Interpolymer composite based on poly(Ε-caprolactone) and various cellulose derivatives for regenerative medicine

Abstract

FIELD: cellulose production.SUBSTANCE: interpolymer composites based on poly(ε-caprolactone) and cellulose derivatives for use as materials in regenerative medicine. The interpolymer composite consists of a matrix based on poly(ε-caprolactone), in which a filler, which is a cellulose derivative, is distributed in an amount of 5–10 wt.%. The cellulose derivative is a methacrylated derivative of hydroxyethyl cellulose and hydroxypropyl methylcellulose, including their conjugates with RGD peptide.EFFECT: use of modified fillers improves the physical and chemical, mechanical and biological properties of poly(ε-caprolactone).5 cl, 9 dwg, 9 ex

Description

[0001] Technical field

[0002] The claimed invention relates to the field of high-molecular compounds, specifically to biocompatible biodegradable composite materials based on polyesters and cellulose derivatives, and is intended for use in the biomedical field.

[0003] Technology Level

[0004] Currently, the field of creating biocompatible and biodegradable polymer composites for bone defect restoration is actively developing [1]. Among the existing variety of polymers capable of degradation in the body, composites based on aliphatic polyesters, such as poly(lactide-co-glycolide) (PLGA), polylactide (PLA), polyhydroxybutyrate (PHB) and poly(ε-caprolactone) (PCL) are of particular interest [2, 3]. These polymers differ in the length of the aliphatic fragment and, as a result, in the rate of degradation. In this series, PCL is the most hydrophobic and has the longest degradation period. This makes this polymer promising for the production of materials intended for the regeneration of large-volume defects. The restoration of such defects requires a longer time, and therefore a longer presence of the implant inside the defect to provide the mechanical support function of the damaged bone area.

[0005] The widespread use of PCL as a material for bone defect restoration is hindered by key disadvantages: high hydrophobicity and inertness, as well as insufficient mechanical strength (the elastic modulus is lower than that of natural bone) [4]. To improve the mechanical properties of PCL, various fillers are used, such as carbon nanomaterials [5] or mineral nanofillers [6]. However, the disadvantages of carbon nanomaterials include their often high hydrophobicity, tendency to aggregation, cytotoxicity and immunogenicity. An example of the production of composite materials for 3D printing of biomedical products is known from patent documentation, which is based on a composition consisting of PCL, hydroxyapatite and polysorbate 20 [7]. Mineral nanoparticles, namely hydroxyapatite nanoparticles, are more suitable as nanofillers for bone composites due to their properties than carbon nanomaterials.However, the introduction of mineral fillers does not contribute to a reduction in the hydrophobicity of PCL-based materials and does not allow, through relatively simple methods, to effectively modify the nanofiller under mild conditions to impart the desired biological properties to the matrix.

[0006] From the prior art, it is known that fillers in the form of various forms of cellulose, including its nano- and microcrystals and nano- and microfibers, are often used to improve the wettability (reduce hydrophobicity) of the surface and enhance the biological properties of PCL-based materials [8]. However, in practice, the use of unmodified cellulose is limited due to its strong tendency to aggregation and poor distribution in hydrophobic polymers, which leads to deterioration of the mechanical properties compared to the pure polymer [8,9]. To improve the distribution of cellulose materials in the matrix of hydrophobic PCL, various modified cellulose derivatives are used. For example, a number of studies have reported the use of methacrylated cellulose (MC)

[10] , hydroxypropyl methylcellulose (HPMC)

[11] and hydroxyethyl cellulose (HEC)

[12] , as well as nanocrystalline cellulose modified with poly(glutamic acid)

[13] .Known cellulose modification methods improve filler distribution within the PCL matrix and, consequently, the composite's mechanical properties by 20-50%. The use of soluble cellulose derivatives, compared to nanocrystals, allows for better filler distribution within the PCL matrix and higher elastic moduli in composite materials. However, such composites lack the osteoinductive properties necessary to improve bone tissue regeneration.

[0007] The following inventions in the field of composite materials containing one or more similar components and aimed at biomedical applications are known from patent documentation. For example, Gunduz et al. developed composite nanofibers consisting of PCL filled with ethylcellulose and collagen, which were obtained by electrospinning for use as artificial vessels

[14] . Chen et al. developed composite porous materials for wound healing based on PCL and a natural polymer including cellulose, alginate, hyaluronic acid, heparin, collagen, etc.

[15] .

[0008] The literature and patent documentation contain information on the production of materials based on aliphatic polyesters containing antibiotics. For example, the development of three-dimensional matrices based on PCL containing the antibiotic ciprofloxacin

[16] or films based on PLA / PCL containing the antibacterial agent 5-(4-pyridyl)-10,15,20-tris(4-n-hexadecyloxyphenyl)porphyrin

[17] has been reported.

[0009] A polymer composite material for bone tissue regeneration is known based on aliphatic polyester and nanocrystalline cellulose modified with poly(glutamic acid), including one containing an RGD peptide, which is closest to the claimed invention and is accepted as a prototype

[18] .

[0010] A disadvantage of the known polymer composition is the use of hydrophilic nanocrystalline cellulose, which is prone to aggregation in the matrix of hydrophobic PCL, which, in turn, leads to an insufficiently uniform distribution of the filler in the matrix, and, as a consequence, low mechanical properties of the material.

[0011] Thus, the development of a composite material with improved properties for bone tissue regeneration remains a pressing challenge. Such a material must be biocompatible and capable of gradual degradation over a long period of time. It must also exhibit improved mechanical properties similar to those of corresponding bone tissues, while exhibiting lower surface hydrophobicity and demonstrating improved biological properties.

[0012] Disclosure of invention

[0013] The technical objective of the claimed invention is to develop biocompatible and degradable composite materials with improved mechanical and biological properties suitable for bone tissue regeneration, as well as antibacterial properties for local antibacterial therapy.

[0014] The technical result of the present invention consists in the development and creation of a new interpolymer composite based on PCL, containing various amphiphilic cellulose derivatives as fillers. Their uniform distribution in the matrix polymer contributes to improved mechanical properties of the composite compared to the prototype, while simultaneously hydrophilizing the surface, which also leads to improved adhesion and proliferation of cells. In some embodiments, the composite material contains a fluoroquinolone antibiotic to achieve local antibacterial therapy. Biocompatibility, degradability, and antibacterial properties of the material are ensured by the properties of the constituent components, of which PCL is a biocompatible degradable matrix polymer, a cellulose derivative is a biocompatible amphiphilic functional filler, and a fluoroquinolone antibiotic is a medicinal substance with antimicrobial properties.

[0015] The said technical result is achieved in that in the composite material the proportion of the filler, a cellulose derivative that is methacrylated hydroxypropyl methylcellulose (HPMC), methacrylated hydroxyethyl cellulose (HEC), a conjugate of methacrylated HPMC with an RGD peptide, or a conjugate of methacrylated HEC with an RGD peptide, varies from 5 to 10 wt.%, while the proportion of the matrix PCL varies within the range of 90-95 wt.%; the antibiotic content varies from 0 to 5 wt.%.

[0016] In particular embodiments, the methacrylated HPMC or methacrylated HEC are HPMC or HEC, respectively, modified with glycidyl methacrylate (GMA) or methacrylic acid residues with a final content of methacrylic derivatives in the product at a level of 2-16 mol.%.

[0017] In particular embodiments, the conjugate of methacrylated HPMC with an RGD peptide or the conjugate of methacrylated HEC with an RGD peptide are methacrylated HPMC or methacrylated HEC, respectively, modified with a peptide from the RGD family containing a terminal thiol group that enters into a radical “thiol-ene” click addition reaction with methacrylated cellulose derivatives with a final content of RGD peptide in the conjugate at the level of 2-11 mol.%.

[0018] In particular embodiments, the antibiotic used is a fluoroquinolone antibiotic from among ciprofloxacin, levofloxacin or moxifloxacin.

[0019] From the claimed composition, composite films are obtained and it is possible to manufacture composite three-dimensional products using the extrusion 3D printing method.

[0020] Analysis of the prior art did not reveal a solution that completely matches the claimed solution in terms of its essential features, which may indicate its novelty.

[0021] Detailed disclosure of the invention

[0022] Definitions (terms)

[0023] For a better understanding of the claimed invention, some terms used in the present description of the invention are provided below.

[0024] Interpolymer composite is a material consisting of two polymer components, one of which is a polymer base (matrix polymer), in which another polymer component, called a filler, is distributed.

[0025] Cellulose derivative – cellulose modified with various molecules in order to obtain certain functional properties.

[0026] Conjugate is a synthesized macromolecule in which other functional molecules are covalently linked to the polymer.

[0027] Other technical and scientific terms used in this application have the standard meanings generally accepted in the scientific and technical literature.

[0028] Description of the invention

[0029] Components for producing modified fillers and interpolymer composites

[0030] Commercial cellulose derivatives, namely, HEC, HPMC, and an aliphatic polyester (PCL), were used as starting components to produce interpolymer composites. The polymers had the following molecular weight characteristics (according to size-exclusion liquid chromatography):

[0031] • GEC:M w = 35000;M n = 41000; Ð = 1.17; degree of substitution 2;

[0032] • HPMC:M w = 1065000;M n = 1225000; Ð = 1.15; degree of substitution 1.5;

[0033] • PCL:M w = 28000;M n = 49000; Ð = 1.75.

[0034] Cellulose derivatives exhibit pronounced hydrophilic properties, which complicates their compatibility with hydrophobic polycaprolactone (PCL) when creating interpolymer composites. To address this issue, HEC and HPMC were hydrophobized using commercially available reagents: methacrylic anhydride (MA) (Fig. 1) or glycidyl methacrylate (GMA) (Fig. 2). These chemical compounds were chosen due to the possibility of performing one-step methacrylation, which does not require harsh conditions or significant reagent consumption, making the process efficient and cost-effective.

[0035] The RGD peptide used had the following sequence: Cys-Gly-Arg-Gly-Asp-Ser-Pro (or CGRGDSP in the single-letter notation code). The presence of a thiol group on the terminal cysteine ​​enabled covalent conjugation with methacrylated derivatives of HEC and HPMC via a thiol-ene addition reaction (Fig. 3).

[0036] Obtaining interpolymer composites and their properties

[0037] Interpolymer composites were produced by rolling a polymer mixture—a PCL matrix and a filler in the form of a cellulose derivative (Fig. 4). This method relies on repeated rolling of the polymers using heated rollers, and composite formation occurs through mechanical mixing. The matrix polymer content varied between 90-95 wt.%, and the filler content between 5-10 wt.%.

[0038] In the case of using HEC and HPMC in their original form, samples of PCL and HEC or HPMC were taken in a given ratio, mechanically mixed, and before rolling, the mixture was kept in a thermostat at a temperature of 60 °C to soften the matrix polymer.

[0039] In the case of using methacrylated derivatives of HEC and HPMC, methacrylation of HEC and HPMC was initially carried out using methacrylic anhydride (MA) or glycidyl methacrylate (GMA). Methacrylation of cellulose derivatives using MA or GMA was achieved by reacting the hydroxyl groups of HEC or HPMC with methacrylic anhydride through an acylation reaction to form an ester bond and the epoxy group of GMA to form an ether bond. Depending on the reaction conditions, the degree of methacrylation ranged from 2 to 16 mol%. Otherwise, the interpolymer composites were prepared in the same way as for the initial HEC and HPMC.

[0040] In the case of using conjugates of methacrylated HEC and HPMC derivatives with the RGD peptide, such conjugates were initially obtained by reacting the double bonds of the methacrylic moiety in the HEC or HPMC chain with the thiol group of the terminal cysteine ​​of the peptide (thiol-ene addition reaction) in the presence of the photoinitiator 2-hydroxy-2-methylpropiophenone and irradiation with broad-spectrum UV light. The RGD peptide content in the conjugate was in the range of 2-11 mol%. Otherwise, the interpolymer composites were prepared in the same way as for the case of using methacrylated HEC and HPMC.

[0041] During the introduction of the filler, the hydrophilic-hydrophobic properties of the material surface changed, which was reflected in a change in the contact angle and swelling capacity. PCL had the most hydrophobic surface, with a contact angle of 103°. The introduction of cellulose derivatives led to a decrease in the contact angles to 70-85°, indicating a decrease in the hydrophobicity of the surface of the interpolymer composites. The smallest contact angles were observed with conjugates of methacrylated HEC or HPC with the RGD peptide, while the highest were observed for highly modified methacrylated forms of HEC and HPC (Fig. 5). Copolymers with the highest contact angles demonstrated the lowest swelling capacity. Thus, for materials based on pure PCL, the swelling degree did not exceed 1.3% (Fig. 6). At the same time, the introduction of cellulose derivatives as a filler contributed to an increase in the degree of swelling from 2.5 to 6.8%, depending on the filler type. The lowest swelling was observed for composites containing methacrylated HMC derivatives, and the highest for composites containing methacrylated HEC conjugates with the RGD peptide. The swelling increased with increasing filler content from 5 to 10 wt%.

[0042] The study of the surface of composite films and films based on pure PCL by atomic force microscopy showed a difference in certain values ​​of average roughness (R a ) and root mean square roughness (R q ) surface. All composites demonstrated a decrease in these parameters in the range of 184-215 nm for R a and 252-285 nm for R q, while for pure PCL these values ​​were 276 nm and 383 nm, respectively. It can be assumed that the inclusion of HEC or HMPC or their modified derivatives in the composite leads to a decrease in surface roughness due to the formation of a smoother amorphous phase.

[0043] Testing the films in the uniaxial tensile mode (Fig. 7) showed that the introduction of unmodified HEC and HMPC into PCL in an amount of 5 wt.% decreased the elastic modulus. Thus, for materials based on pure PCL, the elastic modulus was 441 ± 28 MPa, and for PCL composites with HEC or HMPC 365 ± 37 MPa and 382 ± 24 MPa, respectively. At the same time, the introduction of methacrylated derivatives of HEC or HMPC, or their conjugates with the RGD peptide contributed to a significant increase in the elastic modulus, the values ​​​​of which for the modified forms lay in the range of 667 ± 53 MPa - 820 ± 99 MPa. Moreover, the values ​​of the ultimate stress for the composites (45.8 ± 2.7 MPa – 52.9 ± 6.3 MPa) were close to those for the material based on pure PCL (51.4 ± 1.4 MPa), and the values ​​of the maximum deformation of the materials decreased from 44% for PCL to 20-34% for the composites.The introduction of selected fillers, as shown by the results of the tests, makes it possible to achieve higher mechanical properties in all respects, in comparison with the known world level of technology in this field

[18] .

[0044] The obtained mechanical properties correspond to some types of bone tissue. For example, for trabecular bone tissue, the elastic modulus values ​​in tensile tests range from several tens to 800 MPa depending on the bone sample collection area (tibia, femur, spine, etc.)

[19] ; ​​the tensile strength values ​​of trabecular bone tissue lie in the range of 1-30 MPa

[19] .

[0045] In vitro biological experiments showed an increase in mesenchymal stem cell adhesion from 60 ± 5% for materials based on pure PCL to 80 ± 7% - 88 ± 11% for materials containing conjugates of methacrylated HEC or GMPC with RGD peptide after 24 hours. Moreover, after 5 days, cell proliferation on the surface of the composite materials reached 105 ± 8% - 146 ± 12%, while for PCL-based materials it did not exceed 82 ± 6% (Fig. 8).

[0046] At the stage of obtaining the composite by the rolling milling method, a fluoroquinolone antibiotic such as ciprofloxacin, levofloxacin or moxifloxacin can be additionally included in the composition in an amount of 1 to 5 wt%. A study of the antibiotic release from composite films in 0.01 M phosphate-buffered saline, pH 7.4 (PBS), as well as PBS containing the lipase enzyme, and at a temperature of 37 °C showed that in the absence of the enzyme, the antibiotic release over 14 days was 20-50%, and in the presence of the enzyme - 30-80%, depending on the antibiotic content. The degree of antibiotic release (in %) was higher with a lower loading. The antibacterial properties of the obtained composite materials containing the antibiotic were tested against Pseudomonas aeruginosa. Effective inhibition of bacterial growth was confirmed.

[0047] It is shown that the obtained composites can in principle be used to produce three-dimensional matrices by 3D printing using a low-temperature (up to 100 °C) pneumatic extruder as a 3D printer print head (Fig. 9).

[0048] The claimed technical approach results in materials with improved physicochemical, mechanical, and biological properties for the production of 3D implants suitable for bone tissue regeneration. The known nature of individual components did not a priori guarantee the high-quality and effective characteristics of the claimed composite materials, with properties that meet the fundamental requirements for implants for bone tissue regeneration.

[0049] Brief description of drawings

[0050] The claimed invention is illustrated by Fig. 1 - Fig. 9, which show:

[0051] Fig. 1. 1 H NMR spectra of HEC before and after methacrylation with MA (D2O, 25°C).

[0052] Fig. 2. 1 H NMR spectra of HMPC before and after methacrylation with GMA (D2O, 25°C).

[0053] Fig. 3. Scheme for obtaining a conjugate of RGD-peptide with HPMC-GMA through the reaction of radical “thiol-ene” addition.

[0054] Fig. 4. Scheme for obtaining films by rolling and photo of a composite film based on PCL containing 5 wt.% of a conjugate of methacrylated HEC with an RGD peptide.

[0055] Fig. 5. Contact angles for some interpolymer composite films and control films based on PCL.

[0056] Fig. 6. Swelling degree of some interpolymer composite films and control films based on PCL.

[0057] Fig. 7. Testing films in uniaxial tension mode.

[0058] Fig. 8. Comparison of cell proliferation adhesion on the surface of some composite films and control films based on PCL.

[0059] Fig. 9. Examples of three-dimensional products obtained by extrusion 3D printing for a PCL-based composite containing 5 wt.% of a methacrylated HEC conjugate with an RGD peptide.

[0060] Examples of specific implementation

[0061] Below are presented examples of the preparation of certain cellulose derivatives and their conjugates, as well as interpolymer composites according to the invention. All examples provided in the application materials are provided to illustrate the invention and do not limit the claims.

[0062] Example 1. Preparation of HEC or HMPC derivatives using MA as a methacrylating agent

[0063] 375 mg of HEC or HPMC were dissolved in 125 ml of a bicarbonate buffer solution with pH 8.0 at room temperature. Then, 1 ml of MA was added dropwise with constant stirring and the solution was left for 24 hours. After this, dialyzed against water for 24 hours. The resulting suspension was lyophilized. The product yield was 90% for HEC and 92% for HPMC. The degree of substitution was 8% for HEC and 7% for HPMC. The success of the methacrylation reaction was confirmed by the method 1 H NMR spectroscopy by the appearance of peaks of diastereotopic hydrogen atoms in the region of δ = 5.5-6.5 ppm, as well as the signal of the methyl group of the methacrylic acid residue (δ = 1.81 ppm) (Fig. 1).

[0064] Example 2. Preparation of HEC or HMPC derivatives using GMA as a methacrylating agent

[0065] 375 mg of HEC were dissolved in 125 ml of acetate buffer solution with pH 5.6, or phosphate buffer solution with pH 6.0 or 6.8 at room temperature. Then, 1 ml of MA was added dropwise with constant stirring and the solution was left for 24 hours. After this, dialyzed against water for 24 hours. The resulting suspension was lyophilized. The product yield in all cases was more than 89%, but the highest yield was observed when the reaction was carried out at pH 6.8 and amounted to 94%. The degree of substitution was 2% when carrying out the reaction in a medium with pH 5.6, 4% in a medium with pH 6.0, and 13% in a medium with pH 6.8.

[0066] Methacrylation of HPMC was carried out using GMA according to the procedure described above, in a buffer medium with pH 6.8, resulting in a product with a yield of 93% and a degree of substitution of 16%. As in the case of the MA modification, in all spectra 1 After modification of the GMA, H NMR signals of protons at the double bond and signals of protons of the methyl group of the methacrylic acid residue appear (Fig. 2).

[0067] Example 3. Preparation of conjugates of methacrylated derivatives of HEC and HMPC with RGD peptide

[0068] 35 ml of acetate buffer solution with pH 5.5 were placed in a quartz flask, 250 mg of HEC-GMA with a methacrylation degree of 13% or HPMC-GMA with a methacrylation degree of 16% and 60 mg of RGD peptide were dissolved. Then 2-hydroxy-2-methylpropiophenone (Darocur-1173) was added to the solution in an amount of 1 mol% of the thiol group content. The resulting solution was irradiated with UV light in the range of 320-550 nm (the wavelength of the decomposition of the initiator into free radicals is 365 nm) using a chamber equipped with UV lamps on three sides with a total power of 90 W. Irradiation was carried out for 10 min. After completion of the thiol-ene click reaction (Fig. 3), the product was purified by dialysis against water for 5 days using 3500 cutoff mass dialysis bags. The collected water fractions from the outer chamber were evaporated to a small volume and the content of unreacted peptide was determined using the quantitative Ellman method for thiol groups.Ellman's reagent was prepared by dissolving 4 mg of 5,5'-dinitro-bis-(2-nitrobenzoic acid) in 1 ml of 0.1 M phosphate buffer solution (pH 8.0) containing 1 mol / L EDTA. Then, 2.5 ml of Ellman's reagent were added to a test tube containing 0.5 ml of the test solution. The mixture was left for 15 min at room temperature, and then the optical density of the solution was measured at a wavelength of 412 nm. The concentration of the peptide in the solution (and then the mass) were calculated using a calibration curve previously constructed for the same peptide. The amount of conjugated peptide was calculated from the difference between the initial amount and the amount of unreacted peptide. The degree of substitution was determined by relating the molar amount of the conjugated peptide to the molar amount of GMA substituents in HEC / HMPC. For the given method, the degrees of substitution were 9 and 11 mol.% for HEC and HPMC, respectively.

[0069] Example 4. Obtaining interpolymer composite films by rolling

[0070] 50 or 100 mg of cellulose derivative were added to 950 or 900 mg of PCL to achieve a filler content in the composite of 5 or 10 wt.%, respectively. The resulting mixture was heated in a drying oven until the PCL softened. The sample was then transferred to rollers with the upper roller temperature set at 25 °C and the lower roller temperature at 60 °C and rolled 20 times until the polymers were uniformly mixed. The softened composite film was left until completely solidified and then removed from the rollers. A schematic of the process of producing composite films by rolling, as well as a photograph of the film, are shown in Fig. 4. Control films based on PCL were prepared in the same way, but without the addition of filler.

[0071] Example 5. Evaluation of the physicochemical properties and surface topography of composites

[0072] To assess changes in the surface hydrophilic-hydrophobic properties, a drop of distilled water was placed on the film surface, and contact angles were measured using a goniometer. Five measurements were taken for each sample, and the average angle and standard deviation of the results were determined. The contact angle values ​​for some composites and a control film based on PCL are shown in Figure 5.

[0073] The swelling degree was determined by measuring the increase in film weight after immersion in water and incubation at human physiological temperature (37°C) for 24 h. For this purpose, 50-mg samples were cut from the films, placed in 10 ml of water, and left in a thermostatted orbital shaker (150 rpm). After 24 h, the films were removed from the water, water was carefully removed from the surface with filter paper, and the sample weight was determined. Three parallel measurements were performed for each sample, and the mean swelling degree and standard deviation were determined. The swelling degree values ​​for some samples are summarized in the histogram shown in Figure 6.

[0074] Surface topography was assessed using atomic force microscopy (AFM). Samples with a diameter of 15 mm were cut from the films for the study. Quantitative roughness assessment was performed in several zones measuring 50 μm × 50 μm or 70 μm × 70 μm (W × L).

[0075] Example 6. Evaluation of mechanical properties of composites

[0076] Uniaxial tensile testing (Fig. 7) was performed on film specimens measuring 10 mm × 50 mm. The tensile speed was 10 mm / min. Three parallel measurements were performed for each specimen, the average value of the obtained characteristics was determined, and the standard deviation was calculated.

[0077] Example 7. Biological properties in vitro

[0078] Mesenchymal stem cells (MSCs) were used to evaluate cell adhesion on the surface of the obtained composite films. The cells were cultured in DMEM / F12 culture medium containing fetal bovine serum (10 vol%), L-glutamine (2 mol / L), and gentamicin (10 mg / mL) at 37°C in a humidified atmosphere containing air and 5% CO2. Film samples with a diameter of 6 mm were placed into the wells of a non-adhesive 96-well plate. The films were fixed to the bottom of the plate using BF-6 medical glue. Then, 100 μl of a suspension containing 20,000 cells for an adhesion experiment (1 day) and 10,000 cells for a cell proliferation experiment (5 days) were added to each well. As a positive control, cells were seeded on the plastic surface of the wells of a 96-well adhesion plate. After the incubation period, the medium was removed and 100 μl / well of DMEM / F12 medium containing MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.1 mg / ml) was added.The cells were incubated in a CO2 incubator for 2 h at 37°C. The medium was then removed, and the formazan crystals formed by metabolically viable cells were dissolved in dimethyl sulfoxide (100 μl / well). The formazan solutions in DMSO were transferred to clean wells of a 96-well plate, and the optical density of the solutions was measured at 570 nm using a plate spectrophotometer. Cell adhesion and proliferation in the experimental samples were assessed as a percentage relative to the positive control surface. Comparative histograms of cell adhesion and proliferation on the surface of some interpolymer composite films are shown in Fig. 8.

[0079] Example 8. Study of the release rate of antibiotics and antibacterial properties of composites

[0080] Film samples with a diameter of 10 mm, containing 5% by weight of the antibiotic, were placed in test tubes containing 1 ml of 0.01 M phosphate-buffered saline, pH 7.4 (PBS) or PBS containing lipase from Candida rugosa (1300 IU / mg) at a concentration of 1 mg / ml. The films were incubated at 37 °C for 14 days. At certain intervals, the solution in which the films were incubated was replaced with a fresh portion. The collected solution was analyzed spectrophotometrically to determine the antibiotic concentration using a pre-constructed calibration curve. The medium into which the release was carried out was used as a reference solution. The optical density of ciprofloxacin solutions was measured at a wavelength of 275 nm, levofloxacin solutions at 290 nm, and moxifloxacin solutions at 295 nm.

[0081] To evaluate the antimicrobial activity of the samples, composite films with a diameter of 10 mm containing 5 wt% of the antibiotic were placed in test tubes containing suspensions of P. aeruginosa in nutrient broth with a titer of 10 6 CFU / ml. Films without antibiotics were used as controls. After 24 or 48 hours of incubation of the samples at 37°C, the number of colony-forming units (CFU) in the suspensions was assessed. After 24 hours, the bacterial count had decreased to 10 4 CFU / ml, while after 48 hours, inhibition of bacterial growth to 10 was observed. 2 CFU / ml depending on the antibiotic content in the composite. In the control, which did not contain antibiotics, bacterial growth of up to 10 was observed. 8 CFU / ml.

[0082] Example 9. Obtaining three-dimensional composite matrices by 3D printing

[0083] PCL film and composite samples cut into randomly shaped pieces were loaded into the cartridge of a pneumatic extruder of a 3D printer with a print head diameter of 0.4 mm. After setting the cartridge temperature, the printer waited 20 min before starting printing. Uniform material extrusion was observed at a temperature of 75 °C and a pressure of 450 kPa. Other printing parameters were as follows: print head-to-layer distance of 0.6 mm, substrate temperature of 35 °C, print head speed of 0.8 mm / s, and layer height of 0.33 mm. The print head lift-off movement was performed in the horizontal plane. Examples of printed products are shown in Fig. 9.

[0084] Abbreviations used

[0085] Abbreviation Transcript GEC hydroxyethyl cellulose GMPC hydroxypropyl methylcellulose GMA glycidyl methacrylate MA methacrylic anhydride GEC-GMA HEC covalently modified with GMA GMPC-GMA GMPC covalently modified with GMA GEC-MA HEC covalently modified with MA GMPC-MA GMPC covalently modified with MA RGD peptide RGD group peptide; in this work it has the sequence CGRGDSP PCL poly(ε-caprolactone) DMSO dimethyl sulfoxide MTT reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MSC mesenchymal stem cells

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Claims

1. An interpolymer composite based on poly(ε-caprolactone) and various cellulose derivatives, which are methacrylated hydroxypropyl methylcellulose, methacrylated hydroxyethyl cellulose, a conjugate of methacrylated hydroxypropyl methylcellulose with an RGD peptide, or a conjugate of methacrylated hydroxyethyl cellulose with an RGD peptide, for regenerative medicine (hereinafter: Composite), consisting of a matrix based on poly(ε-caprolactone), in which a filler is distributed, which is a cellulose derivative, in an amount of 5-10 wt.%.

2. The composite according to claim 1, in which hydroxypropyl methylcellulose or hydroxyethylcellulose, methacrylated with methacrylic anhydride or glycidyl methacrylate and characterized by a degree of methacrylation in the range from 2 to 16 mol.%, is used as a filler.

3. The composite according to claim 1, in which a conjugate of methacrylated hydroxypropyl methylcellulose with an RGD peptide or a conjugate of methacrylated hydroxyethyl cellulose with an RGD peptide with an RGD peptide content in the conjugate in the range of 2 to 11 mol.% is used as a filler.

4. The composite according to item 3, additionally containing a fluoroquinolone antibiotic from among ciprofloxacin, levofloxacin and moxifloxacin in an amount of 1 to 5% by weight.

5. A composite according to paragraphs 1-4, manufactured in the form of films or three-dimensional matrices.

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