIN"

FIELD OF INVENTION:

Today the options for small vascular grafts that is grafts less than 6 mm internal diameter range from autologous saphenous vein, homografts, Dacron grafts, PTFE grafts but each one of them is beset with issues like non-availability problem of Thrombogenicity.

PRIOR ART :

Arterial damage typically results from atherosclerotic plaques that restrict the lumen of the blood vessel and subsequently damage the endothelial lining of the artery. This damage ultimately results in the formation of a thrombus and obstruction of blood flow.

Significant research has gone into developing vascular grafts since 1942. Artificial prostheses were introduced into cardiovascular surgery in 1952 when Hufnagel implanted the first artificial heart valve, and in the same year, Voorhees introduced the first artificial vascular graft.

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use.

For reconstruction of large arteries, such as the aorta or iliac artery, synthetic grafts made from ePTFE or Dacron are the vascular grafts of choice. However, synthetic materials are not suitable for reconstruction of smaller diameter arteries ((6 mm diameter), as required for lower extremity bypass and coronary artery bypass grafting procedures, because they carry a substantial risk for thrombosis anastomotic intimal hyperplasia, aneurysm formation, infection, and progression of atherosclerotic disease.

DESCRIPTION OF INVENTION:

For surgeries generally an autologous vein graft is used e.g., saphenous vein, and in the case of inability to use it i.e., internal thoracic artery, gastroepiploic artery, inferior epigastric artery, radial artery can be used.

Cryopreserved allograft have also been used in the clinic as coronary artery bypass conduits, but poor patency rates (i.e., high rates of occlusion) and problems with aneurysm have limited the use of these grafts to situations in which no other autologous conduits are available.

Although venous and arterial autografts currently yield the best results, disadvantages include the need for multiple surgical procedures, with increased risk and cost to the patient. In addition, vein grafts have thin walls that may be damaged when transplanted into the arterial system, and suitable vessels are not available in all patients due to disease, amputation, or previous vessel harvest.

Thus, there remains a clear need for a vascular prosthesis that would be suitable for small-diameter vessel reconstruction. The creation of such a graft has been the focus of considerable research for many years, yet there is still no adequate alternative to the autograft for small-caliber vessel reconstruction procedures.

Photoxidation

Dye-mediated photooxidation is an alternative tissue crosslinking method that requires no harsh chemical such as glutaraldehyde. Certain amino acids such as tryptophan, histidine, tyrosine, and methionine in collagen can be specifically oxidized by irradiation with visible light in the presence of a suitable photosensitizer (e.g., methylene blue and rose Bengal dyes)

This reaction does not cause cleavage of the peptide bond. With respect to photoxidation of histidine, it has been reported that aspartic acid and urea were the final products of this reaction, and a detailed mechanism for the oxidation reaction has been described.

Furthermore, other studies show that photooxidation of collagen solution stabilizes the collagen to denaturation and to enzymatic degradation, presumably by the formation of protein crosslinks More recent studies have shown that dye-mediated photooxidation can be used to stabilize intact collagen based tissues such as bovine or sheep pericardium and small-diameter arteries. For these applications, photoxidation serves as a catalytic process that induces modification and crosslink formation within the existing matrix components, resulting in a more natural material with little added matrix complexity.

Photooxidized pericardium and vascular grafts have supported endothelial cell growth in vitro demonstrating the non-cytotoxic nature of dye-mediated photooxidation. In vivo studies have further demonstrated the potential utility of PhotoFixTM tissue for medical applications. For example, PhotoFixTM bioprosthetic heart valves implanted into sheep are partially endothelialized after 2 years, demonstrating the ability of these tissues to adequately support cell adhesion and migration.

While dye-mediated photooxidation has been shown to be noncytotoxic, nonimmunogenic and to retain natural mechanical properties, the cytotoxicity andmechanical properties have not been fully elucidated for polyepoxide compounds.

Decellularization

In addition to alternative treatments that preserve (i.e., crosslink) natural tissue, there are methods to produce completely acellular tissue matrices by specifically removing cellular components that are believed to promote calcification and to give rise to a residual immunological response.

Saphenous vein is in contact with blood, thereby decalcification is highly important feature to be focused on. These decellularization techniques include chemical, enzymatic and mechanical means of removing cellular components, leaving a, material composed essentially of extracellular matrix components.

For the most part, these acellular tissues retain natural mechanical properties and promote remodeling of the prosthesis by neovascularization and recellularization by the host. Ultimately, the creation of actual living tissue replacements for cardiovascular applications would solve many of the existing problems associated with cardiac heart valve replacements and vascular prostheses.

During typical processing and crosslinking treatment of tissue, cells are ruptured, but the cellular debris is largely retained. Various cell extraction methods (e.g., detergent treatments, enzymatic digestion, and sonication) have been pursued as a means to create completely acellular tissues for use as biomaterial implants, but presence of residual cellular components and lipids within processed tissue may promote undesired effects such as calcification.

Cell extraction procedures, such as detergent treatments, often remove proteoglycans, which also appear to play a significant role in calcification. The removal of free proteoglycans from glutaraldehyde-treated pericardium using the reagent guanidine hydrochloride had resulted in reduced calcification, but increasing the extent of proteoglycan extraction produced a greater accumulation of calcium this may be due to extensive extraction procedures, which presumably removed some of the more tightly bound proteoglycans and resulted in loss of collagen- proteoglycan interactions, generated a porous matrix into which calcium salts were deposited The role of proteoglycans in calcification is not clear, although it appears that the removal of the more tightly bound, collagen associated proteoglycans in tissue is detrimental.

So there has to be a balance between the procedures of decellularization and crosslinking to produce a better preserved tissue after decellularization and other steps and finally preservation.

After extensive research we have found out the methods to produce effective small ^' diameter vascular grafts out of homologous Saphenous vein graft as it was determined that synthetic vascular grafts are thrombogenic.

The need for small diameter vascular graft was for the purposes:-

> Shunt surgeries often require small diameter vascular grafts specially in very young children

> Aorto-visceral bypass (specially kidney or great vessels of the neck)

> Re-operations in CABG is quite frequently done and problem for finding suitable conduit and where varicose veins are a problem

> Neuro-surgical cases 2mm-3mm vascular grafts are dire necessities.

• Peripheral vascular diseases as a vascular conduit

• Redo coronary artery bypass surgery where conduit deficit can happen, because of previous usage

• Aorto visceral bypass (specially kidney or great vessels of the neck)

• Where conduit for CABG is not available due to varicosity or various reasons • In 2002* more than 50000 surgical procedures were performed involving replacement of small caliber blood vessels (<6 mm)

• Primarily autologous arteries and veins are preferred as graft material. In a significant number of cases no suitable autologous vessels were available.

• Alternative grafts in cases of non-availability of vessels options are

> Thrombogenicity a concern in synthetic (PTFE) grafts

> Synthetic biodegradable (PLA, PGA ) not proven

> Immunosuppression concerns in homologous grafts

• Even today saphenous vein remains the standard for small vascular grafts especially in CABG surgery.

The work plan is Decellularization

Crossliηking studies

Mechanical testing &Burst test

Thrombo Igenicity stud ^■y

Biocompatibilty &cytotoxicity test

Animal studies

SMALL DIAMETER VASCULAR GRAFT:(H)

Cadaver saphenous veins harvested from preferably road accident cases and head injury patients in sterile manner, within 24 hours of death, collected in saline solution containing antifungal agent. Brought to GMP laboratory and same as above procedure under laminar airflow they are transferred into antibiotic cocktail.

50gms of tissue treated with 100ml of 1 % Deoxy cholic acid of the solution of for 20- 50 hours, subsequently treated with ribonucleus for enzymatic digestion of the cells.

It is then treated with heparin in glucose solution ( 5-25% Dextrose 500ml with 5000 ^■ units -50,000units of heparin/100 ml of glucose) to reorganize the collagen matrix. To get back the compactness of the tissue.

It is then crosslinked with photo oxidation physico chemical with methylene blue (.01%-10%) and UV irradiation (wavelength of 125-525-nano-metre) For 10-20hours.

It is further additionally treated with heparin in glucose solution to reorganize the collagen matrix with heparin, which acts as an agent adapted to modify the surface properties of the tissue.

Preservation in 80% ethyl alcohol solution with glycerine and water.

No small diameter vascular graft are available commercially out of natural biomaterial. Synthetic Scaffolding And Cell Seeding Methods Are Still At Experimental Level. In neurosurgical cases and cardiovascular surgery they are of immense use. Especially when it comes to repeat surgeries where conduits are lacking . Homologous vein grafts without treatment have been used with little success under the cover of immunosuppression. But thrombosis of the graft is very common.

Available synthetic small diameter grafts are thrombogenic and unpredictable. So far our small diameter vascular grafts are non thrombogenic and having the properties of autologous cell deposition so that it becomes an autograft within a period of 3 months. Our processing procedure can enable any size of human vein to be useful as small diameter vascular graft.

From the experiments conducted for the purpose of this invention, the following conclusion were arrived at:~

• Apparently DCA decellularization with enzymatic digestion appears to be most effective if done for long hours.

• Initial mechanical and cross linking studies studies have shown favorable results.

• Thrombogenicty study results are encouraging .

• Biocompatibility needs to be tested for animal experimentation and subsequently human application.

The process of Preservation:-

• Long term preservation - with alcohol glycerine and water, which further crosslink and preserves the tissues.

Claims

CLAIMS:

1. A process for diminishing the antigenicity and calcification of derived vascular graft when intended to be used as a implant, for rendering such grafts substantially resistant to infection, and for rendering such grafts liable to be stored for a prolonged period of time, comprising:

a. cleansing the harvested vein grafts in balanced salt solution with antifungal agent;

b. treating the vein grafts in a solution of Deoxycholic acid in an aqueous solvent of suitable pH along with ribonucleic enzyme treatment;

c. reorganizing the collagen matrix with heparin in glucose solution;

d. cross linking with photooxidation physico chemical with methylene blue and UV radiation for a duration;

e. reserving with alcohol solution; and

f. implanting the treated grafts into a host body needing repair.

2. The process according to claim 1 , wherein said treating step is effected in a 1 percent solution of deoxycholic acid with RBN enzyme sequentially for 20 to 50 hours.

3. The process according to claim 1 , wherein said treating step is effected during 20-50 hours.

4. The process according to claim 1 , wherein said reorganizing step is effected in 5-25% dextrose solution, with 5,000 to 50,000 ml. of heparin per 100 ml. of glucose.

5. The process according to claim 1 , wherein said step of crosslinking is effected in the 0.01-10% range of methylene blue.

6. The process according to claim 1 , wherein said step of crosslinking further effected with UV radiation of wavelength 125-525 nanometers.

7. The process according to claim 1 , wherein said step of crosslinking further effected for 10-20 hours.

8. The process according to claim 1 , wherein said step of preserving further effected with 80% ethyl alcohol solution.

9. The process according to claim 1 , wherein said step of preserving further effected with glycerin and water.

10. The process according to claim 1 , further including the step of additional reorganizing the collagen matrix with heparin, which acts as an agent adapted to modify the surface properties of the tissue,

1 1. The process according to claim 10, wherein said agent is adapted to attach negative or positive charges to the surface of the tissue.

12. The process according to claim 1 , wherein the graft used is saphenous vein and said implanting step is a vascular graft.

13. The process according to claim 1, wherein the treated graft used is human vein and said implanting step is a vascular graft.

14. A process according to claim 1, wherein the treated graft is an animal vein, and said implanting step is a vascular graft.

15. A process according to claim 1, wherein the treated graft is implanted into a human as vascular graft and was derived from an animal species other than a human.

16. A process according to claim 1 , wherein the treated graft is implanted into a human as vascular graft and was derived from another human.

17. A treated vascular graft as claimed in claim 1 intended to be used as a implant, such that the said treated grafts is substantially resistant to infection and liable to be stored for a prolonged period of time before implantation.

18. A method for implanting a graft into a recipient host treated for damaged arteries.

19. A method as claimed in claim 18, wherein said graft is a vascular conduit.

20. A method as claimed in claim 18, wherein (i) said graft harvested from corpse within 24 hours of death; (ii) the harvested graft are decellularised; (iii) decellularized grafts are cross-linked; and (iv) cross linked grafts are implanted into host body.

21. A method as claimed in claim 18, wherein the said graft has less than 6mm internal diameter. .

22. A method as claimed in claim 18, wherein said graft is mounted onto biologically acceptable carriers prior to implantation onto the recipient host.

23. A method as claimed in claim 22 wherein said carriers is a bio-degradable scaffold.

24. A method as claimed in claim 1 wherein the said grafts are introduced directly into the arteries.

25. A method as claimed in claim 1 , wherein the said graft is derived from homologous saphenous vein.