RU2526429C1 - Method of manufacturing bone implants - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: bone fragment is blown by jet of an ozone-air mixture with the concentration of ozone of 5-50 mg/m³ for 7-10 min. After that, the said bone fragment is mechanically processed by hydrodynamic jet, which results in obtaining a blank. After that the blank is demineralised in an inorganic acid solution. The remains of acid are neutralised. Then, the blank is re-processed by blowing by the said ozone-air mixture in the same mode.

EFFECT: method provides complete sterilisation of bone implants with preservation of their osteoinductive properties.

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Description

The invention relates to biology and medicine and can be used in traumatology, orthopedics in the manufacture of biological mineralized implants and their deep sterilization during procurement, preservation and subsequent storage. Also, the invention can be used in the work of "tissue banks" to provide bone-plastic material for health facilities.

To obtain osteoplastic material equivalent in quality of autobone, the most effective is the physical and (or) physico-chemical effect on the allogeneic plastic material, as a result of which they reduce the antigenic activity of implants and reduce the risk of tissue incompatibility by completely removing bone marrow from them and cells.

One of the most common materials for bone grafting is demineralized bone matrix due to biocompatibility, good osteoinductive and osteoconductive properties. In order to achieve the best clinical result in the demineralized bone matrix, it is necessary to maintain the activity of bone morphogenetic proteins.

Depending on the production method, the demineralized bone matrix may have different properties. But it is important to achieve high sterility of the product, which affects the duration and storage conditions.

Thus, the demineralized bone implant used in clinical practice must have certain standardized parameters. To preserve the activity of bone morphogenetic proteins during storage of the drug, its moisture content should not exceed 3-5%, and the pH should be in the range from 4.5 to 7.5, the total content of bone morphogenetic proteins BMP-2 and BMP-7 250-350 mg / g of product. To reduce the risk of an immune response to the introduction of the material, as well as to maximize the release of protein factors of bone growth, the lipid level should not exceed 2%.

A high degree of sterility is necessary in bioimplantology, in particular, at the final stages of manufacturing a bone implant in tissue banks. When using them, the possibility of infection of recipients with bacterial, fungal and viral infections should be excluded. Therefore, the manufacturing process of any implants of a biological nature should be completed by reliable and adequate sterilization with the maximum possible preservation of the plastic properties of the tissue. The selection of donor material, the choice of technology for the production of biological implants in world practice is regulated by relevant standards and monitored by serological analyzes. In parallel with the solution of these issues, there are others that relate to methods of sterilization of biological tissues. These include the influence of various factors (temperature, chemical, radiation exposure, etc.) on the endo- and exopathogenic flora present in donor tissues, which can lead to the denaturation of their protein structures and minimize the complex plastic properties of implants or completely disappear .

Currently, for the disinfection and sterilization of medical instruments, laboratory equipment, media and biological objects, high-temperature technologies, chemically active preparations, ionizing and ultraviolet (UV) radiation are used (Shkarin V.V., Shafeev M.Sh. Disinfection: A manual for students medical universities and doctors.Nizhny Novgorod, 2003.368 s.).

1. High temperature technology. Disinfection and sterilization is carried out using steam, air and gas sterilizers. The sterilizing agents of gas sterilizers are: a mixture of ethylene oxide and methyl bromide, formaldehyde, ethylene oxide in a mixture with inert gases. High-temperature technologies are characterized by heating and cooling and do not allow the disinfection of temperature-sensitive (especially biological) materials.

2. Chemical reagents. The degree of effectiveness of disinfection with chemically active substances is different and depends both on the chemical activity of disinfectants and on the properties of the processed samples. The range of biological materials disinfected with chemical reagents is much narrower, moreover, all disinfectants are extremely toxic.

3. Radiation. A sufficiently effective disinfecting effect is characterized by radiation exposure. However, a major drawback of the method is the significant effect of radiation on the osteoinductive properties of implants, as well as the high cost of the equipment used, the presence of specially equipped radiation-safe rooms. These installations require highly qualified staff.

Various manufacturing techniques for bone implants are known, depending on the tasks.

So, there is a known method for the manufacture of an allograft, including mechanical processing of a bone tissue blank obtained from a donor, washing it with cold water, and demineralizing it in 1.2-3.6 n. hydrochloric acid solution, washing the demineralized preform in the distillate and in physiological saline, sterilizing and preserving the preform by placing and keeping it in an appropriate sealed container (package), filled with a formaldehyde solution with the addition of an antibiotic (Savelyev V.I. (Demineralized bone as a special kind of bone -plastic material. Collection of scientific works of LNIITO named after PP Vredena. Harvesting and transplantation of demineralized bone tissue in an experiment and clinic. L .: NIIITO, 1983, pp. 3-12). due to bone tissue demineralization, it is possible to obtain allografts with high osteoinductance, which frozen non-demineralized grafts practically do not have, and low antigenicity.However, a significant drawback of allografts obtained using this method is the use of formaldehyde as a preservative and sterilizer, which entails a number of problems caused by limitation transplant storage time (not more than 6 months), the need to wash the prepared transplant Ed clinical use, formaldehyde toxicity and inconvenience of storage and transportation of the graft is immersed in the solution of formaldehyde.

A known method of manufacturing implants from spongy bone tissue (US Pat. RF No. 2172104), including grinding the bone, washing with water, sterilization and preservation. In this case, bone fragments are immersed in a 6% hydrogen peroxide solution for 48 hours at a ratio of one volume of bone fragments to four volumes of hydrogen peroxide solution with a change of solution 4 times every 12 hours, the bone fragments are centrifuged, then immersed in ethanol mixture with chloroform in a ratio of 1: 1 for 48 hours with a ratio of one volume of bone fragments to four volumes of a solution of hydrogen peroxide with a change of solution 4 times every 12 hours, bone fragments are centrifuged again, aired in air for 24 hours, after which the fragments are frozen at -70 ° C for 24 hours, after which they are lyophilized for 48 hours to achieve a residual moisture content of 5%, and then packaged in a standard double bag and sterilized by a stream of fast electrons dose of 18 ± 5 kGy at the LUE-8-5M accelerator. The disadvantages of this method are its significant duration, complexity, the possibility of destruction of bone morphogenetic proteins in the resulting implant during prolonged treatment with hydrogen peroxide, which does not exclude the loss of osteoinductive properties.

A known method of manufacturing a bone implant (US Pat. RF No. 2147800), which consists in sequentially machining and washing the billet from bone material, performing through holes in the billet, demineralizing in hydrochloric acid solution, neutralizing acid residues, preserving the demineralized billet by lyophilization, sterilization after drying, carried out by irradiating a workpiece placed in a sealed package, with a beam of accelerated electrons with a dose of 15-18 kGy for 16-20 from. However, a significant drawback of the implants obtained by this method is the long cycle for obtaining transplants. In addition, the use of radiation exposure as a sterilizing agent, characterized by a minimum processing time of a significant volume of packaged material, however, has disadvantages associated with a decrease in the osteoinductive properties of demineralized bone at a dose of 10 kGy gamma rays and loss of osteoinductive abilities at a dose of 25 kGy . In this case, the minimum dose necessary for the complete destruction of bacteria is 20 kGy, and for spores and viruses - 20-40 kGy.

A known method of producing a bone graft (US Pat. RF 2223104) by cleaning and washing bone tissue, deproteinization of the fragments first in a 0.01% solution of chymopsin, then in a 10% solution of hydrogen peroxide for 48 hours, treatment with liquid ether for 6 hours, drying and processing with a 10% solution of lithium chloride for 16 hours, followed by sterilization of the target product. Allogeneic or xenogenic fragments of long tubular bones are deproteinized in a solution of chymopsin for 96 hours, and when treated with a 10% solution of hydrogen peroxide, they are placed in an alternating magnetic field at 45 ° C. The deproteinization of a fragment of a bone fragment measuring 5-6 cm is carried out twice, and more than 6 cm in size - three times. The disadvantage of this method is the long chemical effect, the absence of indications of the preservation of the osteoinductive properties of the implant, as well as information about controlling the degree of deformation at various stages of processing.

Currently, a method for treating spongy bone tissue is widely used, including washing from blood and myeloid-fat bone marrow with water, sterilization with formaldehyde and preservation by freezing (Harvesting and preservation of spongy and tubular bone matrix. Method. Rivers. Yerevan, 1984. ) The disadvantages of this method can include the following:

- formaldehyde used for sterilization in the form of concentrated vapors has a cytotoxic effect;

- the duration of continuous washing from formaldehyde is not less than a day;

- the shelf life in a frozen state is limited (1 year);

- storage requires refrigerators with an appropriate operating mode and constant control over the storage process;

- there are certain technical difficulties when transporting plastic material in glass flasks.

A known method of producing demineralized bone matrix in the form of crumbs (US Pat. RF 2456003), including grinding the bone, processing bone fragments with a solution of detergent, removing detergent, treatment with a mixture of alcohol-chloroform (1: 1), washing with water, lyophilization, sterilization. The bone is cross-cut into fragments, bone fragments are treated with a 2% Tween-80 solution for 12-24 hours twice, then treated with isopropanol-chloroform (1: 1) mixture for 24-48 hours in an ultrasonic bath, washed with water, treated with a solution of 0.6 M HCl for 30-90 minutes, then the chips are washed with distilled water, placed in phosphate buffer, washed with distilled water, the resulting fragments are poured with ethanol, the chips are dried, and crushed. Sterilization is carried out by the radiation method at the GU-200 facility (FSUE NIIP, Lytkarino). The absorbed dose value is 15 kGy. At the same time, the specified long-term chemical and radiation treatment can have a negative effect on the osteoinductive properties of the resulting material.

A known method of plasma sterilization of small fragments of lyophilized cancellous bone [Shimizu K., Yano H., Nakamura E., Kaku N. Lipid extracted freeze-dried bank bone sterilized with low temperature plasma. // Ann. Transplant - 2001. - Vol.6, N 1. - P.26-31]. The authors of the method did not find differences in the evolution of transplants sterilized by plasma and gaseous ethylene oxide, taken as a control. Unfortunately, the authors did not present the histological evolution of such grafts and information about their osteoinductive activity. In addition, it is known that lyophilized bone in its biological value is significantly inferior to bone tissue preserved by freezing.

A known method in which experimental data is presented characterizing the osteoinductive properties of demineralized bone grafts sterilized with low-temperature hydrogen peroxide plasma [Ferreira S.D., Dernell W.S., Powers B.E. et al. Effect of gas-plasma sterilization on the osteoinductive capacity of demineralized bone matrix. // din. Orthop. - 2001. - N 388. - P.233-239]. In this case, low-temperature plasma deprived demineralized grafts of their useful osteoinductive properties.

A known method of obtaining biomaterial (US Pat. RF No. 2472516) for the replacement of bone defects based on natural coral, cleaned of coral dust and microorganisms with running water, followed by sterilization. Coral, additionally purified with a 3% solution of sodium hypochlorite and ultrasonic treatment with a frequency of 40 kHz for 3-5 minutes, is dried and sterilized by gamma radiation at a total dose of 25 kGy.

A known method of chamber sterilization of biological transplants (US Pat. RF 2317109) with a low-temperature plasma of hydrogen peroxide, which consists in exposing hydrogen peroxide to magnetic radiation with a frequency of 13.576 MHz at a temperature of 46 ± 4 ° C. Immediately before sterilization, the transplants are treated for 2-3 hours at room temperature in a solution consisting of: sulfosalicylic acid (1.0), propanediasaccharol (10.0), absolute ethyl alcohol (90.0), and then dried in a thermostat at 37 ° C. The use of low-temperature plasma of hydrogen peroxide for sterilization of demineralized bone grafts subjected to dehydration does not violate their osteoinductive properties. In addition, as shown by the experiment, tendon implants sterilized by low-temperature plasma of hydrogen peroxide do not cause morphological changes in the surrounding tissues that could serve as a contraindication for their clinical use. These dehydrating effects that occur during heating, as well as when using an alcohol solution, can degrade the initial properties of the implants and reduce their regenerative potential.

The closest prototype to the claimed technical solution is the method according to US Pat. RF №2268060 manufacturing a bone implant, including machining with a hydrodynamic jet of bone tissue and washing the workpiece from bone material, demineralization of the workpiece in 0.7-1.1 N. phosphoric acid solution, neutralization of acid residues, sterilization and preservation of the workpiece. Upon completion of the demineralization of bone fragments, which can be complete (total), partial (superficial) and segmental (selective), the samples are washed to neutralize acids for 1.5 hours in 5% solution of baking soda and 1 hour in physiological saline. For preservation, subsequent sterilization and storage, a 0.1% formaldehyde solution with the addition of gentomycin is used at a rate of 0.5 g per liter. Phosphoric acid is washed from the workpiece with sodium hypochlorite solution, immersing it three times in the solution for 35 minutes. In this form, the implant is ready for use in plastic surgery and can be stored at a temperature of 18-20 ° C for up to 5 years. The disadvantages of sterilization and preservation are the inability to obtain a 100% degree of sterilization from bacterial, fungal and viral infections, as well as the disorganizing effect of sodium hypochlorite.

The technical result of the invention consists in 100% sterilization of bone implants while maintaining the osteoinductive properties of the sample, which leads to morphological and bioplastic safety of the sterilized objects, the possibility of using mass preparation of implants from spongy bone tissue, reducing the time of preparation of implants for clinical use of bone samples.

The achievement of the technical result is possible by using a method for manufacturing a bone implant, including mechanical treatment of a bone tissue fragment with a hydrodynamic jet, demineralization of a workpiece in an inorganic acid solution, neutralization of acid residues, washing of a workpiece from bone material, its sterilization and preservation, while the ozone-air implant is sterilized a mixture with an ozone concentration of 5-50 mg / m ³ for 7-10 minutes before machining and a similar final degree Realization after completion of the implant manufacturing process.

Improvements to the method of manufacturing bone implants proposed according to the claimed invention are the result of a generalization of experimental studies on the creation and practical use of grafts (samples) made using the above improvements, new in relation to the prototype method of actions, conditions and parameters of the modes of their implementation. The obtained results of laboratory tests confirm the possibility of solving the problem posed in the claimed invention.

, поэтому он не попадает в атмосферу, не скапливается в нижних слоях в недопустимом количестве, а полученные химически активные О^* и $$O_2^{*}$$

имеют продолжительность "жизни" десятые и сотые доли секунды и исчезают или используются также в непосредственной близости от обрабатываемого объекта. Преимущества стерилизации озоном - низкотемпературный режим, короткая экспозиция, глубокое проникновение в материал, возможность стерилизации термонеустойчивых изделий, возможный большой объем стерилизационной камеры, отсутствие токсичности, а также безопасность для окружающей среды. Согласно многочисленным исследованиям озон обладает сильно выраженными фунгицидными, бактерицидными, вироцидными свойствами («Клинические аспекты озонотерапии» под ред. А.В. Змызговой и В.А. Максимова, М.: НПЦ Озонотерапии, 2003, 288 с.). Озон способен эффективно уничтожать все виды бактерий, вирусов, грибов и простейших. В проведенных экспериментах показано, например, полное подавление роста колоний протеев, кишечной и синегнойной палочки, клебсиеллы, стафилококка при 10³-10⁴ КОЕ/мл при обработке озонированной дистиллированной водой с концентрацией озона 4 мг/л в условиях in vitro, a также антивирусный эффект озона на культуре лимфоцитов, зараженной ВИЧ-1 с инактивацией вируса как экстракорпорально, так и внутри клеток, и подавлением сопровождающих ВИЧ инфекций, устойчивых к антибиотикам (А.В. Густов, К.Н. Конторщикова, Ю.П. Потехин. Озонотерапия в неврологии. - Нижний Новгород, Издательство Нижегородской гос. Медицинской академии, 2012. - 192 с.).Ozone O ₃ - an excellent modern disinfector - can be obtained for this purpose in the simplest inexpensive way, for example, by blowing a stream of atmospheric air through a torch discharge of electric current voltage of 220 V with an industrial frequency of 50 Hz, i.e. without the use of special generators, high-frequency converters of electric current, oxygen concentrators, etc. It should also be noted that the proposed method facilitates compliance with safety regulations due to the fact that ozone O _{3 is} created in the immediate vicinity of the treated object and is almost completely decomposed here into O ^* and $$O_2^{*}$$

therefore, it does not enter the atmosphere, does not accumulate in the lower layers in an unacceptable amount, and the chemically active O ^* and $$O_2^{*}$$

Tenths and hundredths of a second have a life span and disappear or are also used in the immediate vicinity of the object being processed. The advantages of ozone sterilization are low temperature, short exposure, deep penetration into the material, the possibility of sterilization of thermally unstable products, the possible large volume of the sterilization chamber, the absence of toxicity, and environmental safety. According to numerous studies, ozone has very pronounced fungicidal, bactericidal, virocidal properties (Clinical Aspects of Ozone Therapy, ed. By A.V. Zmyzgova and V.A. Maksimov, M .: Scientific and Practical Center for Ozone Therapy, 2003, 288 pp.). Ozone is able to effectively destroy all types of bacteria, viruses, fungi and protozoa. The experiments showed, for example, complete inhibition of the growth of colonies of proteins, Escherichia coli and Pseudomonas aeruginosa, Klebsiella, staphylococcus at 10 ³ -10 ⁴ CFU / ml when treated with ozonated distilled water with an ozone concentration of 4 mg / l in vitro, as well as antiviral effect of ozone on a lymphocyte culture infected with HIV-1 with inactivation of the virus both extracorporeally and inside cells, and suppression of the accompanying HIV infections that are resistant to antibiotics (A.V. Gustov, K.N. Kontorshchikova, Yu.P. Potekhin. Ozone therapy in neurology. - None Nij Novgorod, Publishing Nizhny Novgorod State Medical Academy, 2012. -. 192)..

Example 1 of the prototype method.

From a fragment of the femur received from the donor (cortical bone of the femoral diaphysis), a hydrodynamic jet is machined to obtain a preform of 25 cm long, 2 cm wide and 0.5 mm thick. Soft tissues and myeloid-fat marrow are removed. Then place the workpiece in a 3% hydrogen peroxide solution for 1 hour to remove blood components from the compact layer. Next, the workpiece is placed in 1.1 N. phosphoric acid solution at 18-20 ° C. The degree of demineralization, controlled by x-ray and morphometric methods, is 50% at the end of the process. Phosphoric acid is washed from the workpiece with sodium hypochlorite solution, immersing it three times in the solution for 35 minutes. For preservation and subsequent sterilization and storage, a 0.1% formaldehyde solution with the addition of gentomycin is used at a rate of 0.5 g per liter. On 3-4 days of incubation in a thermostat at 37 ° C, gas bubbles and turbidity appeared on the surface of the samples immersed in the broth. The next day, turbidity spread evenly throughout the nutrient medium. With shaking, intense gas formation was noted. Sterilization was performed within 7 days. The content of anaerobic bacteria Clostridium spp was detected. The completeness of the removal of bone marrow and bone-covering cells from the bone sample was controlled morphologically according to the results of studying the structure of the samples by scanning microscopy. Shelf life of the sample is one month. After that, the content of anaerobic bacteria Clostridium spp increased. 17% of the original content.

Example 2 of the proposed method.

In the atmospheric air injected into the ozonizer, which passes through a 220 V flare electric discharge with an industrial frequency of 50 Hz, ozone is formed. Next, the ozone-air mixture is fed into the sterilization chamber, in which the original sample from example 1 is placed. The optimal operating range of ozone concentrations is 5-50 mg / m ³ . An object is blown continuously for 7-10 minutes. Then a mechanical treatment is carried out by a hydrodynamic stream, and a workpiece of 25 cm long, 2 cm wide and 0.5 mm thick is obtained. Soft tissues and myeloid-fat marrow are removed. Then place the workpiece in a 3% hydrogen peroxide solution for 1 hour to remove blood components from the compact layer. Next, the workpiece is placed in 1.1 N. phosphoric acid solution at 18-20 ° C. The degree of demineralization, controlled by x-ray and morphometric methods, is 50% at the end of the process. Phosphoric acid is washed from the workpiece with sodium hypochlorite solution, immersing it three times in the solution for 35 minutes. For preservation and subsequent sterilization and storage, the sample is again blown with a stream of ozone-air mixture with an ozone concentration of 5-50 mg / m ³ for 7-10 minutes. The completeness of the removal of bone marrow and bone-covering cells from the bone sample was controlled morphologically according to the results of studying the structure of the samples by scanning microscopy. As a result, complete inactivation of aerobic and anaerobic bacteria was established. This is especially true for anaerobic bacteria Clostridium spp. Using acoustic microscopy, it was found that the structure and physicomechanical properties of the obtained demineralized samples did not change.

The proposed method provides 100% sterilization of bone implants while maintaining the osteoinductive properties of the sample.

Ozone treatment at the stage of preparation of the initial bone fragment is carried out to ensure the safety of personnel directly in contact with the source material at all stages of implant manufacturing.

Tests confirmed the viability of the proposed method for practical use in medicine for sterilization of bone implants.

Claims (1)

A method of manufacturing a bone implant, including machining a fragment of bone tissue with a hydrodynamic jet, demineralizing a workpiece in an inorganic acid solution, neutralizing acid residues, washing a workpiece from bone material, sterilizing and preserving it, characterized in that the implant is sterilized with an ozone-air mixture with an ozone concentration 5-50 mg / m ³ for 7-10 minutes before machining and a similar final sterilization after completion of the process and the manufacture of the implant.