Title of Invention

A METHOD OF OBTAINING BIOMATERIALS COATED IN FIBRIN ADHESIVE

Abstract

This invention relates to a method of obtaining biomaterials coated with growth factor incorporated fibrin adhesive comprising in the steps of : subjecting the surface of the material such as herein described to adsorption of enzyme activity by exposing it with a solution of enzyme, thrombin, to obtain a thrombin adsorbed surface; coating the surface with a protein composition such as herein described and allowing fibrinogen in the mixture to polymerise on thrombi n adsorbed surf ace to obtain a protein coated surface; subjecting the protein coated surface to the step of freeze-drying at a temperature in the range of -40°C to -55°C .

Full Text

This invention relates to amethod of obtaining biomateriais coated to fibrin adhesive. This invention further relates to stable coating of growth factor incorporated bioadhesives onto polymeric materials so as to enhance attachment and proliferation of endothelial cells, and thus to reduce the thrombogenicity of the materials that are implanted as card iovascular prosthesis, BACKGROUND OF THE INVENTION Blood vessel diseases and its blockage have become a major cause of damage of organs. Occlusive arterial disease is commonly treated, by-passing the occluded vessel. Though autogeneous grafts are the best, when suitable vein is not available, synthetic vascular grafts made of polyethylene terephthalate or polytetrafluoro ethylene are substituted. Alternatively, the blocked portion of the vessel may be reopened or replaced by a vascular stent composed of metal or synthetic materials. However, small caliber synthetic vascular grafts for medium or smal1 vessel bypass is limited by frequent graft occlusion. This is mainly because the currently available vascular grafts do not endothelialize and they remain throrobogenic. The seeding of vascular grafts with endothelial cells has been considered as a solution to the problem of thrombogenicity (Mosquera and Goldman, Br. J- Surg. 78:656-660, 1991) , In experimental settings, seeding of endothelial cells has shown beneficial effects. However, seeded grafts suitable for routine use are still not produced- There is a clear need for nonthrombogenic synthetic materials for implant applications. The surface in contact with blood should be able to endothelialize or form neointima in a short period. This will prevent protein deposition, clot formation, platelet adhesion and aggregation on the surface. The thrombus should not grow on the surface to occlude the flow or to get embolized into the flow. While endothelial cell seeding can reduce thrombogenicity of the surface, the major problem is the lack of a suitable protein coating on the surface to which endotheial cells can attach and proliferate. Most of the proteins have problem that their cell attachment domain also attract platelet and thus thrombus forms on the area where endothelial cells haw not grown. The coating of a matrix protein is, however, essential for cells to attach and grow. Another problem in the field is that only limited number of cells can be obtained when cells are harvested from the patient's own blood vessel. The cells should proliferate quickly so that after donating the cells at least in a week's time he can get the endothelialised graft placed, instead of the insured vessel. Thus, the proteins coated on the surface should also be able to promote cell proliferatiron. PRIOR ARTT When a blood vessel is injured, the process of blood clotting generates fibrin, which holds other blood cells and the cytokines and growth factors released by white blood cells, platelets and surrounding cells. The fibrin network then acts as a natural scaffold for cells to migrate and proliferate and thus tissue repair and remodeling takes place. The idea of using fibrinogen or fibrin as a matrix has been tried out [US Patent s 5,324,647 and 5,272,0743. However, the method that has been used to stabilize the fibrinogen to the surface is heating. But heating above 50°C in presence of moisture will denature the proteins and especially the growth factors. Thus these proteins may act as a eel 1 attachment scaffold but may not promote the migration and proliferation of cells. Collagen is also a natural extrace1lular matrix protein which helps migration and cell growth. Gelatin has been routinely used for growing endothelial cells in tissue culture wells as it enhances the migration and proliferatiron - However, stabi1ization of gelatin on the surface is difficult and cells thus grown get easily detached under shear stress. Therefore, we have incorporated gelatin into the fibrin network and stabilized it on the surface to improve the cel1 migration and proliferation. OBJECTS OF THE INVENTION An object of this invention is to propose a polymeric materials having a coating of growth factor incorporated adhesives. Another object of this invention is to propose a method of coating polymeric materials with growth factor incorporated bioadhesives which is shown to resist the shear stress as compared to the endothelial cells grown on gelatin coated material. Still another object of this invention is to propose a method of coating polymeric materials with growth factor incorporated bioadhesives which is stabilize. DESCRIPTION OF INVENTION According to this invention there is provided a method of coating polymeric materials with growth factor incorporated bioadhesives which comprises in the steps of s i) treating the surface of said material to obtain an enzyme activity ii) coating the treated surface with a protein composition and allowing it to cross link and polymerize iii) subjecting the polymerized surface to the step of tyophilization. The present invention provides methods for coating surfaces of polymeric materials using cryoprecipitate obtained from human plasma, bovine thrombin, gelatin from porcine skin and growth factors isolated from calf hypothalamus. After coating the surface with a composition of above proteins, the material is left between 30°C to 40°C for the thrombin to act on fibrinogen in the cryoprecipitate to form fibrin monomers, which is subsequently cross linked by the activated FXIII to form a net work in which gelatin and growth factors are retained - The proteins thus coated on the surface is then subjected to a freeze—drying step so that fibrin network holding the growth factors and gelatin is stabilized on the surface. The composite coated surfaces showed minimal platelet adhesion and throfflbogenesis compared to the bare surfaces. This invention also relates to the accelerated proliferation of the endothelial ceils on the composite coated surface. The cells grown on the surface seemed to be physiologically active, having good nitric oxide production, expressed low density lipoproteins, was not thrombogenic and resisted shear stress when exposed to flow. Details of the Invention; I 1. The present invention provides a composition of different proteins as a composite to coat polymeric surfaces. The polymeric material used is either polystyrene, polyethylene, polyethylene terephthalate, or expanded polytetra fluoroethylene and may be in the form of circular or square pieces or as conduits. 2. According to one aspect of the invention, the polymeric material is initially incubated for 15 to 30 min in a solution of thrombin, 10 to 50 I U per ml, dissolved in 10 mM to 50 mM CaCl2 solution. The thrombin solution is added in a proportion of 0.5 ml to 1ml per cm2 material. After the incubation period, which is done between 30 to 40oC, the excess thrombin solution is aspirated out. 3. According tO; a related aspect of the invention, a fibrinogen concentrate obtained from human plasma as cryoprecipitate and stored as lyophilised powder is dissolved in DAA/ to get a concentration of 10 to 30 mg of total protein per ml. The solution of fibrinogen concentrate is mixed with an equal volume of 2% skin gelatin prepared by autoclaving gelatin in distilled water. To this fibrinogen concentrate 250 to 500 ug of endothelial cell growth factor is also added. The composite solution containing fibrinogen, FXIII, gelatin and endothelial cell growth factors is mixed well. Fifty to 200 ul of this composite solution is then layered over thrombin absorbed surface. The material is then incubated between 30oC to 40oC for 30 min to 60 min. E.g.1. Bovine thrombin (E.Merck, Germany) is adsorbed on the surface. Human fibhnogen concentrate obtained as cryoprecipitate is mixed with porcine skin gelatin (tissue culture grade obtained from Sigma Chem. USA), endothelial cell growth factors isolated by a standard method, from calf hypothalamus. 4. In another aspect of the invention the composite coated material is frozen at temperature ranging from -50oC to -70oC for a period of 2 to 24 h. The frozen material with the composite coating is then transferred to a lyophilizer and freeze dried at a temperature of -40°C to -55°C and negative pressure of 10 ' to 10-2 torr, for a period of 2 to 12 h. The polymeric material surfaces thus coated with the composite and stabilized by lyophilization has a thin uniform network of fibrin enmeshed with the growth factors and gelatin. Since the stabilization is done at a low temperature, the growth factors and the mitogenic fibrinopeptide generated during the action of thrombin on fibrinogen is expected to be biologically active. 5. In order to show that the growth factors are retained for a sufficient period for the seeded endothelial cells to consume, a release kinetics of proteins/peptides from the coating was measured. Chromoproteins such as hemoglobin and cytochrome C were used, to measure the release kinetics, because it has similar molecular weights as the growth factors, and it is easy to quantitate from their Soret band absorbance. In this aspecct of the invention we have incorporated hemoglobin, and cytochrome C, into the composite. The release of the protein into the serum containing medium is estimated using a standard calibration curve that is generated with known concentrations of the chromoproteins. diluted in serum containing medium. The calibration curve is used to estimate the concentration of the protein released into the medium for a period of 120h. at an interval of 24 h. The medium is drawn from the tissue culture wells, first after 5 min after exposure to the medium, and then after every 24h. We found that till 96h, there is a steady release of the chromoproteins into the medium. This result confirmed that the coating technique used is efficient to retain the growth factors within the fibrin network. The uniformity of the network is visualised inder the microscope after staining the coated surface with Coomassie blue. 6. According to another aspect of the invention, we also tested the attachment and proliferation of endothelial cells on the coated composite matnx. The edothelial cellsare isolated from human umbelical cord vein. The vein of the umbelical cord is flushed several times with Hank's Balanced Salt Solution (HBSS) and 0.1% collagenase in serum free MCDB is filled in the cannulated vein. After 5 min cells are resuspended in serum containing MCDB and cells are seeded onto a polystyrene culture flask coated with 0.1% gelatin. After 4 days, cells are harvested by treatment with 0.5% trypsin. The cells are washed and subcultured in another set of gelatin coated culture plates. These second passage ceils are harvested after 3 days and seeded on the composite coated surface. In 48h, a confluent monolayer is formed on the composite. The morphology of the cells grown on gelatin alone, fibrin alone and the composite are compared. 7. In another embodiment of the invention, the growth rate of the endothelial cells from the same passage is compared on gelatin coated, fibrin coated, and composite coated surfaces. The lyophilized surfaces with either fibrin or composite coating is rinsed with culture medium to remove excess thrombin. Equal number of cells harvested from the second passage grown on gelatin coated polystyrene dishes are then seeded on gelatin coated-, fibrin coated-, or composite coated polymeric material such as PTFE or Dacron. The cell attchment and spreading is monitored after 2h. The attachement and spreading are similar on fibrin coated- and composite coated surfaces, whereas on gelatin coated surface spreading is poor. By 48h, it is found that on the composite coated surface, the cells proliferated into a confluent monolayer, whereas on the fibrin-coated and gelatin coated surfaces confluent monolayer is formed by 72h or later. Thus an embodiment of this invention shows that endothelial cells attach similarly on fibrin and composite whereas proliferation on the composite is much better than that on the fibrin alone, 8. According to another aspect of the invention', it is also shown here that the fibrin coated on the surface is stable and is not lysed into fibrin degradation products during cell culture. To estimate fibrin degradation during endothelial cell culture, the tissue culture medium is collected at 24 h intervals during cell culture. Using an agglutination kit obtained from Diagnostica Stage we estimated the FDP in the culture media. There is no detectable level of FDP in the culture media for upto 7 days. 9. To measure the physiological activity of the endothelial cells, the cell monolayer grown on the composite coated surface is incubated with serum-free medium for 24h and the medium is taken for NO assay. A NO production in the range 2-3 nM per 0.5x106 cells are detected. 10. In another aspect of the invention we also demonstrated that the composite coated surface with endothelial cell monolayer is less thombogenic compared to the bare surface. The endothelialized surface is exposed to platelet rich plasma in a parellel plate flow chamber (fabricated according to Baumgartner's design). The perfusion of PRP is attained using a peristaltic pump at a flow rate of 4 ml per min or 8 ml per min. After exposure of the surface to the PRP for 30 mm. the surface is rinsed with serum-free culture medium and the surface is fixed in 2% glutaraldehyde for 2h. The surface is then stained with Leishman's to view the adhered platelets. The adherence of platelets are estimated by counting several fields. The effect of the endothelialised monolayer on platelet activation in the PRP is assessed by measuring the response to agonist of exposed platelet rich plasma using a Chronplog platelet aggregometer. The platelet rich plasma after perfusion through the chamber without any materials served as the control After perfusion through a chamber holding an endothelialised surface, the PRP responded to agonists similar to the control PRP. WE CLAIM: 1. A method of obtaining biomaterials coated in fibrin adhesive comprising in the steps of : subjecting the surface of the material such as herein described to adsorption of enzyme activity by exposing it with a solution of enzyme, thrombin, to obtain a thrombin adsorbed surface; coating the surface with a protein composition such as herein described and allowing fibrinogen in the mixture to polymerise on thrombin adsorbed surface to obtain a protein coated surface; subjecting the protein coated surface to the step of freezing at a temperature in the range of 50 C to 70 C and drying at a temperature in the range of -40 C to -55 C. 2. The method as claimed in claim 1 wherein the biomaterial is exposed for 15 to 30 mins. to a solution of thrombin dissolved in CaCl2 solution and at a temperature of 30 to 40 C. 3. The method as claimed in claim 2 wherein 10 to 50 lU per ml of thrombin solution is dissolved in 10 to 50 mM of CaCl2 solution. 4. The method as claimed in claims 2-3 wherein the thrombi n is 2 added in a proportion of 0-5 to 1 ml/cm of biomaterial. 5. The method as c1aimed in claim 1 wherein the protein composi- tion comprises a mixture of 1 to 5 mg/ml of fibrinogen concen trate, 0.1 to 0.2 mg/ml of gelatin and 0.1 to 0.2 mg/ml of growth factors and allowed to polymerise on the surface between a tempe rature of 35°C to 40oC. 6. The method as claimed in claim 1 wherein the protein-coated surface is subjected to freez ing for a period of 2 to 24 h and then freeze-dried between -40°C to -55°C and at negative pressure between 10-1 to 10-2 for 7, A method as claimed in claim 1 wherein the biomater in is coated are polystyrene, polyethylene, polyterepthalate, expanded poly- tetraf1uoroethylene, ultra high molecular weight po1yethylene, titanium and diamond-like carbon coated titanium. 8. A method of coating of growth factor incorporated fibrin adhesive on biomaterials substantially as herein described. .Dated this 15th day of FEBRUARY, 2000

Documents:

130-mas-2000-abstract.pdf

130-mas-2000-claims.pdf

130-mas-2000-correspondence others.pdf

130-mas-2000-correspondence po.pdf

130-mas-2000-description complete.pdf

130-mas-2000-form 1.pdf

130-mas-2000-form 26.pdf

130-mas-2000-form 4.pdf

130-mas-2000-other documents.pdf