BIORESORBABLE BIOLOGICAL MATRIX FOR REPAIRING BONE TISSUE DEFECTS AND METHOD FOR THE PRODUCTION THEREOF

Abstract

The invention relates to medical biotechnology, medicine, traumatology, orthopedics, dentistry, and orthodontics. A method is proposed for producing a biological matrix intended for the repairing of bone tissue defects; said method may include a plurality of consecutive stages, such as pre-treating biological material, coarse filtering and fractionating, fine filtering and extracting, delipidizing, fermenting, demineralizing, and sterilizing in supercritical fluid. The resulting bioresorbable biological matrix is characterized by increased osteo- and biointegration, an optimal biodegradation rate, high biocompatibility, an absence of recipient immunoreactivity, high osteoconduction capacity, and pronounced osteogenic potential in osteosynthesis and bone grafting. The matrix consists of ossein, hydroxyapatite and/or calcium phosphate, wherein the ossein is in native unreduced form with its three-dimensional structure completely intact, the hydroxyapatite and calcium phosphate are in native amorphous form, and the matrix itself, from which cellular debris, foreign lipids, nucleic acids, and immunogens have been removed, contains residual amounts of bone morphogenetic proteins and is impregnated with vesicular phosphatidylcholine and/or cholesterol containing gelatin (hydrolyzed collagen), and/or bone atelocollagen, and/or poly-(ε-caprolactone) and additionally containing biologically active substances (including bioactive peptides and growth factors).

Claims

1. A method for producing a bioresorbable biological matrix for replacing bone defects from a xenogenic or allogeneic bone tissue, comprising the following successive stages: a. preliminary processing of the xenogenic or allogeneic bone tissue, comprising one or more cycles of freezing at −20 to −80° C. and thawing at +5 to +37° C.; b. deep cleaning and extracting of the material obtained in the previous stage, comprising treatment of this material in a solution of ionic or ionic and amphoteric detergent in a buffer solution, then, optionally, washing in a buffer solution, then further processing in a solution of non-ionic detergent, followed by extracting the material using an ultrasonic disintegrator; c. delipidating the material obtained in the previous stage, comprising processing the material in a solution of ethanol or ethanol-chloroform or ethanol-ethyl acetate and toluene, and then processing with sodium hydroxide; d. demineralizing the material obtained in the previous stage, comprising treating the material with a strong acid solution, followed by neutralizing with sodium hydroxide; e. impregnating the material obtained in the previous stage with a solution containing vesicular phosphatidylcholine or cholesterol, wherein the solution further contains gelatin (hydrolyzed collagen) or bone atelocollagen or poly-(e-caprolactone).

2. The method according to claim 1, wherein between the preliminary processing and deep cleaning stages, there is an additional stage of coarse cleaning and fractionation of the material obtained in the preliminary processing stage, in which a spongy layer is separated partially or completely from a cortical layer, and/or a diaphysis is separated partially or completely from a metaphysis and epiphysis (pineal gland), and/or a periosteal and middle layer is separated partially or completely from an endosteal layer.

3. The method according to claim 2, wherein the partial separation of the periosteal and middle layer from the endosteal layer is carried out to a depth of 8-10 mm.

4. The method according to claim 1, wherein at the stage of deep cleaning and extraction, sodium dodecyl sulfate and/or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in a concentration of 0.1-10% are used as ionic detergent at 37-41° C.; and Triton X-100 at a concentration of 0.1-5% and/or Tween-20 at a concentration of 0.005-1% are used as non-ionic detergents at 1-8° C.

5. The method according to claim 1, wherein between the stages of delipidation and demineralization an additional stage of fermentation of the processed material is carried out, comprising processing of the material in solutions of DNase I or trypsin.

6. The method according to claim 1, wherein there is an additional stage of sterilization of the material obtained at the stage of demineralization between the stages of demineralization and impregnation, which includes processing of the material with a supercritical carbon dioxide.

7. The method according to claim 6, wherein at the sterilization stage, the material is statically saturated with the supercritical carbon dioxide at 35-41° C. for 1-3 hours with a constant supply of carbon dioxide at a speed of 1.5-5 kg/h.

8. The method according to claim 1, wherein the material is additionally impregnated with bioactive peptides or growth factors at the stage of impregnation.

9. The method according to claim 1, wherein at the stage of impregnation, the impregnation of the material is carried out using an ultrasonic disintegrator.

10. A bioresorbable biological matrix for replacement of bone defects obtained by the method according to claim 1, consisting of bone collagen, hydroxyapatite and calcium phosphate, and also containing vesicular phosphatidylcholine or cholesterol, and further containing gelatin (hydrolyzed collagen), or bone atelocollagen, or poly-(ε-caprolactone), wherein the matrix is characterized by the fact that bone collagen is presented in a native form with a preserved three-dimensional structure; hydroxyapatite and calcium phosphate are presented in a native amorphous form; and the matrix itself is essentially free from cell debris, foreign lipids, nucleic acids and immunogens, has osteoconductive and osteoinductive potential, is sterile and can be presented in various forms.

11-23. (canceled)

24. The bioresorbable biological matrix according to claim 10, wherein the matrix additionally contains an autologous stromal-vascular fraction and/or mesenchymal stem cells added during the impregnation stage; and/or human blood plasma enriched with platelets, added during the impregnation stage; and/or autologous blood; and/or bioactive peptides and/or growth factors; and/or an antibiotic, antimycotic, antiseptic and/or anesthetic agents.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] FIG. 1. Generalized scheme of a method for producing bioresorbable biological matrix in a preferred embodiment.

[0113] FIG. 2. Biological matrix surface closeup photography.

[0114] FIG. 3. Histological section of the biological matrix (hematoxylin-eosin staining).

[0115] FIG. 4. Microelectron diffraction pattern of the biological matrix surface.

[0116] FIG. 5. The formation of fibrocartilage callus on Day 30.

[0117] FIG. 6. Complete fusion of bone tissue on Day 60.

[0118] FIG. 7. X-ray of bone fusion: a—control group; b—experimental group.

DETAILED DESCRIPTION OF THE INVENTION

[0119] In the description of this invention, the terms “includes” and “including” are interpreted as “includes, but is not limited to.” These terms are not intended to be construed as “consists of only”.

[0120] Unless defined separately, technical and scientific terms in this application have standard meanings generally accepted in the scientific and technical literature.

[0121] Any fragment of a mammalian organ or tissue can be used as a biological material for the implementation of the invention. In a preferred embodiment, an auto-, allo- or xenomaterial of mammalian bone tissue is used.

[0122] Bioactive peptides or growth factors that can be impregnated to enhance osteoconductive or osteoinductive properties in a purified matrix prepared in accordance with one embodiment include bone morphogenetic proteins (BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18), vascular endothelial growth factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E), transforming growth factors beta (TGF-beta1, TGF-beta2, TGF-beta3), stem cell factors (SCF), platelet-derived growth factors (PDGF-A, PDGF-B, PDGF-C, PDGF-D), insulin-like growth factors (IGF), fibroblast growth factors (FGF), connective tissue growth factors (CTGF-1, CTGF-2, CTGF-3), osteoprotegerin (sRANKL).

[0123] The following examples are provided to disclose the characteristics of this invention and should not be construed as in any way limiting the scope of the invention.

[0124] An example of production of the bioresorbable matrix

[0125] A method consisting of several successive stages was developed and experimentally tested to obtain an effective and safe bioresorbable biological matrix for the replacement of bone tissue defects. It involves the stage of preliminary processing of biological material, rough cleaning and fractionation, deep cleaning and extraction, delipidation, fermentation, demineralization and sterilization in supercritical environments.

[0126] At the pre-treatment stage, to remove the blood, fat and connective tissue residues, the biological sample undergoes several freezing and thawing cycles, processing in a solution of hydrogen peroxide and antibiotics/antimycotics (disinfectants), and freezing is carried out at −20 to −40° C., and thawing—at room temperature in a water bath.

[0127] At the stage of rough cleaning and fractionation in order to separate a biological sample, in this case, the bone tissue of a mammal, the spongy layer is separated from the cortical layer and/or the diaphysis is separated from the metaphysis and epiphysis, and/or the periosteal and middle layer are separated from the endosteal layer, with the periosteal layer separated to a depth of 8-10 mm.

[0128] At the stage of deep cleaning and extraction, for the purpose of decellularization and purification from antigens and debris, the sample is treated in a solution of ionic and amphoteric surfactants at 37-41° C., then, to precipitate surfactants, they are washed in buffer solutions at 1-8 C, then ionic surfactants are neutralized, and sodium dodecyl sulfate and/or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfone are used as ionic surfactants at a concentration of 0.1-10%, to neutralization and renaturation using Triton X-100 at a concentration of 0.1-5% and/or Tween-20 at a concentration of 0.005-1%, and the extraction is carried out using an ultrasonic disintegrator.

[0129] At the delipidation stage, to remove residual fatty acids and lipids, the sample is treated in a solution of ethanol and/or ethanol-chloroform and/or ethanol-ethyl acetate and toluene, and after delipidation, sodium hydroxide is treated at a concentration of 0.5-20 g/L.

[0130] At the fermentation stage, treatment is carried out in solutions of DNase and/or trypsin, and trypsin treatment is carried out in the presence of magnesium chloride at a concentration of 0.05-5 mM.

[0131] At the stage of demineralization, the solution is treated with a strong acid solution, followed by neutralization with sodium hydroxide; at the stage of sterilization in supercritical media, it is treated with supercritical CO.sub.2, and the sample is initially statically saturated with a supercritical solvent at 35-41° C. for 1-3 hours with a further constant supply of CO.sub.2 at a speed of 1.5-5 kg/h.

[0132] To improve the osteoinductive properties of the biological matrix, the prepared purified biological sample is impregnated with emulsified vesicular phosphatidylcholine and cholesterol with additionally included substances. For this purpose, a solution of phosphatidylcholine and/or cholesterol with additives is evaporated in a rotary evaporator to obtain a wall film, resuspended in phosphate-buffered saline, and the impregnation is carried out using an ultrasonic disintegrator. Gelatin (hydrolyzed collagen) and/or bone atelocollagen, and/or poly-(e-caprolactone), and, in a preferred embodiment, biologically active substances such as bioactive peptides or growth factors, are used as substances additionally included in fat vesicles. Another biodegradable synthetic polymer may be used instead of poly(e-caprolactone).

[0133] Thus, during the procedure, a sample of biological tissue undergoes: (1) cleaning and disinfection; (2) manual or automated cleaning of soft tissues and the fibrous layer of the periosteum with subsequent fractionation and disinfecting washing; (3) a step-wise treatment with ionic/amphoteric and nonionic detergents with intermediate and final washing; (4) delipidation with washing if necessary; (5) fermentation followed by washing; (6) demineralization followed by washing; (7) sterilization in supercritical media; and (8) impregnation with emulsified vesicular phosphatidylcholine/cholesterol with biologically active substances (including bioactive peptides and growth factors).

[0134] The above method allows obtaining a biological matrix for the replacement of defects in bone tissue, consisting of bone collagen, hydroxyapatite and/or calcium phosphate, and the bone collagen in this matrix has a native unrestored form with a fully preserved 3D structure, hydroxyapatite and calcium phosphate have a native amorphous form, and the matrix itself, purified from cell debris, foreign lipids, nucleic acids and immunogens, contains residual amounts of bone morphogenetic proteins, has osteoconductive and osteoinductive potential, satisfactory physical and mechanical properties, it is sterile and may have various forms.

[0135] Moreover, the resulting bioresorbable biological matrix can have a form of a block, crumbs, fine powder, paste, membrane, and plate. At various stages of the described method, preferably at the stage of sterilization under supercritical media or immediately after this stage, the synthetic polymer poly(e-caprolactone), hydrolyzed collagen, atelocollagen, bioactive peptides, growth factors, antibiotics, antiseptics, anesthetics individually or combinations thereof) can be impregnated into the biological matrix, while these substances are administered as part of fat vesicles consisting of phosphatidylcholine and/or cholesterol.

[0136] Moreover, the resulting bioresorbable biological matrix immediately before use can be enriched with an autologous stromal-vascular fraction, a culture of mesenchymal stem cells, an air-conditioned culture medium enriched with platelets, plasma or autologous blood. Methods for preparing the stromal-vascular fraction and culture of mesenchymal stem cells are well known in the art and are described in numerous sources (i.e. WO 2016044780 A1).

[0137] The bioresorbable biological matrix obtained using the above method can be used in clinical practice, namely, in traumatology, orthopedics, dentistry and orthodontics to fill bone defects in the treatment of congenital pathologies, various injuries, benign tumors, for use as an implant and as a drug carrier.

[0138] Characterization of the resulting bioresorbable matrix.

[0139] Prototypes of the biological matrix were obtained from the femur of cattle. Samples were washed and treated with disinfectants and prepared according to the above procedure. The bone matrix was fractionated into usable blocks containing both the cortical layer and the spongy layer.

[0140] The morphology of the samples, sterility of the bone matrix based on the number of colony-forming units (CFU) (according to GOST ISO 11737-2-2011), and the content of residual lipids (Bazarnova Yu. G., Theoretical foundations of food research methods, 2014) were evaluated as control parameters; Skurikhina I. M., Chemical composition of food products, 1987), residual DNA content (DNA-Sorb-C, AmpliSens; detection was carried out by fluorometric method), calcium content (according to GOST ISO 12081-2013), protein content (according to Kjeldahl according to GOST 938.7-68 in modification—pharmacopoeial monograph of the Ministry of Health of the Russian Federation), pH and humidity (moisture content analyzer MB35, Ohaus).

[0141] Samples of bone matrix were divided into the following groups: (1) native tissue—bone tissue that has not undergone processing (n=10); (2) BM—demineralized biological matrix (n=10); (3) BM/SCE—demineralized biological matrix, additionally processed in supercritical environments; (4) BM/IMP—a demineralized biological matrix impregnated with vesicular phosphatidylcholine and cholesterol with hydrolyzed collagen. The results of the comparative analysis of the control parameters of the test groups are presented in Table 1. Data are presented as the mean and standard deviation, M (y).

TABLE-US-00001 TABLE 1 Comparative analysis of control parameters of bone matrix. Group Native tissue BM BM/SCE BM/IMP Parameter (1) (2) (3) (4) No. of samples 10 10 10 10 Weight, mg 480 (40.8) 249 (18.7) 130 (21.7) 170 (19.4) CFU, CFU/mL — 0 0 0 DNA, ng/mg 312.5 (21.7) 0.14 (0.4) 0.27 (0.1) 0.2 (0.06) Total protein mg/g 14.96 (3.4) — Calcium, WCa % 37.8 (7.5) 17.6 (3.6) 19.7 (3.4) 16.1 (4.2) Lipids, % 14 (3.4) <2 <1 5.1 (0.9) pH 7.2 (0.6) 6.3 (0.4) 6.8 (0.5) 7.1 (0.4) Humidity, % 75.0 (15.4) 1.8 (0.04) 1.4 (0.7) 5.1 (0.3)

[0142] An ultrastructural study of the surface of the biological matrix was carried out using a scanning electron microscope to assess the preservation of the structure. It was shown that the biological matrix after processing fully retained its 3D organization. The most representative results are shown in FIG. 2, 3, 4.

[0143] The cleaved surface of the sample was a porous structure consisting of: 0.5-1 μm micropores which are intertwined and form a microporous network: 10-20 μm pores, as well as large cavity formations (50-100 μm). Pores permeate the entire thickness of the sample. The pores did not have foreign inclusions. There are no pores on the outer surface; a slit-like and dense material structure was observed.

[0144] An experimental model was used to evaluate the osteoinductance of the biological matrix, described in the reference (Miguleva I. Yu. Two new models of an experimental bone defect on the lower leg of a rat for studying bone tissue regeneration after plasty with various materials, 2015).

[0145] The animal welfare in vivarium was standard and consistent with the requirements of the European Convention (Strasbourg, 1986) and the Helsinki Declaration of the World Medical Association on the Humane Treatment of Animals (1996). All manipulations with animals were carried out in accordance with ISO 10993-1-2003 and GOST R ISO 10993.2-2006. The requirements of Order of the Ministry of Health of the USSR No. 755 of Aug. 12, 1977 “On measures to further improve the organizational forms of work with experimental animals”, the annexes to Order of the Ministry of Health of the USSR No. 755 of Aug. 12, 1977 “Rules for the use of experimental animals” and the Federal Law adopted by the State Duma on Dec. 1, 1999, “On the protection of animals from cruel treatment” were met during the research.

[0146] The tibia of anesthetized animals was accessed, the muscle layer and periosteum were dissected, and a cut was performed on the front surface of the tibia closer to the metaphyseal zone, where the bone has an extension and a thicker wall. The biological matrix was introduced into the defect and the wound was sutured. On Day 30 and Day 60, the animals were taken out of the experiment.

[0147] The results of the study showed that the biological matrix had osteoinductive properties and could cause bone regeneration. The formation of fibrocartilage callus is observed on Day 30 after implantation in all experimental groups. The most representative results are shown in FIG. 5. Complete fusion and regeneration of bone tissue was observed in all experimental groups after 60 days after implantation, in the complete absence of aseptic inflammation. The most representative results are shown in FIG. 6. The X-ray in FIG. 7 shows signs of bone fusion as compared with the control group, and the resulting bone-cartilage callus is similar in density to healthy bone tissue of the tibia.

[0148] Thus, this invention describes a method for producing a biological matrix for the replacement of bone defects, which includes the following stages: (1) pretreatment of biological material; (2) coarse cleaning and fractionation; (3) deep cleaning and extraction; (4) delipidation; (5) fermentation; (6) demineralization; (7) sterilization in supercritical media.

[0149] The biological matrix described above, consisting of bone collagen, hydroxyapatite and/or calcium phosphate, is characterized in that bone collagen has a native unreduced form with a fully preserved 3D structure, hydroxyapatite and calcium phosphate have a native amorphous form, and the matrix itself, purified from cellular debris, foreign lipids, nucleic acids and immunogens, contains residual amounts of bone morphogenetic proteins, has osteoconductive and osteoinductive potential, satisfactory physico-mechanical properties, is sterile and can have various forms, moreover, the biological matrix can be impregnated with vesicular phosphatidylcholine and/or cholesterol, which additionally includes gelatin (hydrolyzed collagen) and/or bone atelocollagen, and/or poly(e-caprolactone) with a complex of biologically active substances (such as bioactive peptides or growth factors), and is additionally sterilized and purified in supercritical environments.

[0150] Although this invention has been described in detail with examples of options that appear to be preferred, it must be taken into account that these examples of the invention are provided only for illustrative purposes. This description should not be construed as limiting the scope of the invention, since the stages of the described methods and devices, etc., may be amended by specialists in the field of medical biotechnology and cell technology to adapt them to specific devices or situations, and not beyond the scope of the claims. Specialist in this field understands that various options and modifications, including equivalent solutions, are possible within the scope of the invention, which is defined by the claims.

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