JPH0159241B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing transplanted tissue that retards mineralization after transplantation, and to a transplantable tissue so produced. Mineralization here refers to the deposition of minerals such as calcium. BACKGROUND OF THE INVENTION In the past, natural tissue from animals, including humans, has been used for the manufacture of prosthetic devices for use in humans; for example, a heart valve made from a pig's aortic root was disclosed in US Pat. Described in No. 3966401. Prostheses in the form of tubes, strips and channels can be manufactured from the arteries and veins of the umbilical cord according to US Pat. No. 3,988,782. A number of stabilization methods are known to increase the longevity of natural tissues in the human body. The process is generally covered by U.S. Patent No.
A tanning agent such as glutaraldehyde as disclosed in US Pat. No. 4,378,224 is used. PROBLEM SOLVED BY THE INVENTION Mineralization after implantation of a bioprosthetic heart valve results in a decrease in the flexibility of the valve tissue and thereby a decrease in the effectiveness of the prosthesis' function in the host's body.
One solution to this problem, presented in U.S. Pat. No. 4,323,358 to Lentz et al., granted April 6, 1982, is the use of sulfated water-soluble salts of higher aliphatic alcohols, particularly sodium dodecyl sulfate. The fixed tissue is treated with a solution. Although this treatment with anionic surfactants retards the onset of mineralization to some extent, the amphoteric surfactants of the present invention provide further retardation as well as inhibition of the rate of mineralization. Means for Solving the Problem According to the invention, transplantable tissue can be produced by soaking the tissue in an aqueous solution of an amphoteric surfactant before, during or after fixation of the tissue. The surfactant used in the present invention is manufactured by Calbiochem-Behring, Four Research Biochemicals and Immuno-Chemicals (Calbiochem-Behring, Inc.).
Behring for Research Biochemicals and
N-decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate [Zubitutargient ^R 3-10 detergent ( Zwittergent ^R 3-10 Detergent) and N
-Hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate [Zwittergent ^R 3-16 Detergent (Zwittergent ^R
3-16 Detergent). Tissues treated according to the invention include dura mater, ligaments,
Bovine or porcine pericardial tissue is typically used, although aortic root, tendon, heart valve tissue, etc. may also be used. Unfixed, unprocessed natural tissue is first carefully washed in buffered saline. The buffer is a known calcium-free buffer such as a suitable phosphate or acetate. Salts are water-soluble salts such as those of alkali metals or alkaline earth metals and halogen compounds such as chlorides. Whenever referred to generally herein, this type of solution refers to buffered saline. More specifically, according to the present invention, the tissue is approximately 0.01
-10 (eg 0.01-2)% by weight aqueous solution of surface active betaine. The aqueous solution is preferably a buffer of the type described above. The tissue is soaked in the surfactant solution for at least 1 hour, such as 4 hours. Longer immersions are acceptable, but typically about 24 hours. After immersion, the tissue is rinsed with buffered saline and then fixed with glutaraldehyde (GA) for a period of about 7-14 days or longer.GA-fixative has a pH of about 4.0-8.5 (preferably A known fixed number such as a buffered aqueous solution of about 0.01-2% GA buffered at about 4.5-6.5, most preferably 5.5). After fixation, the tissue can be used to manufacture prosthetic devices such as heart valves. For final storage, the tissue is aseptically transferred to a phosphate-free formaldehyde buffer with a pH of approximately 4.5-8.5. The effectiveness of the treatments in delaying tissue mineralization (particularly calcium deposition) was determined by animal transfer studies using the following procedure. A New Zealand white rabbit weighing 1 to 3 kg is anesthetized and operated on. A piece of skin (8-10 cm) located at the vertebral end of the scapula was dehaired. I sterilized the rough part and the rough part around it. A 4 cm incision was made in either lateral circle of the spinal column to remove the skin, but the underlying trapezius muscle was not cut. A small depression was created in the subcutaneous area by forcibly separating the connective fascia. Place a 2 x 3 cm sterile sample of pericardial tissue rinsed three times with sterile saline in one well, and a 2 x 3 cm sterile sample of myocardial tissue rinsed three times with surfactant betaine in the other well. Ta. The incision was closed and the animal returned to the cage. After the specified time (transfer time in the table below), kill the animal, reshorn the original skin piece, and
The implanted tissue was removed and placed in a 4% formaldehyde solution for calcium quantification, which was rinsed to reduce the amount of hair surrounding the surgical site. Quantification of calcium was carried out as follows. The transplanted tissue was fixed in 4% formaldehyde acetate buffer at pH 5.5. After cutting out the histological samples, the remaining portion of the original 2 x 3 cm explant was dried in a desiccator at 50°C for 24 hours. The dried sample was weighed accurately to 0.00001 g on an analytical scale and then added with 3 ml of a 1:1 solution of 16N nitric acid and 30% hydrogen peroxide.
The cells were extracted at 23°C for 24 hours or until the tissue was no longer visible. The extracts were measured for calcium using atomic absorption spectrophotometry. Bockman2380 atomic absorption for Ca
Atomic Absorption) The sensitivity of a spectrophotometer is
It was below 0.092 ppm. The linear range of Ca is 4ppm
It was hot. The sample was diluted with 0.2% lanthanum chloride and
The interference by elements forming stable oxygen salts with Ca was reduced. The following examples illustrate the invention. EXAMPLE Unfixed, unprocessed natural bovine pericardial tissue was carefully washed in buffered saline as described below. The washed tissue was immersed in a 0.1% aqueous solution of N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate at 4°C for 22 hours. The zwitterionic detergent solution contains 1.0g of N-hexadecyl-
N,N-dimethyl-3-ammonium-1-propanesulfonate was prepared by dissolving N,N-dimethyl-3-ammonium-1-propanesulfonate in sufficient buffered saline as described below to make one 0.1% w/v detergent solution. The buffered saline was a 0.05M acetate buffer containing 0.35% by weight of sodium acetate and 0.4% by weight of a 2M acetic acid solution along with 0.05% by weight of sodium chloride. The pH of buffered saline is 1N acetic acid solution.
Adjusted to 5.5. The final detergent solution had a pH of 5.5. After exposure to the detergent solution, the tissue was rinsed with the above buffered saline and adjusted to pH 5.5 with the above 0.05 M acetate buffer.
The cells were fixed by placing them in a 0.5% glutaraldehyde solution buffered for 7 days. The results of the above animal translocation test are shown in the table below. Pre-fixation samples are obtained according to the above examples. The difference is that the post-fixation sample is first subjected to a fixation step, then rinsed and then soaked in detergent. Tests using tissue from Hancock T-6 show that prior art treatments are less effective than the present invention in delaying the onset of calcification and less effective in inhibiting the rate of calcification. This is a sample for comparison. Hancock heart valve tissue was obtained from a commercially available Hancock Extra-corporeal heart valve labeled as: Pericardial Heart Valve Processing Hankock T6 Form: T405. Size: 25mm. Serial number: A8682 Manufacture date: October 20, 19982. The Hankostok heart valve tissue has undergone SDS post-fixation treatment in US Pat. No. 4,323,358. 【table】

Claims (1)

[Claims] 1. N-decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate and N-hexadecyl-N,N-dimethyl- before, during or after fixing the transplantable tissue. 3-Ammonium-
A method for producing a transplantable tissue with delayed mineralization, characterized in that said transplantable tissue is immersed in an aqueous solution of a surface-active betaine selected from the group consisting of 1-propanesulfonates. 2. The method of claim 1, wherein the tissue is a tendon, heart valve tissue, dura mater, ligament, or pericardium. 3. The method according to claim 1 or 2, characterized in that the fixation is by glutaraldehyde. 4. A method according to claim 3, characterized in that said fixation is by a 0.01-2% by weight aqueous solution of glutaraldehyde buffered to a pH of about 4.5-8.5. 5. The method of any one of claims 1 to 4, wherein the aqueous solution contains about 0.01 to 2% by weight of the surface active betaine. 6. Treating the tissue with the surfactant at least about 4
6. A method according to any one of claims 1 to 5, characterized in that the contact is carried out for a period of time. 7 The surface active betaine is N-hexadecyl-
7. The method according to any one of claims 1 to 6, characterized in that it is N,N-dimethyl-3-ammonium-1-propanesulfonate. 8. The tissue is washed with buffered saline after the immersion step and before storage or fixation in glutaraldehyde. The method described in Section 1.