RU2633544C1 - Method for manufacture of valve-containing conduit from cattle jugular vein - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for manufacture of a valve-containing conduit from the cattle jugular vein involves biomaterial selection, mechanical cleaning and washing, followed by its preservation with an epoxy crosslinking agent. At the stage of biomaterial selection and at the final inspection of the finished product, an endoscopic examination of the internal structures of the conduit with a functional hydraulic test of valve closing function is carried out. After the conservation stage, a histological check of the structure of the biomaterial of each product is carried out and then subjected to an anticalcification treatment with a 1.2-5% solution of a bisphosphonic compound containing a free amino group at a temperature of 35-40°C for 4-8 hours.

EFFECT: method of manufacture of a valve-containing conduit provides creation of a xenovenous conduit to replace the outflow of the right ventricle, valve and pulmonary artery in children with a high reliability of the bioprosthesis valve apparatus and a reduced risk of dysfunctions due to cytotoxic reactions and calcification.

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Description

The invention relates to medicine, namely to reconstructive surgery of congenital heart defects, and can be used in the manufacture of bioprostheses to replace the output section of the right ventricle, valve and trunk of the pulmonary artery in children.

Yuguo Ichikawa (Ichikawa Y. A New RV-PA Conduit) conduits based on the jugular vein of cattle (YRS cattle), namely its segments containing a tricuspid valve, preserved with polyglycidyl ether of glycerol (Denacol-313). with a Natural Valve Made of Bovine Jugular Vein. // ASAIO Journal. - 1992. - v. 38. - p. M266-M270.). Subsequently, he and his co-authors substantiated in the experiment the expediency of its use for replacing the excretory section of the right ventricle, valve, and pulmonary artery trunk (Ichikawa Y., Noishiki Y., Soma T., Ishii M., Yamamoto K., Takahashi K., Mo M ., Kosuge T., Kondo J., Matsumoto A. A new antithrombogenic RV-PA valved conduit. // ASAIO Journal. - 1994. - v. 40 (3). - p. M714-M718; Ichikawa Y., Noishiki Y., Kosuge T., Yamamoto K., Kondo J., Matsumoto A. Use of a bovine jugular vein graft with natural valve for right ventricular outflow tract reconstruction: a one-year animal study. // J. Thorac. Cardiovasc. Surg. - 1997. - v. 114. - p. 224-233.).

Y. Ichikawa et al. Proposed using Denacol-313 as a preservative for biomaterial, since the main component of this preparation, polyglycidyl ether of glycerol, in comparison with aldehyde preservatives, gives biotissue improved elastic elastic properties and low calcium binding activity. In addition, in order to increase thrombotic resistance, the authors proposed to immobilize heparin on the biomaterial - either covalently, due to direct binding of the glycerol polyglycidyl ester remaining free after cross-linking by epoxy groups, or through the formation of complexes with protamine sulfate (followed by release of heparin into the bloodstream).

The disadvantages of the method proposed by Y. Ichikawa et al. Are:

1) The use of technical (used in the manufacture of adhesives, paper and rubber) epoxy preparation Denacol-313, containing in large quantities, in addition to polyglycidyl ether glycerol, three impurities, two of which are chlorine-containing and, therefore, potentially toxic;

2) Additional antithrombotic modification with heparin is inappropriate for this type of bioprosthesis, because thrombotic complications practically do not occur in the intracardial position of the pulmonary valve. At the same time, the high frequency of calcification of biomaterials in children for whom this medical product is intended is the main limiting factor in the use of bioprostheses in this age group. Therefore, it would be advisable to include anti-calcium treatment in the technology.

Since 2001, Medtronic Inc. produces a non-patented commercial product - Contegra® conduit - made from valve-containing cattle segments preserved with glutaraldehyde and stored until use in a mixture of 1% glutaraldehyde and 20% isopropyl alcohol. Conduit Contegra® is a tubular segment of UW cattle up to 15 cm long, containing inside, at the level of approximately the middle of the length of the tubular part, a tricuspid natural valve.

To date, experimental and clinical studies have proved that cattle JS is an adequate material for the conduit "right ventricle - pulmonary artery" for several reasons:

- the variability of the diameter of UW cattle - from 12 to 24 mm - anatomically allows its use in children in a wide age range - from the neonatal period (12 mm) to 18 years (24 mm);

- the wall thickness of the cattle's UE is comparable in thickness to the wall of the pulmonary artery in children;

- the venous valve is designed to function in the conditions of the right departments and is competent at low blood pressures; while its flaps are very thin and elastic, which does not create a gradient on the valve (Carrel T. Bovine valved jugular vein (Contegra ™) to reconstruct the right ventricular outflow tract. // Expert Rev. Medical Devices. - 2004. - v. 1 (1). - p. 11-19).

The disadvantages of Contegra® Conduit, widely used in clinical practice, are:

1) a large number of long-term complications caused by stenosis of a distal anastomosis (40-60% in the structure of dysfunctions);

2) high frequency of calcification of the wall (tubular part) of the conduit (17-25% of the removed conduits are calcined) (Yuan S. - M. The Contegra Valved Bovine Conduit: A Biomaterial for the Surgical Treatment of Congenital Heart Defects. // Arq. Bras. Cardiol. - 2012 .-- v. 99 (6). - p. L159-1165 .; Urso S., Rega F., Meuris B., Gewillig M., Eyskens B., Daenen W., Heying R., Meyns B. The Contegra conduit in the right ventricular outflow tract is an independent risk factor for graft replacement. // Eur. J. Cardio-thorac. Surg. - 2011. - v. 40. - p. 603-609.).

These disadvantages are due to the use of glutaraldehyde as the main preservative of biomaterial. When preparing a bioprosthesis for implantation, glutaraldehyde is poorly washed from the tissue of the prosthesis, which leads to cytotoxic effects at the recipient tissue / bioprosthesis boundary, accompanied by the formation of a hyperplastic neointima with severe cellular infiltration. In addition, it is well known that chemical bonds formed by glutaraldehyde in biomaterial are nucleation centers of calcium phosphates from blood plasma, followed by the growth of hydroxyapatite crystals, that is, they cause calcification of the bioprosthesis.

From the existing level of technology there is known a method of manufacturing a valve-containing conduit from cattle core (CN No. 104095692 (A), IPC A61F 2/06), which includes the following stages:

1. The selection of animal donors: at the age of 2-5 years, healthy, not in contact with sick animals;

2. Isolation and excision with a jugular vein knife along the entire length for 1 minute;

3. Repeated washing with water until the rinsing liquid is completely discolored, then temporary (no more than 3 hours) storage in saline at a temperature of 4-10 ° С;

4. Initial treatment - cutting off adipose and connective tissue from the outer surface of the vein, carefully avoiding injury to the vessel wall;

5. Rinsing in a balanced salt solution with gentle stirring for 6 hours with a change of solution every hour;

6. Preservation of 0.6% glutaraldehyde for 2 days at a temperature of 4-12 ° C, then 0.3% with amyl dialdehyde for a week;

7. The formation of the outer shell of polyester fabric.

The disadvantages of the method according to CN patent No. 104095692 (A) are the above disadvantages related to Contegra® conduit, since glutaral and amyl dialdehydes are used as the main preservative. In addition, prolonged (3 hours) washing in water leads to tissue swelling, and the total long washing time before preservation - 9 hours (3 hours in water and 6 hours in saline) - may cause the onset of autolytic processes with a violation of structural integrity biomaterial in the pre-conservation period.

The lack of methods for the manufacture of Contegra® Conduit and Conduit according to CN patent No. 104095692 (A) is also the absence of a stage for monitoring the state of the valve apparatus both at the stage of raw material selection and at the stage of output inspection of the product. Given the small diameter and significant length of the proximal (prevalvular) and distal (supravalvular) tubular parts - 6-8 cm, an adequate assessment of the valve with the naked eye is not possible. Assessment of valve function using a hydrodynamic stent for this type of bioprosthesis is a destructive control method and, accordingly, also impossible.

In addition, a disadvantage of the known methods (proposed by Y. Ichikawa, Contegra®, CN Patent No. 1,04095692) is the lack of anti-calcium treatment of biomaterial in the manufacturing technology of conduit. It is known that tissues containing a large amount of elastin, to which the wall of cattle ’s belong, accumulate calcium in a much larger amount than collagen-containing ones - pericardium and valve flaps. Moreover, if the calcification of collagen can be significantly inhibited by replacing the aldehyde crosslinking agents with epoxy ones, then the calcification of elastin does not depend on the choice of a crosslinking agent. The risk of calcification of xenovenous conduit in the postoperative period is all the more so because the age group for which it is intended (children, including the first years of life) is most susceptible to this complication. Accordingly, for the prevention of calcification of biomaterial, additional anti-calcium treatment is necessary.

A known method of anticalcium treatment of biomaterials previously preserved with epoxy preservatives by placing bioprostheses in a 0.05-1.0% solution of diphosphonate containing an amino group in the structure for 3-5 hours at a temperature of 18-25 ° C, followed by washing and placing for storage in a sterilizing solution (RU 2374843, IPC A01N 1/02).

The disadvantage of this method is a number of points that impede the covalent interaction of the amino group of diphosphonate (bisphosphonate) and the residual free epoxy group that did not bind to the biomaterial during the conservation process (the technology according to the corresponding patent is based on this mechanism):

1) Covalent binding reactions proceed faster with increasing temperature. When processing biomaterial, very high temperatures are impossible, but 37-40 ° C is quite acceptable. In this regard, the temperature of 18-25 ° C, as claimed in patent No. 2374843, is not optimal.

2) An increase in the concentration of the immobilized substance with the remaining unchanged parameters also increases the reaction rate, and therefore 0.05-1.0% concentration of the solution of diphosphonate according to patent No. 2374843 is not optimal.

The technical result of the invention is the creation of xenovenous conduit to replace the output section of the right ventricle, valve and trunk of the pulmonary artery in children, which has a high reliability of the valve apparatus of the bioprosthesis, as well as a reduced risk of dysfunctions due to cytotoxic reactions and calcification.

This result is achieved by improving the methodology for the manufacture of conduit from cattle core, including multi-stage quality control of biomaterial, as well as its multi-stage chemical processing. For the incoming control of raw materials, an endoscopic study of the morphological features of the wall of the cattle core in the sub- and supravalve part, as well as the quality of the casement device, is used. At this stage, cattle core segments are rejected that have xenoven wall surface defects, a two- or four-leaf valve, uneven area, insufficient coaptation, leaflet structure uneven in thickness, or other internal defects that can be detected by endoscopy. In this case, a hydraulic functional test is carried out: the tubular part is pinched with any “soft” instrument in the proximal part itself, the segment is filled with sterile saline solution, and then the clamp is removed. At this point, the valve should close and retain the fluid filling the distal section. Such a valve is considered competent. Otherwise (if the valve is incompetent), the segment is also subject to rejection. The segments of the livestock of cattle that have passed the control stage are subject to final preparation followed by preservation with a 5% buffer solution of an epoxy crosslinking agent for 14 days, with the solution being replaced with a fresh one after 24 hours from the time of preservation. The duration of the period from the moment of collection of UW cattle to the beginning of preservation should not exceed 6 hours. After 14 days, UW cattle are subjected to histological control, for which 0.5-1 cm of the vein wall is cut from the distal and proximal edges, standard wiring and staining are performed followed by evaluation of drugs by light microscopy. Criteria for rejecting xenovenous conduits at this stage are: tissue edema, loosening and fragmentation of collagen and elastin elements, the presence of unwanted tissue elements (excessive vascularization, smooth muscle bundles, inflammatory cell infiltration, etc.), pathological inclusions (e.g. finely divided calcium) . Conduits that underwent histological control are subjected to anticalcium treatment by covalent immobilization of a bisphosphonic compound containing a free amino group in the structure. As the bisphosphonic compound, pamidronic, or non-hadronic, or 2,2-carboxyethylaminoethidronic acid or salts thereof are used. The treatment is carried out by immersion of segments of the cattle ores in a 1.2-5% solution of bisphosphonate for 5-8 hours at 37 ° C. It should be borne in mind that the lower the concentration of the bisphosphonate solution, the longer the exposure should be in it, and vice versa. After processing, a final endoscopic control of the xenovenous conduit with a functional breakdown for the closure function is performed, re-evaluating the morphology of the inner surface of the xenoven wall and the sash apparatus. Particular attention is paid to the hydraulic functional test, as it is known that under the action of a crosslinking agent, the thin valves of the venous valve can shrink, deform, as a result of which the valve loses its competence. Such conduits are rejected. Conduits that have passed endoscopic inspection are sealed in a storage solution (biocidal preservative that does not contain crosslinking agents).

The invention is illustrated by drawings:

FIG. 1A, 1B. Verification of visual inspection data of segments with the naked eye. The valve-containing segment of the jugular vein of cattle cut parallel to the longitudinal axis. A - tricuspid valve; B - bicuspid valve with reduced wings.

FIG. 2. Endoscopic picture of a closed xenovenous valve. AB — variants of defects of the venous valve: A — atresia of one of the valves; B - lack of area of one of the wings; In - the excess length of the free edge of the two valves of the venous valve. G - competent 3-leaf valve with a homogeneous structure and anatomically correct arrangement of the valves.

FIG. 3. Microscopic structure of the xenovenous wall: A - normal structure, tight packing of collagen and elastin fibers (hematoxylin-eosin staining); B - pronounced edema of the biomaterial, impaired perception of the dye (hematoxylin-eosin staining). Ref. SW. × 400.

The following are examples of the implementation of the proposed method.

Example 1

The valve-containing segments of the jugular vein of cattle, selected at the meat processing plant and cleaned according to existing methods, are evaluated visually with the naked eye for the presence of a 3-leaf valve. At this stage, the xenovenous segments only 30% of the xenovenous segments containing the 3-leaf valve turned out to be potentially suitable for creating conduit (Fig. 1A). The remaining segments contained a 2- or 4-leaf valve; the valves were located at different levels or significantly varied in area (Fig. 1B). Primarily selected segments are viewed using an endoscope (Hopkins Telescope 45 °, Karl Storz, Germany). When visualizing pay attention to the number of valves, their anatomical structure and structure, axisymmetry and quality of closure (conducting a hydraulic test).

For endoscopy, the xenovenous valve-containing segment is filled with a sterile 0.9% sodium chloride solution and squeezed with a soft clip with protected branches in the most proximal (subvalvular) part. After the introduction of the endoscope, it is advanced in the direction of the valve so as to provide the best visualization of the wall and the sash. After obtaining and documenting the image of the valve (or detected wall defect), the clip is removed from the subvalvular part and the locking function of the valve is evaluated. A valve is considered competent if it remains closed and holds a column of fluid above the valve.

The results of an endoscopic assessment of tricuspid valves missed during the visual inspection stage revealed various defects in almost 50% of the valves: the area of one or several valves (insufficient for full closure) (Fig. 2A), anatomical openings (Fig. 2B), or defects in the free edge of the valves ( Fig. 2B). Thus, no more than 15% of the feedstock can be used to create conduits. These are segments having a sufficient length of the sub- and supravalvular parts, a competent 3-leaf valve with a homogeneous structure and anatomically correct arrangement of the valves (Fig. 2G).

Segments that have successfully passed the input control are subjected to preservation with an epoxy crosslinking agent according to existing methods, namely, a 5% solution of ethylene glycol diglycidyl ether prepared in phosphate buffer (pH 7.4).

After the end of the conservation period (14 days), histological monitoring of conduits is performed. For this, 0.5 cm of the most distal and most proximal parts of the xenovenous wall is cut, standard histological wiring and block filling are performed, using a microtome, standard serial sections of 4 μm thickness are obtained, which are stained with hematoxylin-eosin and picrofuxin according to Van Gieson. When viewing drugs, the state of fibrous structures and the presence / absence of foreign tissue and / or inorganic inclusions are revealed. The normal structure of the xenovenous wall does not contain foreign inorganic inclusions, muscle elements; collagen and elastin fibers are tightly packed, without signs of swelling and loosening (Fig. 3A). The most common criterion for rejection (5-10%) is tissue edema (Fig. 3B).

Conduits who received a positive conclusion on the results of histological control are subjected to anticalcium modification with pamidronic acid. The treatment is carried out in a 1.2% aqueous solution of pamidronic acid at a temperature of 37 ° C for 8 hours. After that, the conduits are washed from unbound bisphosphonates in a 0.9% NaCl solution for 20 minutes with a double change of solution and a final endoscopic control is performed with functional hydraulic breakdown as described above.

Conduits who have a positive conclusion on the results of endoscopic control are placed for storage in a solution of a biocidal composition that preserves the sterility of the product for a long time.

Example 2

All stages of the manufacture of conduits were carried out analogously to example 1, but for anti-calcium modification as a bisphosphonic compound selected non-uronic acid. The treatment is carried out in a 2.5% aqueous solution of non-hadronic acid at a temperature of 37 ° C for 6 hours

Example 3

All stages of the manufacture of conduits are performed analogously to example 1, but 2,2-carboxyethyl-aminoethidronic acid is used for the anti-calcium modification. The treatment is carried out in a 5% aqueous solution of 2,2-carboxyethyl-aminoethidronic acid at a temperature of 37 ° C for 5 hours

The amount of immobilized bisphosphonates when using the above acids for calcium modification is presented in table. one.

For a comparative assessment of the effectiveness of the anticalcium modification, conduit samples were prepared in accordance with the method according to the patent RU 2374843. For this, cattle core was preserved with a 5% solution of diglycidyl ether of ethylene glycol. After 14 days of preservation, the biomaterial was washed in a 0.9% NaCl solution for 1 h with a three-fold change of solution and placed for 4 h at room temperature in 0.9% solutions of pamidronic or 2,2-carboxyethylaminoethidronic acid, prepared on phosphate buffer at pH 7.4. After this time, the bioprostheses were washed in a 0.9% NaCl solution for 40 minutes with a two-time change of the solution and placed in a 1% solution, for example, in 2-bromo-2-nitropropane-1,3 diol.

The number of immobilized bisphosphonates for anticalcium modification (examples according to the claimed method and patent RU 2374843) are presented in table. one.

The biological effectiveness of anti-calcium treatment was assessed using the standard model of accelerated calcification by subcutaneous implantation of the test material to Vistar rats for 60 days, followed by a quantitative analysis of the calcium content in the tissue. As control samples, biomaterial samples preserved with glutaraldehyde (HA) and ethylene glycol diglycidyl ether (DEE) without calcium treatment were used. The smallest accumulation of calcium in the samples of the xenovenous wall was achieved when it was preserved with an epoxy compound (diglycidyl ether of ethylene glycol, DEE) with the immobilization of 2,2-carboxyethyl aminoethidronic, pamidronic and non-iridronic acids according to the proposed method (Table 2).

Claims (3)

1. A method of manufacturing a valve-containing conduit from the jugular vein of cattle, including the selection, mechanical cleaning and washing of the biomaterial, followed by its preservation with an epoxy crosslinking agent and anti-calcium treatment, characterized in that at the stage of biomaterial selection and during the final inspection of the finished product, an endoscopic examination of the internal structures of a conduit with a functional hydraulic breakdown on the valve closure function; after the conservation phase, histological monitoring of the structure of the biomaterial of each product, and then subjected to anti-calcium treatment with a 1.2-5% solution of a bisphosphonic compound containing a free amino group at a temperature of 35-40 ° C for 5-8 hours. 2. The method according to p. 1, characterized in that a functional test for the valve closure function is carried out under endoscopic control by filling with liquid a xenovenous valve-containing segment, squeezed in the most proximal part with subsequent removal of the clamp and visual control of the valve closure. 3. The method according to p. 1, characterized in that the bisphosphonic compound is selected from the range of pamidronic, non-iridronic, 2,2-carboxyethyl-aminoethidronic acids or their salts.

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