Method for the preparation of biological tissue for dry use in an implant

Abstract

A method of preparing biological tissue for use as a component of an implant, in particular as part of a vascular implant, more particularly as part of a heart valve prosthesis, which can be implanted by a catheter. The biological tissue is decellularized using a detergent, which includes surfactin and deoxycholic acid (DCA).

Claims

1. A method of preparing biological tissue for use as a component of an implant, the method comprising decellularizing biological tissue with a detergent comprising surfactin and deoxycholic acid (DCA).

2. The method according to claim 1, wherein the method is a method of preparing biological tissue for use as a vascular implant.

3. The method according to claim 1, wherein the detergent comprises about 0.06% surfactin.

4. The method according to claim 1, wherein the method is a method of preparing biological tissue for use as a heart valve prosthesis.

5. The method according to claim 1, wherein the detergent comprises about 0.5% DCA.

6. The method according to claim 5, wherein the detergent comprises about 0.06% surfactin.

7. The method according to claim 1, the method further comprising cross-linking the decellularized biological tissue with a cross-linking agent.

8. The method according to claim 7, wherein the cross-linking agent is an aldehyde-containing solution.

9. The method according to claim 7, the method further comprising performing a structural stabilization step on the decellularized tissue before or after cross-linking.

10. The method according to claim 9, wherein the structure stabilization step is performed on the decellularized tissue after cross-linking.

11. The method according to claim 9, wherein the structure stabilization step comprises exposing the decellularized tissue to at least two different solutions, wherein one solution comprises polyethylene glycol and another solution comprises glycerol.

12. The method according to claim 8, wherein the cross-linking agent is a glutaraldehyde or a formaldehyde containing solution.

13. The method according to claim 8, wherein the cross-linking agent is glutaraldehyde.

14. The method according to claim 11, further comprising drying the tissue in a climate chamber by reducing relative humidity.

15. The method according to claim 11, further comprising drying the tissue in a climate chamber by reducing relative humidity from 95% to 10% over 12 hours at 37° C.

16. The method according to claim 11, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having a mean molecular weight between 200 g/mol and 600 g/mol; and a second solution is an aqueous solution of polyethylene glycol having a mean molecular weight between 200 g/mol and 6,000 g/mol and glycerol.

17. The method according to claim 11, wherein the at least two different solutions comprise three different solutions.

18. The method according to claim 17, wherein the first solution comprises glycerol, the second solution comprises polyethylene glycol having a mean molecular weight of or about 200 g/mol and the third solution comprises polyethylene glycol having a mean molecular weight of or about 400 g/mol, wherein the tissue is exposed to the first and second solutions before the third solution, and wherein the mean molecular weight of the polyethylene glycol is higher in the third solution than the second.

19. The method according to claim 1, the method further comprising performing a structural stabilization step on the decellularized tissue.

20. The method according to claim 1, further comprising exposing the decellularized tissue to at least two different solutions, wherein one solution comprises polyethylene glycol and another solution comprises glycerol.

21. The method according to claim 20, further comprising drying the tissue in a climate chamber by reducing relative humidity.

22. The method according to claim 20, further comprising drying the tissue in a climate chamber by reducing relative humidity from 95% to 10% over 12 hours at 37° C.

23. The method according to claim 20, wherein a first solution comprises polyethylene glycol having a mean molecular weight between 200 g/mol and 400 g/mol.

24. The method according to claim 20, wherein the at least two different solutions comprise three different solutions.

25. The method according to claim 24, wherein the polyethylene glycol in the third solution has a mean molecular weight of 300 to 1500 g/mol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an electron microscopic image of a porcine pericardial tissue according to Example I.

(2) FIG. 2 shows an untreated, cross-linked pericardium, as a reference for comparison to the pericardial tissue shown in FIG. 1.

(3) FIG. 3 is a table summarizing the amount of free protein measured after decellularization of porcine pericardium with surfactin or surfactin and DCA over time compared to native tissue.

(4) FIG. 4 is a graphical depiction of the amount of free protein after decellularization of porcine pericardium with surfactin or surfactin and DCA over time compared to native tissue.

(5) FIG. 5 is a graphical depiction of the amount of residual free protein after decellularization of porcine pericardium with surfactin or surfactin and DCA over time compared to native tissue.

(6) FIG. 6 is a table summarizing the amount of residual DNA measured after decellularization of porcine pericardium with DCA or surfactin and DCA compared to native tissue.

(7) FIG. 7 is a graphical depiction of the amount of residual DNA after decellularization of porcine pericardium with DCA or surfactin and DCA compared to native tissue.

(8) FIG. 8 is a graphical depiction of the percentage of residual DNA after decellularization of porcine pericardium with DCA or surfactin and DCA compared to native tissue.

(9) FIGS. 9A-C are graphical depictions of the saturation of glutaraldehyde crosslinked porcine pericardium with 30% glycerol (FIG. 9A), 40% PEG200 (FIG. 9B), and 40% PEG400 (FIG. 9C).

(10) FIG. 10 is a table summarizing the saturation time of porcine pericardium with different stabilizers.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(11) The invention will be explained in greater detail on the basis of exemplary embodiments and a comparison—depicted in FIGS. 1 and 2—of a dried biological tissue according to one exemplary embodiment of the invention with the prior art.

Example I

Preparation and Drying of Pericardial Tissue

(12) Example I discloses an embodiment of the method according to the invention for the preparation of porcine pericardium with subsequent drying. FIG. 1 shows a porcine pericardial tissue in the dried state after a treatment according to example I. A sealing and stabilization of the surface structure resulting from the treatment with the stabilizers is shown very clearly in the electron microscopic image. The untreated reference tissue shown in FIG. 2 comprises exposed collagen structures, which are present in the unsealed state.

(13) First, a pericardium is removed from a freshly slaughtered pig in a slaughterhouse and is stored in a solution of 0.9 weight % of sodium chloride, which contains penicillin and/or streptomycin, for 2 hours at a temperature of 4° C. In the next step, fat and connective tissue are separated from the pericardial tissue in moist conditions (solution of 0.9 weight % of sodium chloride), and the pericardial tissue is trimmed to the proper size.

(14) Next, the tissue is rinsed (100 ml solution of 0.9 weight % of sodium chloride, accompanied by gentle movement), cross-linked (48 hours in 100 ml solution of 0.6 weight % of glutaraldehyde solution (glutaraldehyde in buffered saline solution at 4-8° C. (DPBS solution from the company Lonza; DPBS w/o Ca++/Mg++; Art. No. 17-512)), wherein this solution then acts for 14 days at room temperature and is replaced with a similar, fresh solution once every 48 hours), and is then rinsed again (rinsed for 10 min in 100 ml solution of 0.9 weight % of sodium chloride at 37° C., accompanied by gentle movement, repeated 6 times).

(15) The thusly treated, cross-linked biological tissue is then subjected to an embodiment of the dimensional and structural stabilization step according to the invention.

(16) In this embodiment, the biological tissue is subjected to a first rinsing for 10 min in 100 ml of an aqueous solution of 25 vol % of polyethylene glycol (containing polyethylene glycol having a mean molecular weight of 400 g/mol) at 37° C., repeated 3 times.

(17) Next, the biological tissue is exposed to a second solution mixture for 2 hours at a temperature of 37° C. In this embodiment of the invention, the second solution mixture comprises an aqueous solution containing polyethylene glycol having a mean molecular weight of 400 g/mol in a concentration of 15 vol % of polyethylene glycol having a mean molecular weight of 6000 g/mol in a concentration of 10 vol % and glycerol in a concentration of 5 vol %.

(18) The thusly stabilized biological tissue is then dried in a desiccator for 24 hours, using a silica gel as the desiccant. The thusly obtained, dried, biological tissue can either be further processed or stored in the desiccator.

(19) Within the scope of this application, a desiccator refers to a closed vessel, which contains an active desiccant and has minimal humidity in the interior thereof.

(20) As an alternative, the drying can also take place in a climate-controlled chamber having an adjustable temperature and humidity.

Example II

Preparation and Drying of Pericardial Tissue

(21) Similar to example I, a pericardium is removed from a pig, stored for 2 hours at a temperature of 4° C. in a solution of 0.9 weight % of sodium chloride, which contains penicillin and/or streptomycin, is prepared in moist conditions (solution of 0.9 weight % of sodium chloride) with removal of fat and connective tissue, is trimmed to size, and is subsequently rinsed with 100 ml of a solution of 0.9 weight % of sodium chloride, accompanied by gentle movement.

(22) The thusly obtained pericardium is then subjected to gentle decellularization and subsequent cross-linking. The following steps are carried out: decellularization in 100 ml surfactin/deoxycholic acid solution (0.06 weight % of surfactin and 0.5 weight % of deoxycholic acid in a solution of 0.9 weight % of sodium chloride) for 20 hours at 37° C., rinsing with 100 ml of a solution of 0.9 weight % of sodium chloride (6 times, accompanied by gentle movement, for 10 min) treatment with a DNase solution for 12 hours at 37° C. rinsing with 100 ml of a solution of 0.9 weight % of sodium chloride (8 times, accompanied by gentle movement, for 10 min) rinsing with 100 ml of a solution of 70 vol % of ethanol (once, for 10 min) rinsing with 100 ml of a solution of 0.9 weight % of sodium chloride cross-linking with glutaraldehyde (48 hours in 100 ml of a solution of 0.6 weight % of glutaraldehyde (glutaraldehyde in buffered saline solution at 4-8° C. (DPBS solution from the company Lonza; DPBS w/o Ca++/Mg++; Art. No. 17-512)), wherein this solution then acts for 14 days at room temperature and is replaced with a similar, fresh solution once every 48 hours) rinsing with 100 ml of a solution of 0.9 weight % of sodium chloride (6 times, accompanied by gentle movement, for 10 min)

(23) In this embodiment of the invention, the thusly produced, decellularized and cross-linked pericardial tissue is stabilized in three steps. First, the tissue is rinsed with 100 ml of an aqueous solution of 25 vol % of polyethylene glycol (containing polyethylene glycol having a mean molecular weight of 400 g/mol at 37° C., 3 times for 10 min). Next, the tissue is exposed to an aqueous solution containing 20 vol % of polyethylene glycol having a mean molecular weight of 400 g/mol and 10 vol % of glycerol for 2 hours at 37° C., accompanied by gentle movement. This is followed by a treatment with an aqueous solution containing 20 vol % of polyethylene glycol having a mean molecular weight of 6000 g/mol and 10 vol % of glycerol, accompanied by gentle movement, at a constant temperature for 2 hours.

(24) The thusly stabilized biological tissue is then dried in a desiccator for 24 hours, using a silica gel as the desiccant, and is then further processed.

Example III

Stabilization and Drying of Cross-Linked Pericardial Tissue

(25) In the embodiment according to example III, already cross-linked porcine pericardial tissue is prepared (stabilized and dried) using the following method: rinsing with 100 ml of a solution of 0.9 weight % of sodium chloride (6 times, accompanied by gentle movement, for 10 min, at room temperature), rinsing with 100 ml of an aqueous solution of 40 vol % of glycerol (3 times, accompanied by gentle movement, for 20 min at 37° C.), placing the pericardial tissue in an aqueous solution containing 30 vol % of polyethylene glycol having a mean molecular weight of 400 g/mol and 10 vol % of glycerol for 2 hours at 37° C., accompanied by gentle movement, placing the pericardial tissue in an aqueous solution containing 30 vol % of polyethylene glycol having a mean molecular weight of 6000 g/mol and 10 vol % of glycerol for 2 hours at 37° C., accompanied by gentle movement, and drying in the desiccator for 24 hours, using silica gel as the desiccant.

Example IV

Decellularization of Pericardial Tissue Using a Mixture of Surfactin and DCA as Decellularizing Agent Significantly Reduces Residual Free Protein

(26) Free proteins in biological tissue are mostly intracellular proteins that are released when the cell membrane is destroyed. In contrast to the fibrous proteins in the extracellular matrix, free proteins can be extracted and quantified in solution after an extraction. The lower the amount of free proteins in the extraction solution, the better the quality of the decellularization.

(27) For analysis, punched (circle diameter 16 mm) and then freeze-dried tissue samples of porcine pericardium are placed in individual 2 ml reaction vessels for extraction. The dry mass is first determined on a precision balance. Extraction is performed in 1.2 ml DPBS (Dulbeccos phosphate buffered saline solution without Ca/Mg) at 37° C. for 72 hours under continuous agitation. After extraction, the samples are centrifuged at 13000 rpm for five minutes. The sample to be measured with the free proteins is taken from the upper part of the extraction solution.

(28) The amount of free protein before/after decellularization was measured by fluorescence labeling with the “Qubit protein assay kit” in the fluorometer “Qubit 2.0” (both from Invitrogen) according to the standard instructions. The amount of free proteins is related to the sample mass according to the following formula:
Free proteins (μg/mg)=measured concentration (μg/ml)×extraction volume (ml)/sample mass (mg)

(29) In order to investigate the influence of the combination of surfactin and deoxycholic acid on decellularization, porcine pericardium was examined on the amount of remaining free proteins in its native state as a reference and after decellularization in only 0.06% surfactin and the mixture of 0.06% surfactin and 1% deoxycholic acid (DCA). The surfactin concentration of 0.06% is lower than the solubility in water and higher than the critical micelle formation concentration, whereby the surface tension remains constant during decellularization. The results are shown in FIGS. 3-5.

(30) The measured values with only surfactin 0.06% show no big differences for the different times. However, they scatter more strongly at the shortest exposure time, which indicates inconsistent decellularization. With only 0.06% surfactin, on average only 55% of the soluble proteins are removed compared to native tissue.

(31) This can be significantly improved by using the combination surfactin 0.06%/DCA 1%, whereby more free proteins are removed with increasing decellularization time. After decellularization for 12 hours, about 47.10% of the free proteins remain in the tissue. After 24 hours, this value is reduced to 19.09%, and after 36 hours, to 16.43%. Thus, additional 28.10% of the free proteins are removed from the tissue during decellularization over 24 hours compared to 12 hours. In contrast, the value decreases by only 2.66% during further 12 hours.

(32) The mixture of surfactin and deoxycholic acid therefore allows effective decellularization of porcine pericardium within 24 hours.

Example V

Decellularization of Pericardial Tissue Using a Mixture of Surfactin and DCA as Decellularizing Agent Significantly Reduces Residual DNA

(33) Animal DNA in biological tissue used as implant material activates the immune system. This reaction promotes the tendency of porcine pericardium to calcify, contributing to the limited long-term stability of biological valve replacement materials. The reduction of residual DNA in animal tissue is therefore also a goal of decellularization. The smaller the amount of residual DNA the better the quality of decellularization.

(34) For analysis, punched (circle diameter 16 mm) and then freeze-dried tissue samples of porcine pericardium are placed in individual 2 ml reaction vessels. The dry mass is first determined on a precision balance. To determine the residual DNA, the samples are dissolved enzymatically using the enzyme Proteinase K (60° C. in 1000 μl TRIS reaction buffer at pH 8 with 40 μl enzyme solution). After complete dissolution of the tissue, the samples are centrifuged at 13000 rpm for five minutes. The sample with the residual DNA to be measured is taken from the upper part of the solution.

(35) The amount of residual DNA before/after decellularization was measured by fluorescence labeling with the “Qubit dsDNA HS kit” in the fluorometer “Qubit 2.0” (both from Invitrogen) according to the standard instructions. The amount of residual DNA is related to the sample mass according to the following formula:
Residual DNA (ng/ml)=measured value (ng/ml)×200 μl/sample volume (μl)×1040 μl/sample mass (mg)

(36) In order to investigate the influence of the combination of surfactin and deoxycholic acid on decellularization, porcine pericardium was examined on the amount of residual DNA in its native state as a reference and after 24 hours decellularization in only 0.5% and 1% deoxycholic acid (DCA), respectively, as well as the mixture of 0.06% surfactin and 0.5% deoxycholic acid. The surfactin concentration of 0.06% is smaller than the solubility in water and larger than the critical micelle formation concentration, whereby the surface tension remains constant during decellularization. The results are shown in FIGS. 6-8.

(37) The measured values with only deoxycholic acid show a reduction of the residual DNA compared to native tissue for both concentrations 0.5% and 1%. The reduction is greater at the higher concentration.

(38) By using the combination Surfactin 0.06%/DCA 0.5%, the residual DNA content can be further reduced by a factor of five compared to DCA 1%. After decellularization for 24 hours, the residual DNA content is only 0.22%. This is significantly lower than the amount of residual DNA when using deoxycholic acid alone.

(39) The mixture of surfactin and deoxycholic acid therefore allows effective decellularization of porcine pericardium within 24 hours.

(40) Given the exemplary results of the above example portion, the inventors unexpectedly found that a combination of surfactin and DCA for decellularization has a significant advantage over the use of surfactin or DCA alone. In particular, when decellularizing only with surfactin, more soluble proteins remain in the tissue than with a combination with DCA. When decellularizing only with DCA, more residual DNA remains in the tissue than in combination with surfactin. It has been thus surprisingly found that decellularization of biological tissue with a combination of surfactin and DCA permits particularly thorough removal of all these undesired remaining tissue components. Experimentally, it has been shown that a combination of surfactin and DCA produces a particularly good decellularizing agent which is better at removing unwanted tissue components than either agent alone.

Example VI

Stabilization of Biological Tissue with Glycerol and Polyethylene Glycol

(41) In order to stabilize the dimension and structure of biological tissue, the tissue is exposed to several solutions containing glycerol and polyethylene glycol (PEG). Measurements of the actual saturation time for different solutions were accomplished using Fourier transformed infrared spectroscopy (FTIR) combined with an attenuated total reflection (ATR) cell. Since only the tissue surface is considered in the measurement set-up, the saturation of the pericardial tissue over time can be obtained.

(42) Fourier transform infrared (FTIR) spectra were recorded using a Jasco FT/IR-460 Plus spectrometer equipped with a liquid nitrogen cooled solid-state detector (mercury cadmium telluride; MCT) and an attenuated total reflection (ATR) sampling accessory (MIRacle ATR, PIKE Tech). For CO.sub.2 correction the spectrometer was purged with a constant flow of nitrogen (0.1 bar). FTIR spectra were acquired from (4000-700) cm.sup.−1 with 2 cm.sup.−1 spectral resolution.

(43) Pericardial tissue was harvested, cleaned and cross-linked with glutaraldehyde. The glutaraldehyde cross-linked pericardial tissue was rinsed (2×10 min with saline solution; 1×5 min with purified water) and die-cut in samples circular in size with an 8 mm diameter. Excess water was removed by dipping on a filter paper (Sartorius, FT-3-208-090). Tissue samples were placed with rough side down on the bottom of a cylindrical plastic tube (6 mm diameter, 14 mm height), mounted through press fitting with a slightly wider cylindrical plastic tube and placed on the ATR-diamond (rough side facing the ATR-diamond). 50 μL of glycerol (30% (aqueous solution); 100%), respectively PEG (40% (aqueous solution); 100%), solution was added on top of the tissue sample in the tube. To ensure the maximum contact area between tissue and diamond the tube was fixated with the pressure arm of the spectrometer. FTIR spectra were recorded every 2 min during 1 h. For glycerol the 1140-950 cm.sup.−1 region was identified as suitable for peak analysis as well as the 1170-970 cm.sup.−1 region for PEG.

(44) The intensity of the absorption band increases with the saturation of the pericardial tissue. Thus, the peak area was determined and plotted as a function of the incubation time, whereas a normalization was done towards the last ten measurement values (total measurement time: 1 h) assuming that a saturation is reached. The progress of saturation was fitted with a 4-parameter-logistic-function using the Levenberg-Marquardt-Algorithm. The time of saturation (95%) is determined according to Equation 1, the deviation was calculated through error propagation as shown in Equation 2

(45) $\begin{array}{cc}t\left(y\right)={x_0\left(\frac{A_1-A_2}{y-A_2}-1\right)}^{1/p}& \mathrm{Equation}\left(1\right)\end{array}$

(46) Where: A.sub.1: lower asymptote A.sub.2 upper asymptote x.sub.0: point of inflection p: slope at the point of inflection y: normalized measurement data

(47) $\begin{array}{cc}\Delta t\left(y\right)=\frac{\partial t\left(y\right)}{\partial y}\Delta y& \mathrm{Equation}\left(2\right)\end{array}$

(48) Saturation as a function of incubation time is shown exemplary for glutaraldehyde cross-linked porcine pericardium in FIGS. 9-A-C, and the results for the saturation time are represented in FIG. 10. Exposure occurred at 37° C. while shaking.

(49) Experimentally, it was found that tissue saturation using stabilizing solutions containing glycerol and polyethylene glycol requires far less than 1 hour.