Sterilization of tissue implants using iodine

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

Many types of implantable devices for repairing or improving the function of human body parts are known. Examples are vascular prostheses and grafts, tissue valves, and even completely artificial organs. The material that is used to make these implants or prostheses may be synthetic or it may be actual tissue derived from man or from some other species. For example, tissue implants often are derived from porcine or bovine sources. When an implant is made of actual tissue, the tissue may be used fresh from the donor; however, it is preferable to preserve the implant tissue for later use.

One primary obstacle to successful implantation of actual tissue implants is immune response against the implant by the recipient. Immune response against an implant is a result of antigenic differences between cells of the recipient and cells of the implant material. The recipient's natural immune response is to attack the foreign antigens on the cells of the tissue implant.

One widespread means used to overcome immune reactions against a tissue implant is to fix and preserve the tissue implant using glutaraldehyde before implantation. Theoretically, glutaraldehyde is believed to coat, bind and cross-link the antigens on the surface of the tissue implant. As a result, the number of antigens on the implant that are capable of inducing an immune response in the recipient are reduced.

Glutaraldehyde-preserved tissue implants are relatively inert biologically and have demonstrated long-term durability in some instances even though the glutaraldehyde renders them somewhat cytotoxic. However, glutaraldehyde treated implants also have demonstrated serious drawbacks, such as tissue-fatigue and a propensity toward calcification. Glutaraldehyde tends to leach out of a tissue implant into both the surrounding tissue and into the bloodstream. Also, because glutaraldehyde is cytotoxic, the cells exposed to the leached glutaraldehyde can be damaged. Cells damaged by glutaraldehyde often die and/or rupture. Dead and/or ruptured cells often serve as a nidus for calcification. In fact, calcification has proven to be one of the primary reasons for failure of glutaraldehyde-treated implants.

One solution to this calcification problem has been to fix and preserve tissue implants using photooxidation rather than glutaraldehyde. Photooxidation involves placing the tissue implant in saline, exposing the implant to a photocatalytic dye, and then subjecting the implant to fluorescent light. Photooxidation also modifies the structure of the collagen and appears to provide new cross-links in the collagenous tissue. However, implants that have been fixed using photooxidation do not exhibit the same tendency to calcify as glutaraldehyde-treated implants.

Although photooxidative fixing of tissue implants shows great promise, the implant still must be sterilized before it can be implanted in the recipient. Unfortunately, the most common method used to sterilize a tissue implant is to treat the implant with glutaraldehyde. Sterilization with glutaraldehyde, even after the tissue implant has been fixed, still could create a calcification problem. Therefore, it would be advantageous if tissue implants could be sterilized without using glutaraldehyde.

Historically, many germicidal or disinfectant solutions have been used to sterilize various objects and materials. The majority of such solutions have been used to disinfect solid surfaces. However, some disinfectant solutions have been used to disinfect soft surfaces, including human skin. Some of the disinfectant solutions previously used to sterilize human tissue, for example, the preparation known by the trademark "Betadine.TM.," have been iodine-based. Although iodine-based disinfectants have been used safely and effectively to sterilize the surface of living tissue, iodine-based solutions have not been used to sterilize non-living tissue such as the tissue found in a tissue implant.

Whether or not an iodine-based disinfectant solution can safely and effectively sterilize the non-living tissue in a tissue implant is a valid concern. Living tissue can survive relatively rigorous conditions because living tissue is capable of repairing any damage that may result from such conditions. In contrast, non-living tissue cannot repair itself. When the proteins in non-living tissue are subjected to rigorous conditions, they tend to denature. Denaturation of the protein in the tissue implant, which cannot be repaired by the non-living tissue, detrimentally affects the physical properties of the tissue implant.

A method of sterilizing tissue implants which does not cause protein denaturation and which does not induce calcification in vivo would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides an iodine-based solution, and a method of using that solution, which sterilizes tissue implants without denaturing the proteins in the implant and without inducing calcification of the implant in vivo. Preferably, the tissue implants sterilized using the present invention are fixed without using glutaraldehyde. Most preferably, the tissue implants are fixed using photooxidation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 chart the results of the experiments discussed below.

FIG. 1A indicates the temperature at which sterilization took place and charts the change in shrink temperature of a tissue implant dependent upon the pH of the iodine sterilant used.

FIG. 1B indicates the pH at which sterilization took place and charts the change in shrink temperature of a tissue implant dependent upon the temperature at which the sterilization procedure took place.

FIG. 2A and 2B compare shrink temperatures, when ethanol was either present (2B, 10%) or not present (2A) in the solution used to sterilize the tissue implants, as a function of iodide:iodine ratios present in the sterilant.

FIG. 3A charts the change in shrink temperature dependent upon the iodine concentration in a sterilization solution that has been buffered to a pH of 6.5.

FIG. 3B charts the change in shrink temperature dependent upon the iodine concentration in a sterilization solution that has been buffered to a pH of 5.0.

FIG. 3C charts the change in shrink temperature dependent upon the incubation temperature used with sterilization solutions having various iodine concentrations and which have been buffered to a pH of 6.5.

FIG. 3D charts the change in shrink temperature dependent upon the incubation temperature used with sterilization solutions having various iodine concentrations and which have been buffered to a pH of 5.0.

FIG. 4 indicates sterilization conditions and charts the change in shrink temperature dependent upon the time of incubation and the presence (4B, 10%) or absence (4A) of ethanol.

FIG. 5A is a transmission electron micrograph of collagen which has been damaged by treatment with iodine at a relatively low pH (3.5) and high temperature (42.degree. C.).

FIG. 5B is a transmission electron micrograph of substantially undamaged collagen treated with iodine at a pH of 6.5 at 42.degree. C.

FIGS. 6A and 6B are photographs of x-rays of explanted tissue valves which were treated according to the present invention which shows no evidence of calcification.

FIGS. 7A, 7B, and 7C are photographs of x-rays of explanted tissue valves which were treated with glutaraldehyde, and which show evidence of calcification.

FIGS. 8A and 8B are photographs of x-rays of explanted Carpentier-Edwards Porcine tissue valves which were treated with glutaraldehyde, and which show evidence of calcification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the sterilization of tissue implants which preferably have not been treated with glutaraldehyde. Most preferably, the tissue implants used in the present invention will have been fixed photooxidatively using the procedure described in U.S. patent application Ser. No. 07/388,003.

In a preferred embodiment of the present invention, the tissue implants are sterilized using an iodine based germicidal solution under conditions having four primary variables: time of incubation; temperature of incubation; chemical content; and, pH of the germicidal solution. If any one of these four parameters is too stringent, the tissue implant may be damaged or the sterilization may be insufficient. Therefore, these parameters must be carefully controlled.

Although it is possible not to add stabilizing salts to the iodine based solution of the present invention, the solution preferably contains iodine stabilizing salts which are believed to help maintain the elemental iodine in a microbiocidal condition. A number of salts might be suitable for this function, preferably halide salts, and most preferably iodide salts, such as potassium and sodium iodide.

Another variable which tends to increase the microbial kill efficacy of the present solutions is to refresh the solution at approximately 24 hour intervals. If the solution is refreshed, solutions containing lower concentrations of iodine appear to be more efficacious.

In order to avoid damaging the tissue implants, as the stringency of any one of the following treatment parameters is increased within the following ranges, the stringency of the other parameters should be decreased:

______________________________________ Parameter Low High ______________________________________ elemental iodine 0.01% 0.20% (I2) potassium iodide 0% 0.40% (KI) sodium iodide (NaI) 0% 0.40% ethanol 0% 20% pH (aqueous 5.0 6.8 solution) NaCl -- 4M time of incubation 3 hours 2 weeks incubation 20.degree. C. 50.degree. C. temperature ______________________________________

A preferred embodiment of the present invention involves incubation of a tissue implant at a temperature between about 35.degree.-39.degree. C. for a period of time sufficient to sterilize the implant, typically between about 46-50 hours, in a solution containing between about 0.09-0.11% each of elemental iodine, potassium iodide, and sodium iodide and between about 1.8-2.0% ethanol, the solution having been buffered to a pH between about 6.4-6.6 using purified water and phosphate buffered saline. The ethanol content of the solution described in these preferred embodiments is a result of residual ethanol from the solution used to prepare the iodine stock. Although the present invention is functional if additional ethanol is present, no additional ethanol is added in these preferred embodiments.

A particularly preferred embodiment of the present invention involves incubation of the implant at approximately 37.degree. C. for about two days in a solution containing about 0.1% each of elemental iodine, potassium iodide, and sodium iodide, about 1.9% ethanol, the solution having been buffered to a pH of about 6.5 using purified water and phosphate buffered saline.

In a preferred method of preparing the germicidal solution of the present invention, a concentrated (10X) phosphate buffered saline solution ("PBS") is prepared by combining about 76 gm NaCl, 35 gm Na.sub.2 HPO.sub.4, and approximately 950 mL distilled or purified water (to a total volume of one liter), and stirring the resulting mixture until the ingredients are dissolved. Additional NaH.sub.2 PO.sub.4 is added (approximately 13.6-14 gm) until the pH of the resulting solution is about 6.5. Unless otherwise specified, the chemicals used herein may be obtained from a number of commercial sources, such as Sigma Chemical Company, St. Louis, Mo., or Aldrich, 1001 West Saint Paul Avenue, Milwaukee, Wiss. 53233.

A preferred iodine stock solution is prepared by combining about 5.0 gm each of elemental iodine (I.sub.2), potassium iodide, and sodium iodide, with about 100 ml of 95% ethanol which has been prewarmed to about 37.degree. C. The mixture is swirled until the iodine is completely dissolved. The resulting solution may be stored in the dark for up to one month.

To prepare the solution for actual use to sterilize a tissue implant, the following should be dissolved in about 850 mL distilled or purified water in a 2 liter beaker or flask: about 100 mL 10X PBS stock solution; 20.0 mL 5% iodine stock solution. After all of the components have dissolved, the pH of the solution should be adjusted to about 6.5 using the appropriate sodium mono- and dibasic-phosphates, as needed. The solution then should be brought to 1.0 liter using additional distilled or purified water, as needed. The resulting solution should be filtered through a 0.2 micron sterilizing filter into a sterile container which may be capped, sealed, labeled, and stored at room temperature in the dark for up to a maximum of one week.

In order to sterilize a tissue implant using the foregoing iodine solution, the following preferred procedure should be performed within a laminar flow hood. All personnel should wear appropriate attire, including gowns, mask, hat, and sterile gloves. The following items should be placed within the laminar flow hood: sterile 5 inch forceps; clean plastic jars; prepared labels; prepared iodine solution. The implant container should be opened and the tissue implant and test sample or "swatch leaflet" removed using the sterile forceps. The containers should be opened one at a time and the transfer should be completed before another tissue implant is removed from another container. The tissue implant and swatch leaflet should be placed in a clean plastic jar (such as a 3 oz. polypropylene jar) and the prepared iodine sterilant should be poured into the jar until the solution completely covers the valve (approximately 100 mL). The Jars containing the implant, the test swatch, and the iodine solution should be closed tightly and the outside of the jar should be wiped with a lint free cloth and ethyl alcohol and labelled. Two empty jars then should be filled with approximately the same volume of iodine and labeled as controls, and all of the jars should be placed in an incubator which has been calibrated to the desired temperature, preferably about 37.degree. C., and incubated for 48.+-.2 hours, or for 24 hour increments after which the iodine solution was refreshed.

After incubation, the jars should be removed from the incubator and placed in the laminar flow hood, along with a sterile five inch forceps, clean plastic jars, labels, and sterile 50% ethanol. The container should be opened and the tissue implant and swatch leaflet removed from the iodine/ethanol solution using the sterile forceps. The containers should be opened one at a time. The implant should be placed into one clean jar and the swatch leaflet placed into another, and 50% ethanol should be aseptically poured into the jars until the implant and the swatch leaflet are covered completely (approx. 100 mL). The jars then should be closed tightly, and the outside of the jar should be wiped with a lint free cloth and ethyl alcohol and labeled. Two control jars for later sterilization testing then should be prepared by filling two empty jars with approximately the same volume of 50% ethanol and labeling the jar