RU2475273C1 - Method of obtaining polymeric cement of medical purpose - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine. Described is method of obtaining polymeric cement of medical purpose, which includes mixing of prescription quantity of powdery polymer, liquid monomer, antibacterial component and mixture mixing, as antibacterial componen applied is solution, which contains bacteriophage or mixture of several different bacteriophages, which have lytic activity, in concentration not less than 10⁶ PFU/ml, in amount 5.0-6.5 wt %, which is introduced into cement after mixture cooling to temperature 4-10°C. Mainly applied is/are bacteriophage(s) against bacteria of genus Pseudomonas, Staphylococcus, Bacillus. In particular case, applied is/are bacteriophage(s), individually selected for particular pathogen, for this purpose typing of causative agent of infection process of bacterial etiology agent in particular patient is preliminarily carried out.

EFFECT: method insures incretion quality of target product by incretion of antibacterial properties of polymeric cement.

4 cl, 1 dwg, 6 ex

Description

The invention relates to medicine and can be used to obtain a polymer cement with antibacterial properties, used in arthroplasty, fixation of bone fragments, to replace missing bone tissue, as well as in maxillofacial surgery, plastic surgery and dentistry.

A known method of producing polymer cement, comprising mixing a prescription amount of a powdered polymer, a liquid monomer with the addition of an antibiotic gentamicin hydrochloride, or gentamicin sulfate (patent US 4059684, op. 22.11.1977). Obtained in a known manner, polymer cement is intended for the formation of bone-like structures of artificial origin of arbitrary shape and at the same time preventing the development of an infectious process in the contact zone between polymer cement in the cement-bone contact zone due to the presence of an antibiotic in the composition of the product.

The disadvantage of this method is the low quality of the target product, namely low antibacterial properties associated with the presence in the polymer cement of the antibiotic gentamicin hydrochloride or gentamicin sulfate, which in modern conditions may be ineffective due to a number of factors, including the emission of the antibiotic into the surrounding tissues and resistance of microorganisms to a specific antibiotic.

Closest to the claimed method, the prototype, is a method of producing polymer cement, comprising mixing a powdered polymer and liquid monomer with the addition of the antibiotic gentamicin hydrochloride in an amount of 0.5-1.0 wt.% Of the total number of components of the composition (patent RU 2195320, op. 12/27/2002).

The disadvantage of this method is the low quality of the target product, due to the following reasons:

1) Lack of diffusion of antibiotic in the tissue with well-homogenized cement due to the absence of pores in the structure of polymer cement (ASBaker, LW Greenham. Release of gentamicin from acrylic bone cement. Elution and diffusion studies. J. Bone Joint Surg. Am. 1988; 70: 1551-1557).

2) The insufficient effectiveness of the antibiotics used in the presence of a microbiota resistant to them, or a low concentration of the antibiotic impregnated in cement or its non-linear decrease over time, which can lead to the formation of resistant strains of pathogenic and conditionally pathogenic microorganisms.

3) The need to increase the concentration of the antibiotic with the ineffective elimination of pathogenic microbiota, which leads to a violation of the homogeneity of the cement structure and the deterioration of its strength properties.

4) The possibility of toxic side effects associated with the presence in the composition of polymer cement of an impregnated antibiotic, in particular gentamicin.

5) If there are allergic reactions to a specific antibiotic that is part of the cement, it is not possible to quickly remove the allergen from the macroorganism.

The objective of the invention is to improve the quality of the target product by increasing the antibacterial properties of polymer cement.

The technical result of solving the problem is to provide the possibility of obtaining polymer cement with increased antibacterial activity against strains of pathogenic and conditionally pathogenic microorganisms responsible for the development of the infectious and inflammatory process at the site of implantation of a cement product.

The problem is achieved by the claimed method, which consists in the following.

The components of the polymer cement composition, commercially available and approved for medical use, are mixed, including powder polymer and liquid monomer, the mixture is mixed, cooled to reduce the thermal yield of the exothermic polymerization reaction to a temperature of 4-10 ° C, and a solution containing a bacteriophage or a mixture of several different bacteriophages is added possessing lytic activity against the pathogen (s) of the infectious process of bacterial etiology with a concentration of bacteriophage particles equal to not enee June ¹⁰ PFU / ml (plaque forming units per milliliter) in an amount of 5.0-6.5 wt.%. Next, polymer cement is re-mixed and the desired shape of the product.

In a preferred embodiment, a specific bacteriophage (s) is selected, for which a typing (determination) of the causative agent of the bacterial etiology of an infectious process is carried out in a particular patient by culturing a microorganism (s) obtained from a patient’s sample of biomaterial with a test set of bacteriophages.

In the particular case, a pre-prepared bacteriophage or a mixture of several different strains of bacteriophages is used, which are active against the most common infectious agents at the site of implantation of polymer cement products, such as bacteria of the genus Pseudomonas, Staphylococcus and Bacillus. In this case, either a commercially available bacteriophage (s) or a bacteriophage (s) located (deposited) in accessible collections of microorganisms are used.

The raw material for the antibacterial component is phagolysates of microbial cultures, sterilized by membrane filtration through filters with a pore diameter of 0.22 μm.

The result is a polymer cement for medical purposes with micropore sizes of the order of 100-200 nm, which has antibacterial activity against strains of pathogenic and opportunistic microorganisms responsible for the development of the infectious and inflammatory process at the site of implantation of the product or medical system. The prepared polymer cement can be stored for 10 days at a temperature of 6-10 ° C without loss of antibacterial activity.

The decisive distinguishing features of the proposed method in comparison with the prototype are:

1. As an antibacterial component, a solution is added to the cement containing a bacteriophage or a mixture of several different bacteriophages with lytic activity against the pathogen (s) of the bacterial etiology infectious process with a concentration of bacteriophage particles equal to at least 10 ⁶ PFU / ml in an amount of 5.0 -6.5 wt.%, Which allows to obtain polymer cement with enhanced antibacterial properties, while having qualities such as the absence of allergic reactions to bacteriophage, termination I am a bacteriophage after lysis of pathogenic bacteria followed by self-elimination from a macroorganism.

2. A solution containing a bacteriophage or a mixture of several different bacteriophages with lytic activity is introduced into the reaction mixture after it is cooled to 4-10 ° C, which allows to lower the temperature of the exothermic polymerization reaction from 110-120 ° C to 45-50 ° C and exclude the inactivation of the activity of the bacteriophage (s) against the pathogen (s) of the infectious process.

3. As bacteriophage (s) with lytic activity, bacteriophage (s) are used against bacteria of the genus Pseudomonas, Staphylococcus or Bacillus, which allows to expand the spectrum and specificity of the action of the bacteriophage (s) used.

4. A bacteriophage or a mixture of several different bacteriophages with lytic activity, which is individually selected for a particular pathogen, is predominantly used. For this purpose, the causative agent of the bacterial etiology of the infectious process is preliminarily typed in a particular patient to produce the corresponding lytic bacteriophage, which enhances the antibacterial properties of the target product and provide individual selection of lytic bacteriophage for specific phagotherapy and phagop prevention of the infectious and inflammatory process of bacterial etiology.

The presence in the structure of polymer cement of micropores with dimensions of the order of 100-200 nm allows the use of such cement as a carrier of bacteriophage (s) with (ly) activity, the pore sizes of which are sufficient to accommodate bacteriophage (s) of polymer cement, which subsequently ensures the emission of bacteriophage ( o) from polymer cement both at the cement-bone border and into surrounding tissues in concentrations sufficient to maintain a minimum, inhibiting infection, bacteriophage concentration in the tissues surrounding implantation my device or medical system.

Figure 1 shows the image of the internal structure of polymer cement in a 2 × 2 μm sample obtained using atomic force microscopy, the composition of which does not contain modifying components, such as bacteriophages, but clearly shows a porosity sufficient to accommodate bacteriophages representing are nanosized particles.

The invention is illustrated by the following examples of specific performance.

Example 1

Previously, typing of the causative agent of the bacterial etiology of the infectious process was carried out for a particular patient and the corresponding bacteriophage with lytic activity. Next, 1 g of a sterile powdered polymer of polymethyl methacrylate [Poly [(Methyl Methacrylate) -Co-Styrene] Copolymer], 0.5 ml liquid methyl methacrylate monomer (Methyl methacrylate) was mixed. The mixture was stirred and cooled in an ice bath for 5 minutes to a temperature of 7 ° C. Then added 0.079 ml of a solution containing a pathogen-specific commercially available "staphylococcal bacteriophage" against Staphylococcus aureus 209 (manufactured by FSUE NPO Mikrogen, Russia) with a particle concentration of the bacteriophage 1.1 × 10 ⁷ PFU / ml in an amount of 5.0 wt.% . They thoroughly mixed and gave the desired shape to the polymer cement.

The result was a polymer cement of the following composition, wt.%:

Polymethylmethacrylate (powder polymer) 63.3

Methyl methacrylate (monomer) 31.7

Bacteriophage against Staphylococcus aureus 209 with the number of particles of the bacteriophage 1.1 × 10 ⁷ PFU 5.0

Example 2

Previously, typing of the pathogen of the bacterial etiology of the infectious process for a particular patient was carried out and the corresponding bacteriophage with lytic activity was produced. Next, 1 g of a sterile powdered polymer of polymethyl methacrylate [Methyl methacrylate - styrene copolymer and Poly methacrylate], 0.5 ml of liquid methyl methacrylate monomer (Methyl methacrylate) was mixed, the mixture was stirred and cooled in an ice bath for 10 minutes to a temperature of 4 ° C. Then added 0.1 ml of a solution containing a pathogen-specific bacteriophage against Pseudomonas aeruginosa deposited in the collection of the Research Institute of KKM SSC VB "Vector", Novosibirsk under registration number V-357 with a particle concentration of the bacteriophage of 1.4 × 10 ⁷ PFU / ml in the amount 6.25 wt.%. They thoroughly mixed and gave the desired shape to the polymer cement.

The result was a polymer cement of the following composition, wt.%:

Polymethylmethacrylate (powder polymer) 62.5

Methyl methacrylate (monomer) 31.25

Bacteriophage against Pseudomonas aeruginosa V-357 with the number of particles of the bacteriophage 1.4 × 10 ⁷ PFU 6.25

Example 3

The preparation of polymer cement was carried out analogously to example 2, except that a bacteriophage mixture containing 0.052 ml of bacteriophage against Pseudomonas aeruginosa deposited in the collection of the Research Institute of KKM SSC VB "Vector", Novosibirsk under registration number V-357 and 0.052 was used as a bacteriophage. ml of a commercially available "staphylococcal bacteriophage" against Staphylococcus aureus 209 with a concentration of bacteriophage particles of 1.25 × 10 ⁷ PFU / ml.

The result was a polymer cement of the following composition, wt.%:

Polymethyl methacrylate (powder polymer) 62.3

Methyl methacrylate (monomer) 31.2

A mixture of bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus 209 with the number of particles of the bacteriophage 1.25 × 10 ⁷ PFU 6.5

Example 4

Previously, typing of the causative agent of the infectious process of bacterial etiology for a specific patient of the corresponding bacteriophage with lytic activity was performed. Next, 1 g of a sterile powdered polymer of polymethyl methacrylate [Methyl methacrylate - styrene copolymer and Poly methacrylate], 0.5 ml of liquid methyl methacrylate monomer (Methyl methacrylate) was mixed, the mixture was stirred and cooled in an ice bath for 5 minutes to a temperature of 10 ° C. Then, 0.1 ml of a solution containing a pathogen-specific bacteriophage against Bacillus species, deposited in the collection of the Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, was added under registration number ph41 with a concentration of particles of the bacteriophage 6.8 × 10 ⁸ PFU / ml in the amount of 6.25 wt. % They thoroughly mixed and gave the desired shape to the polymer cement.

The result was a polymer cement of the following composition, wt.%:

Polymethylmethacrylate (powder polymer) 62.5

Methyl methacrylate (monomer) 31.25

Bacteriophage against Bacillus species ph41 with the number of particles of bacteriophage 6.8 × 10 ⁸ PFU 6.25

Example 5

To study the prolonged action of the bacteriophage, a Pseudomonas aeruginosa cell culture and medical grade polymer cement containing a bacteriophage against this culture with a bacteriophage particle number of 8.0 × 10 ⁶ PFU were used. Incubation of the cell culture of Pseudomonas aeruginosa with polymer cement containing a bacteriophage was carried out at a temperature of 37 ° C for 18-20 hours. Viral particles were counted using a two-layer agar method. The number of viral particles of the bacteriophage added to the cement was determined in the culture fluid. The concentration of particles of the bacteriophage was 1.8 × 10 ⁸ PFU / ml.

Then the cement containing bacteriophage was washed ten times in 1 ml of a 0.9% NaCl solution with vigorous shaking on a vortex for 1-2 minutes. Residual liquid was removed with sterile filter paper. Fractions from aliquots of 10 samples obtained after washing the polymer cement were examined for the presence of phage particles. All aliquots contained the above bacteriophage. Cement washed from the bacteriophage was placed in a test tube with a Pseudomonas aeruginosa culture. The incubation of the cell culture of Pseudomonas aeruginosa with polymer cement washed from the bacteriophage was carried out at a temperature of 37 ° C for 18-20 hours. The viral particles were counted by the method of two-layer agar. The concentration of bacteriophage particles from the washed polymer cement was 1 × 10 ⁸ PFU / ml.

Thus, it was shown that the bacteriophage actively elutes from the pores of polymer cement into the environment, while the lytic activity of the bacteriophage is preserved.

Example 6

The duration of the persistence of bactericidal activity in a sample of polymer cement for medical purposes containing a bacteriophage was determined. A sample of polymer cement containing a bacteriophage against Pseudomonas aeruginosa V-357 with the number of particles of the bacteriophage 8.0 × 10 ⁶ PFU was kept for ten days in air at a temperature of + 10 ° C, and then the number of particles of the bacteriophage was determined. For this, the sample was placed in a test tube with a Pseudomonas aeruginosa culture and cultured at 37 ° C for 18-20 hours. The concentration of bacteriophage particles in the sample with cement stored for 10 days was 4.4 × 10 ⁹ PFU / ml.

From example 5 it is seen that the bacteriophage Pseudomonas aeruginosa V-357, added to the cement, retains its lytic activity for ten days at a temperature of + 10 ° C. Substances that make up polymer cement do not inactivate the bacteriophage.

The proposed method can be used to prepare polymer cements for medical purposes containing a bacteriophage, as well as a mixture of bacteriophages against various infectious agents, such as bacteria of the genera Pseudomonas, Staphylococcus, Bacillus, etc. Using the proposed method provides polymer cement, which simultaneously possesses qualities such as the absence of allergic reactions bacteriophage, lack of resistance mechanisms from pathogenic and opportunistic microorganisms isolated from a sample of biomaterial m patient, the possibility of exposure of the bacteriophage to bacterial biofilm with its partial destruction, termination bacteriophage action after lysis of pathogenic bacteria, followed by self-elimination of the microorganism.

Claims (4)

1. A method of obtaining a polymer cement for medical use, comprising mixing a prescription amount of a powdered polymer, a liquid monomer, an antibacterial component and mixing the mixture, characterized in that the solution containing a bacteriophage or a mixture of several different bacteriophages with lytic activity is used as an antibacterial component concentration of at least June ¹⁰ pfu / ml, in an amount of 5.0-6.5 wt.%, wherein the antibacterial component is introduced into the cement after cooling the mixture to Temperature 4-10 ° C. 2. The method according to claim 1, characterized in that as a powdery polymer and liquid monomer use any commercially available components that are approved for use in medicine. 3. The method according to claim 1, characterized in that as a bacteriophage (s) with lytic activity, bacteriophage (s) are used against bacteria of the genus Pseudomonas, Staphylococcus, Bacillus. 4. The method according to claim 1, characterized in that a bacteriophage or a mixture of several different bacteriophages with lytic activity is used, individually selected for a particular pathogen, for which purpose the pathogen of the bacterial etiology of the infectious process in a particular patient is preliminarily typed.