RU2362589C1 - Way of sterilisation of biological tissues for transplantation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely to transplantology and can be used at preparation of biological tissues with high transplantation properties. A way of sterilisation of biological tissues for transplantation by an admixture including peroxide of hydrogen, is performed in the packed kind without asepsis observance at +4°…+5°C within 48 hours using an admixture from hydrogen peroxide, bromine hydride and salicylic acid at a following parity of components, wt %: bromine hydride acid - 2.0; salicylic acid - 0.1; hydrogen peroxide - 3.0; distilled water - the rest.

EFFECT: working out of a way which provides reliable sterilising effect at preparation of the grafts received in unsterile conditions, does not demand long time and the special technics of observance of works in sterile premises, and excludes cleaning on sterilisation end.

1 tbl, 9 dwg, 1 ex

Description

The invention relates to medicine and can be used in the procurement of biological grafts used in traumatology - orthopedics and in other areas of reconstructive surgery.

A known method of sterilizing biotransplants with an antiseptic complex [1], which can be included in the composition of solution 9 of TSOLIPK, in Tyrode's solution or in 11% sucrose solution. For every 100 ml of these solutions, 200 thousand units of neomycin, 200 thousand units of polymyxin, 0.005 g of furazolidone and 0.1 g of pipolfen (or 0.2 g of sorbic acid) are taken. The sterilization time of biological tissues with an antiseptic complex at 37 ° C is 2 hours, at + 2 ° ... + 4 ° C - one day. Obviously, the composition of the above method is quite complicated. To date, it has been abandoned due to the practical absence of the above antibiotics and pipolfen.

A known method of sterilization of transplants with beta-propiolactone [1]. For every 100 ml of liquid medium, 2.14-2.67 g of sodium phosphoric acid or 4-6 g of sodium bicarbonate is taken (pH, respectively, 7.4-7.8-8.2). The sterilizing agent is injected with a syringe in such an amount that a 1% solution is obtained, after which the dishes with tissues are hermetically sealed, shaken and placed in a thermostat (37 ° C) for 2 hours. After the specified time in a sterile environment, the spent solution is discharged, the grafts are transferred to a sterile container, sealed and subjected to preservation. This method is highly effective and at one time was considered the most promising - in its simplicity and reliability. Unfortunately, the release of this valuable sterilant has been discontinued both in our country and abroad due to its supposedly mutagenicity.

A known method of sterilization of demineralized bone tissue [2] in a closed system under reduced pressure and constant shaking for 4 hours. The sterilizing medium consists of two volumes of peracetic acid (5%), one volume of ethanol and one volume of distilled water. The ratio of the sterilized bone mass to the sterilized solution is 1: 4. At the end of sterilization, the mass is washed at least three times with Sorensen buffer in an isotonic sodium chloride solution. The layout of the material thus obtained is carried out in glass containers under aseptic conditions. The material is preserved by lyophilization. The disadvantages of the method: a) work in a closed system with reduced pressure and mandatory, very long-term shaking, which requires the use of special equipment; b) the need for thorough washing of the sterilized material in a difficult to prepare buffer solution of sodium chloride; c) compliance with strict asepsis at all stages of material processing; d) the method is intended only for granular (crushed) tissues; it is not recommended for sterilization of monolithic grafts.

A known method (prototype) of sterilization of biological tissues with a mixture of hydrogen peroxide with formic acid [3]. In a clean glass flask, first pour 160 ml of perhydrol, then 80 ml of 85% formic acid. The mixture is thoroughly mixed and placed in a refrigerator with a temperature of + 4 ° ... + 6 ° C or in cold water for 1-1.5 hours. After which the mixture is diluted with distilled water at room temperature (18 ° ... 20 ° C) to 5 liters. In this form, an antiseptic solution is ready for use. The process of sterilization of plastic material is carried out in special sterilizers having an outlet valve at the bottom for draining the waste liquid. A mesh is placed at the bottom of the sterilizer, on which prepared grafts are placed. The fabrics on the mesh must be laid in such a way that they do not touch each other. After that, a freshly prepared antiseptic solution is poured into the sterilizer. After 30 minutes, it is drained, and the transplants are filled with physiological saline at room temperature for another 20-30 minutes. At the end of the washing process, the tissues are laid out in glass ampoules or bags and canned by lyophilization or freezing. It is important to note that all manipulations on the sterilization of biological transplants are carried out in a sterile box.

Despite the obvious suitability of the method and the availability of its working ingredients, it is not without drawbacks. So, the preparation of the antimicrobial mixture takes more than 1.5 hours, which increases the time for harvesting the grafts. For the "maturation" of such a mixture requires refrigeration, which complicates the technique. The method is impossible without a special device - a sterilizer with an exhaust valve. Finally, the method does not preclude secondary infection of the transplants, for example, at the time of their washing or during the placement of the transplants in a sterile container. This danger becomes especially real while harvesting a large number of transplants.

The technical result of the present invention is to simplify the method of preparing a working mixture of antiseptics, the exclusion of asepsis from the process of harvesting biological tissues and washing the latter upon completion of sterilization.

The method is as follows. Samples of bone, cartilage, tendon and other tissues prepared for sterilization are placed in individual plastic bags and filled without sterilization with aseptic mixture. For its preparation, the flask with distilled water is sequentially added for every 95 ml of volume: hydrobromic acid - 2.0; salicylic acid - 0.1; hydrogen peroxide - 3.0. The solution is thoroughly mixed and poured into packages with transplants so that 5-7 cm high free space remains above the solution. The bags are sealed by thermal pulse welding, shaken and transferred to a household refrigerator with a temperature of + 4 ° ... + 5 ° C. After 48 hours, the packets are removed from the refrigerator and freed of the working fluid either by puncture with a sterile thick needle or by cutting off a small part of the packet in the region of its angle. The puncture or cut-off place is treated with iodine tincture and brewed. To control sterility, 1 out of 10 harvested transplants, after cutting small fragments from it in several places, is sent to a bacteriological laboratory. The remaining grafts are subjected to conservation either at low temperatures, or by lyophilization.

The table shows:

The results of sterilization of biotransplants of the proposed method.

Note: + microflora growth

- lack of growth

The illustrations show:

Fig 1. The x-ray result of ectopic transplantation of demineralized and sterilized by the proposed method of bone allografts in rats on the 15th day after surgery. The graft deprived of the mineral base is not visible.

Fig 2. The x-ray result of ectopic transplantation of demineralized and sterilized by the proposed method, bone allografts in rats on the 60th day after surgery. A new bone has formed at the site of the former graft.

Fig 3. The x-ray result of orthotopic transplantation of demineralized and sterilized by the proposed method of bone graft in the defect of the radius of the rabbit on the 30th day after surgery. The defect of the radius and signs of the recovery process are visible.

Figure 4. The x-ray result of the orthotopic transplantation of a demineralized and sterilized bone graft by the proposed method into a defect of a rabbit radial bone on the 90th day after the operation. The integrity of the radius is fully restored.

Figure 5. Microphoto. The histological result of orthotopic transplantation of a sterilized bone graft into a defect in the rabbit radial bone 90 days after surgery. On the pictures: the newly formed bone tissue of the lamellar structure with somewhat expanded havers channels. Hematoxylin and eosin stain, X100.

6. X-ray result of surgical treatment of patient P., 13 years old, with a diagnosis of multichamber cyst of the proximal third of the diaphysis of the right humerus.

but). Before surgery;

b) 3 months after surgery;

at). 6 months after surgery;

d). 1.5 years after surgery.

Thus, unlike the prototype:

1. We offer a new sterilizing mixture of chemical antiseptics that provide a reliable sterilizing effect when harvesting biological tissues obtained under non-sterile conditions and used in transplantology. In addition to the bactericidal action, salicylic acid, which is part of the medium, has, as the literature suggests [4], membrane-sealing, that is, preserving collagen properties, and also promotes cell proliferation and the development of regenerative phenomena. In addition, it inhibits the spontaneous decomposition of hydrogen peroxide.

2. The preparation of the proposed mixture of antiseptics in contrast to the prototype does not require a long time, which is important when harvesting a large number of transplants.

3. Sterilization of transplants in the proposed method is carried out in closed systems that do not require special equipment and compliance with work in a sterile room (box).

4. In the proposed method, the washing of the grafts after their sterilization is excluded, since the residual medium does not cause them unwanted damage.

To confirm the above, we present the results of experimental studies.

1. The sterilizing activity of the method

For the experiments, we used tissue samples that were most infected under natural conditions: fragments of skin grafts obtained from donor corpses stored at room temperature for 12-18 hours, as well as tail fragments of rats killed after various experiments. In total, 200 biological test objects infected prior to sterilization with mixed microflora were studied, in which they were most often found: staphylococcus (97.2%), spore bacillus (70.4%), mold (60.8%), E. coli (52 , 7%), streptococcus (20.9%), protea (19.1%), Pseudomonas aeruginosa (10.9%), sarcinol (2.7%), yeast-like fungi (0.9%), diphtheroids (0 , 45%). The sterilization results are shown in table 1.

As can be seen from the table, the antiseptic complex proposed in the method provided reliable results of sterilization of infected test objects after 48 hours of incubation in a refrigerator at + 5 ° C. The established fact allowed us to proceed to the next stage - the study of the bioplastic properties of bone grafts prepared using the proposed method .

2. The study of osteoinductive activity of bone grafts sterilized by the proposed method.

Osteoinductive activity of bone allografts, as the most objective criterion for their quality, was studied in an experiment in 12 rats and 7 rabbits. The first was performed ectopic (in the muscles of the thigh), and the second - orthotopic (in the defect of the radius) bone grafts obtained from slaughtered rabbits in non-sterile conditions. Before sterilization, fragments of the tubular bones of animal donors were freed from soft tissues and bone marrow. Then, after demineralization, they were placed in plastic bags and poured with a sterilizing solution containing 2.0 bromic acid, 0.1 g salicylic acid, 3.0 hydrogen peroxide for every 100 ml of distilled water. The bags with the preparations and the solution were sealed and refrigerated (+ 5 ° С) for 48 hours, after which the solution was drained through a puncture with a thick sterile needle and the puncture site was again sealed.

Graft preservation before surgery was carried out at the same temperature for one to two weeks. In surgery, the grafts were removed from the packaging and placed in a sterile physiological solution of sodium chloride. Bacteriological studies performed prior to surgery showed that all transplants remained sterile. Surgery techniques in rats and rabbits were standard. In the postoperative period, no complications were observed, the temperature of the animals did not exceed normal values, their behavior did not differ from the behavior of healthy animals. 3 months after the operation, the animals were withdrawn from the experiment in accordance with the rules set out in the order of the Ministry of Health of the USSR No. 755 of 08/12/77. The preparations taken from them were examined by X-ray and histological methods. Histological preparations were stained with hematoxylin and eosin.

The results of the studies showed that by the end of the observations, 10 out of 12 rats had x-ray at the site of transplantation, newly formed bone regenerates were found, the density of which was still inferior to the neighboring sections of the recipient's bones (Figs. 1 and 2).

When orthotopic transplantation in all 7 rabbits after 3 months on x-rays (Figures 3 and 4), the radial bone defect was replaced by a newly formed bone regenerate of complex structure. On histological preparations, by this time, a new bone of mainly lamellar structure was found at the site of the defect. The Haversian canals of the new bone had a chaotic arrangement, in places they were somewhat enlarged and contained mainly osteogenic cellular elements (Figure 5).

Thus, the experiment confirmed the clinical suitability of demineralized bone allografts sterilized by the proposed method. Their successful use can be associated not only with their sterility, but also with the positive effect that the hydrobromic and salicylic acids present in the medium have on them. The first is due to the surface demineralization of the bone, providing its osteoinductive properties, the second due to the ability to increase the proliferative activity of connective tissue cells.

Clinical example.

Patient P., 13 years old. Received in LNIDOI them. G.I. Turner with a diagnosis of multicamera cyst of the proximal third of the diaphysis of the right humerus (Fig.6-a). Operation: processing the inner walls of the cyst with a grooved chisel and fenestrated milling cutter. The resulting cavity is filled with strips of allogeneic demineralized and mineralized bone, sterilized by the proposed method. The wound is sutured tightly, the limb is fixed with a plaster cast. The postoperative period was uneventful. Sutures were removed on the 10th day. Discharged for outpatient treatment. Three months after the operation, an active reconstruction of the grafts is visible on the radiographs (Fig. 6-b), after 6 months, signs of the formation of the medullary canal and the cortical layer of the bone are visible (Fig. 6-c). After 1.5 years after surgery, the structure of the affected bone is completely restored (Fig.6-g).

Bibliography

1. Methodological fundamentals of mass procurement of tissue transplants using sterilizing agents of a chemical nature / Methodical letter. - Novosibirsk, 1968 .-- 21 p.

2. Von Verzen R. Preparation of a demineralized bone matrix for clinical use / Demineralized bone graft and its use: Sat. - SPb., 1993. - S.4-11.

3. Chemical sterilization of biological tissues with subsequent freezing / Methodological recommendations. - Astrakhan, 1978.- 26 p.

4. Syzganov A.N. About the treatment of purulent surgical diseases with salicylic acid / A.N. Syzganov, S.N. Levchenko // Alma-Ata. - "Kazakhstan". - 1980. - 142 p.

The method of sterilization of biological tissues for transplantation Table Sterilization time Clock 12 24 48 72 Sterilization result + - + - + - + - Number of studies fifty 0 19 31 0 fifty 0 fifty

Claims (1)

A method of sterilizing biological tissues for transplantation with a mixture including hydrogen peroxide, characterized in that the sterilization of tissues is carried out in packaged form without observing asepsis at + 4 ° ... + 5 ° C for 48 hours with a mixture of hydrogen peroxide, hydrobromic and salicylic acid in the following ratio components, wt.%:
hydrobromic acid 2.0 salicylic acid 0.1 hydrogen peroxide 3.0 distilled water rest

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