Bioactive resorbed porous 3d-matrix for regenerative medicine and method for preparing it
SUBSTANCE: bioactive porous 3D-matrix for tissue engineering involves a resorbed partially crystalline polymer having a porosity of 60-80% and a pore size of 2 to 100 mcm. A biopolymer gel having a particle size of 30-100 mcm is incorporated into a portion of the pores. A polymer/gel ratio makes 99:1 to 50:50 wt %. The matrix is prepared by grinding a mixture of gel and polymer powder having an average particle size of 100 mcm, and the prepared mixture fills prepared moulds to be placed in a high-pressure chamber wherein the temperature is increased to 25-40°C first, and then the CO2 pressure is increased to 4.0-25.0 MPa. The system is kept in the above environment for 1 hour, and then the chamber pressure is discharged to an atmospheric one for 30-120 minutes; thereafter the temperature is decreased to a room value, and the patterns are removed.
EFFECT: ensuring flexibility of using the matrix in various organs and systems, no toxicity, higher ability to tissue regeneration stimulation, prolonged effect of biostimulation.
6 cl, 5 ex, 1 tbl, 4 dwg
The invention relates to medicine, in particular to the replacement and regenerative medicine, and can be used in tissue engineering and replacement of bone defects and soft tissue as bioactive resorbable porous 3D-matrix (BCM).
A fundamental problem of modern clinical transplantation is a widespread shortage of donor organs, which are projected for the coming years, will only increase.
In recent years the search for alternative methods of compensation or replacement of damaged vital organs and tissues, the main emphasis in solving these problems is to use technology regenerative cell medicine [Atala a, Lanza R, Thomson J., Nerem R., Principles of regenerative medicine. Academic Press is an imprint of Elsevier, First edition, 2008, 1473 p.].
Technology regenerative cell medicine can be divided into three groups:
cell therapy using stem cells or signaling biomolecules to stimulate tissue regeneration processes;
- biostimulation of tissue regeneration patient with bioactive biopolymer materials;
- tissue engineering - tissue-engineered constructs (TECS) of organs and tissues.
In turn, the TEC includes the following components:
- autologous or allogeneic cells capable of forming fu is krioneri extracellular matrix;
- suitable biodegradable carrier (matrix frame) for transplantation of cells;
- bioactive molecules (cytokines, growth factors)that stimulate the cells of the damaged tissue [Plase E. S., Evans D. N., Stevens, M. M., Complexity in biomaterials for tissue engineering, Nature Materials, 2009, 8, pp.457-470; Sevastyanov Century. And., Perov H. B., German E. A., Sorochenko C. A., Ponomarev A. C., Examples of experimental and clinical application of biocompatible materials in regenerative medicine. In the book: Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, Part II, Chapter 3, S. 237-252].
Three-dimensional bioresorbable porous matrices are the basic elements in the replacement and regenerative medicine, will provide for the organization and maintenance of growth, proliferation and differentiation multipotent stromal cells in the formation of certain types of living tissues [Hench L., Jones D. Biomaterials, artificial organs and tissue engineering. The series "the World of biology and medicine", Moscow, Technosphere, 2007, 305 S.]. They contribute to the localization of cells in the area of implantation, at the same time as their media and acting as an analogue of the natural extracellular matrix (ACM) [Shumakov Century. And., Sevastyanov Century. And. Biopolymer matrices for artificial organs and tissues. Health is their and medical equipment. 2003, No. 4, S. 30-33].
One of the main problems of development and creation of the TEC necessary physico-chemical and biological properties is the choice of materials and technologies of forming a three-dimensional bioresorbable porous matrices of the desired architecture [Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, 544 pages]. For materials intended for implantation in a living organism, makes stringent and varied requirements. First and foremost is the biological compatibility. The material should not provoke an invalid cell response, to demonstrate immunogenicity and toxicity. Mechanical properties of the material should match the characteristics of the replaced tissue. The material needs to provide mechanical strength and resistance patterns in the implantation process, and then when the remodeling of the tissue. As with all materials in contact with the human body, the material of the matrices must be easily sterilized to prevent the invasion and spread of infection.
The matrices for the TEC is made from materials of synthetic and natural origin (metals and their alloys, synthetic polymers, ceramics, biostable, composite materials, chitosan, collagen, gelatin, elastin, fibronectin, alginate and other) soo the relevant medico-technical characteristics [of Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, 544 pages]. The matrices can also be obtained from biological tissue specific types through the removal of immunogenic components and preserves the original three-dimensional structure [of Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, 544 pages].
The use of bioresorbable polymers for forming the matrices are particularly attractive. In modern literature uses several terms that characterize the degree and mechanism of degradation of polymeric materials. According to the definition of M Werth [Vert M, Li m S,. Spenlehauer G, Guerin P., Bioresorbability and biocompatibility of aliphatic polyesters, J. Mater. Science: Mater. Med., 1992, 3, 432-436], biodegradiruemym are solid polymeric materials which disintegrate in the interaction with biological systems at the molecular fragments in the macromolecular degradation, dissipating in living tissues and, as a rule, remaining in them in the form of by-products. To include bioresorbable polymers that dissolve in the body and then they are absorbed as a result of biochemical reactions or processes of metabolism. Thus, bioresource leads eventually to the disappearance of a foreign body (implant) without any side effects.Implantable matrix from a bioresorbable material that acts as a temporary frame, contributing to the formation of Mature tissue. The speed of resorption of the material of the matrix should be correlated in a certain way with the rate of regeneration of tissue in each case. This process, ideally, should lead to the formation of new tissues of the required types, fully integrated with the tissues of the body that previously existed.
In sum, the matrices for the TICK must have the following crucial properties:
- versatility to perform simultaneously the functions of the frame, substrate and nutrient medium for cell cultures);
mechanical strength and elasticity sufficient to surgical manipulations;
- biocompatibility on the protein and cellular level;
- the ability to stimulate the proliferation and differentiation of cells;
porous structure that processes neovascularization;
- the possibility of sterilization standard ways without changing their physical-chemical and biochemical characteristics.
Widespread bioresorbable the matrices on the basis of biopolymer materials and synthetic resorbable polymers.
Among the natural materials it is necessary to pay attention to the collagen, which is I a major component of the extracellular matrix, its denatured form gelatin, alginate, a natural polysaccharide), which is like picolonate extracellular matrix, the solution of which can form hydrogels in the presence of divalent ions and chitosan and hyaluronic acid. [Sorochenko C. A., Matrices for tissue engineering and hybrid bodies. In the book: a Biocompatible material. Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, Part II, Chapter 1, S. 199-228; Chen L., Jones D., Biomaterials, artificial organs and tissue engineering. The series "the World of biology and medicine", Moscow, Technosphere, 2007, 305 S.].
Collagen has an extremely weak antigenic properties, also has an extremely weak anaphylactogenic and toxic properties. However, besides these advantages, a major disadvantage of collagen matrices is unregulated biodegradation time and limited term functioning of the collagen products (up to 1 month) in the context of a living organism, which is insufficient for full recovery and leads to the formation of scar tissue.
To reduce the speed of biodegradation have been developed methods of forming the heterogeneous supramolecular structure of hydrogels containing the main components of the extracellular matrix of farm animals [RF Patent №224946, RF patent №2433828].
The main disadvantage of biopolymer hydrogel materials is the impossibility of the creation of these frames configured with the necessary physical and mechanical properties.
Among synthetic biodegradable materials are widely used polymers and copolymers of lactic and glycolic acids, polycaprolactone, polypropylenimine, monongalia, polyarteritis and others [Sorochenko C. A., Matrices for tissue engineering and hybrid bodies. In the book: a Biocompatible material. Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, Part II, Chapter 1, S. 199-228., Vert M, Li M S,. Spenlehauer G, Guerin P., Bioresorbability and biocompatibility of aliphatic polyesters, J. Mater. Science: Mater. Med., 1992,. 3,. 432-436].
Analyzing the results of a large number of different experimental works devoted to the study of processes of interaction of the above-described polymer compounds with living tissue, you can get an idea of the average time of their bioresorption in the body [Hutmacher D., Markus M. S., Hurzeler Century, Schliephake H., A review of material properties of biodegradable and bioresorbable polymers and devices for GTR and GBR applications. Intern. J Oral&Maxillofacial Implants, 1996, v.11, pp.667-678]:
|1. poly(L-lactide)||18-36 months|
|2. poly(D,L-lactide)||4-6 months|
|3. polyglycolide||3-4 months|
|4. poly(D,L-lactide-glycolide) (50:50)||2-4 months|
|5. poly(D,L-lactide-glycolide) (85:15)||2-3 months|
|6. poly(D,L-lactide-ε-caprolacton) (90:10)||2-3 months|
The main disadvantage of synthetic aliphatic polymers is their high hydrophobicity, which negatively affects their interaction with cell cultures. In addition, weakly expressed bioactive properties of polylactide and its copolymers relative to cell proliferation and tissue regeneration significantly limit their use in replacement and regenerative medicine.
Analogs of the invention the inventive group can be considered different collagen substance, biocomposite and methods for their production, which can be used for surgical and bioplastic purposes: for example, the method of obtaining funds, stimulating reparative processes (patent RF №2065745, 1996); wound-healing coating (patent RF №2085217, 1997), the material for the plastic tissue (RF patent No. 2137441, 1999), reinforced graft for scleroplastic operations (patent RF №2140242, 1999), release agent (patent RF №2155592, 2000), biocompatible the polymer material and method thereof (patent RF №2162343, 2001), collagen containing material to keratinolytic (U.S. patent No. 6197330, 2001), the porous composite chitosan-gelatin scaffold for filling bone defects (patent RF №2421229, 2010).
In these developments is contained in the main description of the use of homogeneous collagen and denatured forms of gelatin in combination with resorbable or biostability polymers.
As the closest analogue (prototype), as in part BRPM, and the method of its production can be considered patent RF №2464987, where we are talking about how to get resorbable polylactide matrix for culturing and implantation of cells destined for the healing of wounds.
Known prototype method involves obtaining resorbable hydrophilic porous polylactide matrix uniformly covered microfibrillar collagen 1st type. Pre-receive hydrophobic porous polylactide matrix with a thickness of 13 μm to 15 μm and a pore diameter of from 2 μm to 3 μm. This is followed by the application of a hydrophobic porous polylactide matrix of 0.01%solution of collagen 1st type in 0.1%acetic acid, incubated for 30 minutes at room temperature, then the surface of the matrix was washed with phosphate-saline buffer pH of 7.4 to remove unbound with the substrate protein. This% is the fool are investigated. Hydrophilic porous surface polylactide matrix uniformly covered microfibrillar structures of collagen in diameter from 10 nm to 20 nm, is used for the cultivation of human keratinocytes.
The main disadvantages of this method of producing a porous composite matrix are:
- the use of toxic organic solvent (methylene chloride) when creating polylactide matrix;
- there is limited potential for use as a TICK, since the received resorbable matrix of polylactide and collagen type I (not thicker than 15 μm, the pore diameter of 2-3 microns) are not intended for use as 3D matrices in tissue-engineered constructs, except for the TICK of the skin;
- poorly expressed regenerative properties due to the use of only one component VKM - collagen type I.
There are different techniques of manufacturing highly porous (60-90 %by volume) of biocompatible matrices (media frames) for tissue engineering: a method of ultraresponsive hydrogels with subsequent stitching; non-woven binding polymer filaments; leaching method; pore formation using gases; methods with phase separation; electrospinning; method biointerface; formation of porous matrices using technologies such as supercritical fluids (SCF) [You, the LEC Century. N. Methods for the manufacture of matrices. In the book: Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, Part II, Chapter 2, S. 229-236].
However, the analysis of scientific literature we were unable to find information about BCM representing a composite material of synthetic partially-crystalline polymer medical devices and incorporated in part of its pores heterogeneous biopolymer gel, which could be used as a universal TICK for replacement and regenerative medicine of various organs and systems. Accordingly, in the prior art there is no information about how to obtain such BRPM.
We were set a task to create a solid BRPM with high bio-stimulating properties, which can be used as a universal TICK in regenerative and substitution medicine of various organs and systems.
Technical result achieved using the proposed group of inventions is:
- achieving the universality of application of the created RPM in TEAK various organs and systems by 1) combining the positive properties of resorbable polymers - receiving material with the desired mechanical and biochemical properties, and biopolymer gels - hydrop lnost, expressed biostimulation of tissue, 2) the possibility of obtaining BRPM any size and shape, 3) use as a feedstock for a wide range of synthetic polymers; 4) formation on the surface of the pores of the matrix of hydrophilic-hydrophobic patterns that mimic the hydrophilic-hydrophobic surface of cell membranes;
in the absence of toxicity obtained BCM by eliminating the toxic action of organic solvents and remove soluble in supercritical CO2(SC-CO2toxic compounds;
- strengthen the capacity to stimulate the regeneration of body tissues due to the use in the composition of the matrix bioactive gel components;
- increase the duration of effect of biostimulation due to the slower speed of resorption of one of the component BBM-heterogeneous gel.
The proposed method involves the removal of soluble in SC-CO2toxic compounds (unreacted monomers and low molecular weight oligomers, polymerization initiators, plasticizers, and so on), thereby increasing the biocompatibility of the emerging matrix.
It is important to note that the formation of the matrices in our method is carried out at temperatures close to room that allows you to use as a gel thermolabile components of biologically active is heterogeneous biopolymer gel, and does not require the use of additional organic solvents. In addition, SC-CO2easily and almost without residue is removed from the polymer after completion of the process is simple discharge pressure below a critical value.
The essence of the proposed group of inventions is as follows.
BBM for tissue engineering is a composite material made of resorbable synthetic partially-crystalline polymer for medical purposes in the form of a 3D matrix and heterogeneous biopolymer gel. Porosity PPM ranges from 60 to 90%, and the pore size ranges from 2 to 100 μm. When this heterogeneous biopolymer gel with a particle size of 30-100 μm, the viscosity of 15-70 PA and a modulus of elasticity 45-1200 PA incorporated into part time. The ratio of polymer/gel is from 99:1 to 50:50 wt.%, the value of the contact angle of wetting the surface of the matrix in water from 60 degrees to 20 degrees, respectively. In the particular case of heterogeneous biopolymer gel includes a functional additive selected from the group: hydroxyapatite, growth factors, cytokines, antioxidants, or mixtures thereof. The method of obtaining a patent pending bioactive resorbable porous 3D scaffold for tissue engineering includes the following operations. Mechanically triturated mixture of powder resorbable synthetic h is partially-crystalline polymer medical purpose with an average particle size of 100 μm and heterogeneous biopolymer gel in the ratio of polymer/gel from 99:1 to 50:50 wt.%. Fill the prepared mixture of the mold. Pre-mold degrease and clean in SC-CO2at 60°C, a pressure of 20 MPa for 1 hour. The filled mold is placed in a high pressure chamber, where the first increase of the temperature up to 25-40°C, and then the pressure of CO2to 4.0-25,0 MPa. The system is maintained under these conditions for 1 hour. Then within 30-120 minutes to relieve the pressure in the reaction chamber to atmospheric values, and then lower the temperature to room and extract samples.
In the particular case of samples sterilized by γ-irradiation at a dose of 15 kGy.
In the particular case of the powder polymer is a copolymer polielektrolita brand PDLG7507 with a particle size of from 50 to 200 microns.
In the particular case of heterogeneous biopolymer gel contains a functional additive selected from the group comprising hydroxyapatite, growth factors, cytokines, antioxidants, or mixtures thereof.
The essence of the invention is illustrated in the following figures:
Fig.1A is a General view of the microstructure resorbable porous matrix of PDLG7507. Scanning electron microscope JSM-6360 LA (Jeol, Japan), the marker 200 microns.
Fig.1B is a detailed microstructure resorbable porous 3-D matrix of PDLG7507 showing the distribution of pore size. Scanning electron microscope JSM-6360 LA (Jeol, Japan), the marker 100 microns;
Fig.2A - General view of the microstructure of bioactive resorbable porous 3-D matrix of PDLG7507 and heterogeneous biopolymer gel (Spherogel). Scanning electron microscope JSM-6360 LA (Jeol, Japan), the marker 100 microns;
Fig.2B is a detailed microstructure of bioactive resorbable porous 3-D matrix of PDLG7507 and heterogeneous biopolymer gel (Spherogel), illustrating the presence of free pores and pores filled with a biopolymer gel. Scanning electron microscope JSM-6360 LA (Jeol, Japan), marker - 2 microns;
Fig.3 - the results of the study the metabolic activity of fibroblasts mouse line NIH 3T3, adhered on the surface of the resorbable porous 3-D matrix of PDLG7507 and bioactive resorbable porous 3-D matrix of PDLG7507 and heterogeneous biopolymer gel (Spherogel).
Fig.4 - the results of the reaction of the tissues adjacent to the implant (implantation site) at 14 days after subcutaneous implantation of a resorbable porous 3-D matrix of PDLG7507 and bioactive resorbable porous 3-D matrix of PDLG7507 and heterogeneous biopolymer gel (Spherogel). And control (PDLG7507), B - samples BRPM, where 1 - fragments of a porous matrix; 2 - proliferating fibroblast-like cells of the mouse. Staining with hematoxylin and eosin. Optical microscope. Increase 400.
The inventive method of obtaining the proposed us BBM OS is p, as follows.
As the source resorbable polymeric materials can be used any resorbable synthetic partially-crystalline polymers intended for biomedical research and clinical applications. These include, in particular, include:
- aliphatic polyesters, including polylactic and polyglycolic acid, and their copolymers, copolymers of polylactic and polyglycolic acids with polyethylene glycol (PEG), polycaprolactone (such as poly-ε-caprolactone and poly-γ-caprolactone), poly-p-dioxanone, polydioxanone, polypropylene fumarate, and others;
- polyphosphazene, including polychlorobenzene, preorganisation and others;
- aliphatic polyanhydride;
the polyacrylates, including polimetilmetakrilat and their copolymers;
- polyvinyl, including polyvinyl acetate, polyvinyl alcohol and its copolymers, polivinilpirolidon and others;
The above polymers can be used to create a three-dimensional porous matrices using technologies such as supercritical fluids.
As the powder of the polymer may be selected, for example, the copolymer polielektrolita brand PDLG7507 with a particle size of from 50 to 200 microns.
Hydrophilic bioactive component of the matrix may be, for example, injecting the composition of heterogeneous implantable gel sphere®GEL is (produced by CJSC "BIOMER service", Moscow), with pronounced biostimulating properties and approved for clinical use (no SDF 2012/13033 from 01.02.2012 year).
In powder synthetic resorbable partially-crystalline polymer type heterogeneous biopolymer hydrogel with subsequent mechanical grinding of the mixture. Mechanical grinding may be performed, for example, in an agate or porcelain mortar.
Pre-fat and refined in SC-CO2at 60°C and 20 MPa for one hour of the mold fill with the prepared mixture.
The filled mold is placed in a high pressure chamber, where the first increase of the temperature up to 25-40°C, and then the pressure of CO2to 4.0-25,0 MPa. The system is maintained under these conditions for 1 hour, after which within 30-120 minutes to relieve the pressure in the reaction chamber to atmospheric values, and then lower the temperature to room and extract samples.
As the high-pressure chamber can be used, for example, installation SCF synthesis [Popov, C. K., Implants in the replacement and regenerative medicine bone tissues. In the book: Biocompatible materials (training manual). Ed. by C. I. Sevastianov and M. P. Kirpichnikov. Publishing house "MIA", M., 2011, Part II, Chapter 4, S. 253-294].
Samples sterilized by γ-irradiation at a dose of 15 kGy.
For radiation sterilization when enaut:
- radiation technology unit (RTU) with electron accelerators for industrial purposes with a maximum energy of 10 MeV;
- radiation production equipment with radioactive sources of ionizing radiation closed-type60Co.
Radiation sterilization is carried out in accordance with GOST R 50325-92 "medical devices. Methods of dosimetry during the process of radiation sterilization and MI 2548-99 "GSE. Installation of a radiation technology with radionuclide sources for sterilization of medical products. Methodology certification or MI 2549-99 "GSE. Installation of a radiation technology with electron accelerators for sterilization of medical products. Methodology certification". The fact of irradiation products confirm the color indicators of the absorbed dose, which must comply with the requirements of GOST R ISO 11140-1-2000 "Sterilization of medical products. Chemical indicators. Part 1. General requirements, control of the sterilization process should be carried out according to GOST R ISO 13485-2004, GOST R 50325-92, GOST R ISO 11137-1-2008, GOST R ISO 11137-3-2008.
Biopolymer heterogeneous gel may contain a functional additive selected from the group of water-soluble biologically active substances, including growth factors, cytokines, antioxidants or sm is si, for example, nanosized powder hydroxapatite, tumor necrosis factor, insulin-like growth factors (IGF-1 and IGF-2), fibroblast growth factors, dihydroquercetin, which before by mechanical mixing of the polymer and hydrogel component is dissolved or dispersed in a certain ratio in the hydrogel phase.
For the production of specimens of a particular configuration used Teflon mold of a given size. For example, for the manufacture of three-dimensional samples in the form of discs can be used molds, consisting of a set of solid PTFE disks with a thickness of 2 mm, and a disk thickness of 6 mm, hole 5 mm in diameter, arranged concentrically relative to the center, which strung alternately on the stud, stainless steel threaded on the ends. To simultaneously obtain multiple samples of the mold was assembled into strips, consisting of 8 molds.
Holder of several molds are placed in a high pressure chamber setup for SCF processing of polymeric materials.
The chamber is closed, compacted and checked for leaks, napuka it carbon dioxide and gradually increasing the pressure up to 5.5 MPa (initial pressure in the cylinder with CO2) at room temperature (~20°C). Then include the heater and increase the temperature of the system is neither to 25-40°C. Upon reaching a predetermined temperature to increase the pressure of CO2to 4.0-25,0 MPa and maintain the system in these conditions for 1 hour. After that, within 30-120 minutes to relieve the pressure in the reaction chamber to atmospheric values, and then lower the temperature to room and extract samples.
For evidence of a possible implementation of the stated purpose and achievement of the technical result here is the following data.
In the private case of the implementation of the present invention receive BBM for tissue engineering in the following way. In an agate mortar agatova pestle, pulverize the mixture powder of the copolymer polielektrolita brand PDLG7507 (lactide/glycolide = 75:25, Mw~20 kDa and Mw~100 kDa, respectively) with an average particle size of 100 μm and a composition of heterogeneous implantable gel Spherogel with an average particle size of 150 microns, modulus of viscosity 15-70 PA and a modulus of elasticity 45-1200 PA ratio of polymer:gel, 75:25 wt.%. The size of the particles Spherules previously reduced to 30-100 μm. The prepared mixture was filled pre-fat and treated in supercritical CO2(60°C, 20 MPa, 1 h) of the mold (inner diameter 5 mm and height 2 mm) followed by a space holder of several molds in the high pressure chamber installation GFR is INTESO. The camera closed, condensed and checked for leaks, napuka it carbon dioxide and gradually increasing the pressure up to 5.5 MPa (initial pressure in the cylinder with CO2) at room temperature (~20°C). Part of the gas was stravovali for final flushing and cleaning of the cell from the atmosphere of air. Then turn on the heater and the temperature of the system to a pre-selected temperature to 40°C. Upon reaching the desired temperature, increased pressure of CO2to the value of 25.0 MPa and kept the system under these conditions for 1 hour. Then, within 30 minutes, gradually threw off the pressure to the atmospheric value and left to cool down the mixture in the chamber to room temperature, then extracted samples in the form of porous disks right cylindrical shape with a diameter of 5 mm and height 2 mm
Similarly, received resorbable porous 3D-matrix only from a powder of a copolymer polyacetale brand PDLG7507, which served as control. All the analyzed samples BCM control and sterilized by γ-irradiation at a dose of 15 kGy.
Micrograph of the surface morphology of the samples resorbable porous matrix of polielektrolita (control) and BRPM shown in Fig.1A, B and 2A, B, respectively.
All samples have uniform porosity of from 60% to 90%, with pore size in the range from 2 μm to 100 m the m The porosity of the samples was determined by the formula Ω=1-m/m0where m is the mass of the porous sample, and m0- weight monolithic (source) of the polymer is equal to the volume density ρ=1,21 g/cm3.
The values of the contact angle of wetting on the water, measured at the device for the measurement of boundary corners 101 ITSELF (KSV Instruments, Germany) for BCM and control were equal (36±14)° and (68±9)°, respectively.
Tests in vitro cytotoxicity claimed BRPM.
On the culture of mouse fibroblasts line NIH 3T3 investigated the cytotoxicity of three samples (code No. 1, 2, 3) porous polymer disks in the form of discs of diameter 5 mm and height 2 mm, produced according to the method described in Example 1. As control samples were the same shape, made of polielektrolita brand PDLG7507 the same way. Negative control served as cultural DMEM containing no serum, and positive standard solution of zinc in nitric acid (Zn 1-2 wt. % HNO3dilution 1:200 0.9% NaCl for injection).
Tests for cytotoxicity conducted in accordance with the following:
- GOST R ISO 10993.1-99 "medical Products. Assessment of biological effects of medical products. Part 1. Evaluation and research"
- GOST R ISO 10993.5-99 "medical Products. Assessment of biological effects of medical products. Part 5. Investigated the e on the in vitro cytotoxicity".
The results showed (table)that all samples offered by us BRPM (n=3) under the conditions of this experiment do not have a cytotoxic effect on the culture of mouse fibroblasts line NIH 3T3.
|The degree of response of fibroblasts mouse line NIH 3T3 on the analyzed sample|
|PPM (n=3)||No||Not cytotoxic|
|Negative control||No||Not cytotoxic|
|The positive control||Sharp||Cytotoxic|
Tests in vitro matrix properties BRPM.
On the culture of mouse fibroblasts line NIH 3T3 investigated matrix properties RPM disks in the form of discs of diameter 5 mm and height 2 mm, produced according to the method described in Example 1. As control samples were the same shape, made of polielektrolita brand PDLG7507 the same way. Under the matrix t is you samples refers to the ability to support the attachment and proliferation of fibroblasts mouse line NIH 3T3, i.e. bio-stimulating properties of samples.
Under aseptic conditions, fibroblasts mouse line NIH 3T3 were sown on the surface of the samples at a concentration of 1×105/sample, were incubated for 2 h at 37°C under standard conditions: humid atmosphere containing (5±1) % CO2giving the cells to penetrate into the pores and to register, after which the samples were placed in a microtube containing complete growth medium (ORS).
Samples with cells were incubated at 37°C in a humid atmosphere containing 5±1% CO2. After 24 h, 48 h, 72 h and 96 h were evaluated by metabolic activity of the cells with vital reagent prestoBlue® (Invitrogen™, USA).
The results of the study the metabolic activity of fibroblasts mouse line NIH 3T3 shown in Fig.3, which shows data characterizing the metabolic activity of fibroblasts mouse line NIH 3T3 after sowing on samples of control and BRPM (∗ - significant differences, p<0,05).
From a comparison of the two curves (Fig.3) shows that after 24 h metabolic activity in mouse fibroblasts, which characterizes the number desirously cells in the first hours of incubation, significantly more for BBM. At the subsequent timing of cell culturing samples on the metabolic activity of fibroblasts mouse, proportional to their proliferative activity was significantly (p<0.5) is higher for clicks is scov PPM compared with control, which proves the bioactive properties of the proposed composite matrix (the increase in the number of adhered cells and increase their rate of proliferation), due to the presence in its composition of bioactive biopolymer gel.
Tests in vivo BRPM.
Investigated samples made according to the method described in Example 1.
Irritating effect in a single installation into the conjunctival eye bag rabbit no. On the scale of the reaction corresponded to zero degrees.
Study of the reactions of General anaphylaxis and active cutaneous anaphylaxis in Guinea pigs showed no allergic reactions anaphylactic type.
Sensitizing effect on white rats is not detected, the reaction degranulation of mast cells is negative.
Implantation performed on rats (intramuscularly). The observation period of 3 months. Morphological studies of the pathological changes in the surrounding tissue was not detected.
Sterility: the samples Tested sterile.
Progenote: Extracts prepared with 0.9% sodium chloride for injection, pyrogenic reactions when administered intravenously to rabbits did not show. The total temperature rise did not exceed 1.4°C, the samples are not progeny.
Tests in vivo bioactive properties of the RPM.
Investigated samples made according to the method described in Example 1.
To study in vivo bioactive properties BRPM samples BRPM implanted subcutaneously 10 mice-males line DBA. The control group (3 mice-male line DBA) implanted samples of porous disks made polielektrolita brand PDLG7507 (control). After 14 and 28 days after implantation, animals were taken from experiment, and place the implant with the implant were fixed in 10% formalin and sent for histology.
The samples were obezvozhivani in ethanol with ascending concentrations were degreased in a mixture of absolute alcohol and chloroform or xylene and embedded in paraffin with the addition of beeswax. Slices with a thickness of 4-5 μm was obtained by using a Leica microtome, model RM 3255, Germany. The resulting slices were transferred to a glass slide. Histological specimens were stained with hematoxylin and eosin, alcian blue, milori and collagen type II.
In Fig.4 shows the results of the reaction of the tissues adjacent to the implant (implantation site) at 14 days after subcutaneous implantation of porous samples. And control (PDLG7507), B - samples BRPM, where 1 - fragments of a porous matrix; 2 - proliferating fibroblast-like cells of the mouse. Staining with hematoxylin and eosin. Increase 400.
From the comparison of two histol the environmental sections of the sites of implantation shows in the control and in BRPM on the 14th day are proliferating fibroblast-like cells around the fragments of the porous matrix. However, the number of proliferating fibroblast-like cells for BBM significantly higher compared to control, indicating a biostimulating properties BRPM.
Thus, we have derived bioactive solid matrix with a high bio-stimulating properties, which can be used in regenerative and substitution medicine different organs systems. On the basis of experiments conducted in vitro and in vivo, it can be argued that the analyzed samples BRPM are biocompatible and bioactive.
The priority scope of the proposed BCM is using it as a replacement for defects of bone and soft tissue and in tissue engineering and regenerative medicine to activate the regeneration of the patient's own tissue, creating a tissue-engineered structures of bone and soft tissue involving autologous or allogeneic cell cultures. In addition, this product can serve as a depot of biologically active substances when creating prolonged dosage forms.
The advantages of the proposed method to obtain BCM are as follows:
1. as a source of polymeric materials can be used with any of sorbiruemyh partially-crystalline polymers for medical purposes;
2. the ability to obtain 3D matrices of various sizes and shapes, which are determined only by the size of the molds and reaction chambers that can be used to create tissue-engineered structures of various organs and tissues.
3. the ability to form solid bioactive resorbable matrix by incorporation into the porous structure of the polymer material is a biopolymer hydrogel component of the extracellular matrix of tissues of farm animals, preserving its bioactive (regenerative) properties.
4. No toxic organic solvents to obtain the composite matrix.
1. Bioactive resorbable porous 3D scaffold for tissue engineering, representing a composite material made of resorbable synthetic partially-crystalline polymer for medical purposes in the form of a 3D matrix with a pore size in the range from 2 to 100 μm, a porosity of 60-80% and incorporated in part of its pores heterogeneous biopolymer gel particle size of 30-100 μm, the viscosity of 15-70 PA and a modulus of elasticity 45-1200 PA in the ratio of polymer/gel from 99:1 to 50:50 wt.%, with the value of the contact angle of wetting the surface of the matrix in water from 60 degrees to 20 degrees, respectively.
2. Bioactive resorbable porous 3D-matrix DL is tissue engineering by p. 1, in which heterogeneous biopolymer gel includes a functional additive selected from the group: hydroxyapatite, growth factors, cytokines, antioxidants, or mixtures thereof.
3. Method for the production of bioactive resorbable porous 3D scaffold for tissue engineering by p. 1, wherein mechanically triturated mixture of powder resorbable synthetic partially-crystalline polymer medical purpose with an average particle size of 100 μm and heterogeneous biopolymer gel in the ratio of polymer/gel from 99:1 to 50:50 wt.%, fill the prepared mixture of the mold, pre-fat and treated in supercritical CO2at 60°C, a pressure of 20 MPa for 1 hour, then filled the mold is placed in a high pressure chamber, where the first increase of the temperature up to 25-40°C, and then the pressure of CO2to 4.0-25,0 MPa and maintain the system in these conditions for 1 hour, after which within 30-120 minutes to relieve the pressure in the high pressure chamber to atmospheric values, and then lower the temperature to room and extract samples.
4. The method according to p. 3, in which the sample is sterilized by γ-irradiation at a dose of 15 kGy.
5. The method according to p. 3, in which the powder of the polymer is a copolymer polielektrolita brand PDLG7507 with a particle size of from 50 to 200 microns.
6. The method according to any of paragraphs.3, 4, 5, colorability heterogeneous gel contains functional additive, selected from the group comprising hydroxyapatite, growth factors, cytokines, antioxidants, or mixtures thereof.
SUBSTANCE: tissue regeneration or healing is stimulated when using a structure comprising a multilayer plate of a collagen membrane material, which contains a lamellated barrier material of pure collagen prepared of a natural collagen tissue; the lamellated barrier material containing a barrier layer with an outer smooth barrier surface and a fibre surface, which is opposite the outer smooth barrier surface. The structure additionally contains a matrix layer of a collagen sponge material adjoining the fibre surface.
EFFECT: matrix layer of the collagen sponge material is absorbed by an individual's body at a higher rate, than the lamellated barrier material.
20 cl, 3 dwg, 5 ex
SUBSTANCE: invention refers to medicine. There are described methods for making implantable medical devices, preferentially of PEEK, having antimicrobial properties. The antimicrobial action is ensured by implantation of ceramic particles containing antimicrobial metal cations into the molten PEEK resin to be cooled and finally shaped by injection moulding, cutting and mechanical treatment or by other processing methods.
EFFECT: implants possess effective antimicrobial action for reducing a bacterial growth and a risk of infection.
12 cl, 1 dwg, 3 tbl
SUBSTANCE: invention refers to medicine. What is described is a method for preparing a cell-free organic tissue of a human or animal origin for the vitality recovery, particularly for introducing living cells, involving a stage of making a number of holes (4; 14) in the cell-free organic tissue (2; 12) through its surface (8; 18) and setting in the tissue (2; 12); wherein the said number of holes (4; 14) is formed using a needle or a kit of needles. The holes (4; 14) are partially intersected thereby forming partially connected holes (4; 14).
EFFECT: invention also refers to a respective cell-free organic tissue (2; 12) of the human or animal origin.
17 cl, 3 dwg
FIELD: process engineering.
SUBSTANCE: invention relates to medicine. Proposed method can be used in stomatology and orthopedics for production of medical materials stimulating recovery of bone tissue defects, for making dental stopping and dental pastes. It comprises preparation of mix containing compounds of calcium, phosphorus, silicon and sodium, impregnation of bioinertial incombustible porous matrix with made mix, matrix is composed of ceramics from aluminium or zirconium oxides followed by calcination. Note here that silicon compound represents tetraethoxysilane. Note also that phosphoric acid ether is used as phosphorus compound. Calcium and sodium compounds are represented by their carboxylates in polar organic solvent. This method includes making the thin layers on more strong bioinertial porous ceramics. Note also that said process involves no special complicated equipment and expensive reagents.
EFFECT: production of glass ceramics directly from solution omitting sol preparation stage, simplified and accelerated process.
7 cl, 5 ex
SUBSTANCE: invention refers to porous microsphere granules with the adjusted particle size for bone tissue regeneration. The above microspheres have a size within the range of 1-1000 mcm, have through pores of the size of 1-100 mcm and total porosity 40-75%. The declared microsphere granules are prepared by granulation by electrospinning, and heat-treated. A mixture used to form the granules by electrospinning contains a mixture of magnesium orthophosphate and biological hydroxyapatite of bovine demineralised bones in ratio 0.5:1.0, as well as 1-3% sodium alginate in distilled water and a hardener representing saturated calcium chloride.
EFFECT: invention provides preparing the microsphere granules possessing biocompatibility, biodegradation, osteoinduction and osteoconduction properties and able to be substituted by the bone tissue.
SUBSTANCE: invention relates to medicine, namely to ophthalmosurgery, and in particular to scleroplasty. Transplant for scleroplasty has polymeric base, covered with porous layer of the same polymer. As polymer base, transplant includes layer, made from porous stretched polytetrafluoroethylene, which has nodular-fibrillar structure. As porous layer, it includes layer of porous polytetrafluoroethylene, which has volume fraction of void space 15-40%, specific surface of void space 0.25-0.55 mcm2/mcm3, average distance between voids in volume 25-30 mcm and average chord volume 8-25 mcm, with the total width of transplant constituting 0.15-0.35 mm (first version). Transplant for scleroplasty can also include porous layer of polymer, whose surface is processed to add compatibility with sclera tissue. Transplant surface is processed by application of allogenic dermal fibroblasts of 3-5 passages of culturing, with the total width of transplant being 0.15-0.35 mm (second version).
EFFECT: chemically and biologically inert transplant, which ensures effect of invasion of sclera tissues, is obtained.
SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents artificial dura mater, produced from electrospinning layers by technology of electorspinning, with electrospinning layer, consisting of, at least, hydrophobic electrospining layer, which is produced from one or several hydrophobic polymers, selected from polylatic acid and polycaprolactone.
EFFECT: invention ensures creation of artificial dura mater, which has good tissue compatibility, anti-adhesiveness and possibility of introducing medications, preventing cerebrospinal fluid outflow during regeneration of person's own dura mater.
30 cl, 7 ex, 11 dwg
SUBSTANCE: invention refers to medicine, particularly to ophthalmic and maxillofacial surgery, and aims at repairing post-traumatic defects and deformations of the eye-pit bottom and walls. What is described is an implant in the form of a solid perforated plate which is formed by photocuring of a light curing composition; when heated the implant (a cure temperature 70-90°C) keeps the shape after self-cooling that provides high strength and biocompatible properties of the material. The cure temperature of the material is much higher than a temperature of a human body that ensures maintaining the physical and mechanical properties of the implant inserted in the human body, namely the material remains strong, when fixed it shows no cutting and breaking. Before the implantation, the plate is heated to the required temperature and bent so that it is congruently repeats the eye-pit shape in a place of the defect, overlaps the defect and supports the eye in the right anatomical position.
EFFECT: reducing the likelihood of graft rejection and reducing a quantity of various complications by improving the biocompatible properties of the material.
1 tbl, 5 ex
SUBSTANCE: group of inventions refers to medicine. In a method for making an antimicrobial silver-containing endoprosthesis according to the 1 version, a mesh material is made of synthetic polymer fibres with a silver-containing composition in a solution, dried; the endoprosthesis made of polyvinylidene fluoride monofilaments of the diameter of 0.09-0.15 mm in the form of a form-stable combined weaved knitted cloth at surface density of the endoprosthesis 80-200 g/m2, processed for 2-4 hours in 3-15% silver nitrate solution in dimethylsulphoxide, dried in air for 17-20 hours, processed again, dried, additionally processed in the composition consisting of 4-8 wt % of aqueous sodium hydroxide, 6-10 wt % of aqueous ammonium and 0.3-0.7 wt % of aqueous glucose in proportions 100:4:4, at temperature 20-25°C, and then washed in water, and dried. According to the 2 version, the endoprosthesis made of polypropylene monofilaments of the diameter of 0.07-0.15 mm, at surface density 20-120 g/m2, processed in 5-15% silver nitrate solution in dimethylsulphoxide, dried for 5-8 hours, processed again, dried, processed in the composition (as provided by the first version) in proportions 100:7:9 at temperature 20-25°C, and then washed in water, and dried.
EFFECT: group of inventions provides prolonged antimicrobial action of the endoprosthesis.
2 cl, 2 tbl, 3 dwg
SUBSTANCE: invention refers to medicine, particularly traumatology and orthopedics, and can find application in treating false joints of long bones. What is described is a method for preparing a non-toxic porous implant of polylactic acid with controlled pore size for building of long bone defects involving melting the polylactic acid granules in a melting pot immersed in thermally stable oil with porogen presented by sodium chloride crystals 100-250 nm to be washed out by aqueous solutions of antiseptics. The implant is prepared with no toxic solvents added and has a pre-set pore diameter.
EFFECT: improved efficiency of the method.
SUBSTANCE: conduit wall is presented by a material of random micro- and nanofibres of a bioresorptive polymer of poly(ε-caprolactone), and the content is presented by a self-assembled nanostructured hydrogel of acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™) oligopeptide. The above conduit is implanted in a complex with the direct local delivery of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) genes to be introduced into the proximal and distal nerve segments, while the formed conduit is implanted into a nerve rupture, and its ends are fixed with epineural sutures.
EFFECT: invention provides a stimulating effect on the invasion of regenerative medullated fibres, on the recovery of motor and sensitive nerve function, and enables improving the effect of the recovery of the nerve structure and function after the extended ruptures.
SUBSTANCE: claimed invention is aimed at manufacturing intraocular lens (IOL), for introduction of posterior eye chamber in form of PC Phakic lens. IOL is formed from hydrogel material, formed by cross-linked polymer and copolymer component. Lens includes UV chromophore, which is benzotriazole.
EFFECT: IOL hydrogel material usually has relatively high index of refraction and/or possesses desirable degree of protection against irradiation.
12 cl, 3 tbl
SUBSTANCE: invention relates to medicine. Described is implant, which can be injected in subcutaneous or intracutaneous way in form of monophase hydrogel, which contains gel, obtained from cross-linked hyaluronic acid and one of its physiologically acceptable salts.
EFFECT: obtaining subcutaneous implant used for filling wrinkles and stimulation of epidermal cells and/or supporting mechanical properties of skin density and elasticity.
15 cl, 2 ex
SUBSTANCE: invention relates to field of medicine, in particular to method of obtaining form-preserving aggregates of gel particles, in which aggregates are held together by physical forces of non-covalent bonds, such as hydrophobic-hydrophilic interactions and hydrogen bonds. Method of obtaining form-preserving aggregates of gel particles includes introduction of preliminarily obtained suspension of gel particles in polar liquid, where gel particles have absolute electrochemical potential, into receiving medium, in which absolute electrochemical potential of gel particles decreases, which results in fusion of gel particles into form-preserving aggregate.
EFFECT: invention allows to obtain form-preserving gel aggregates in situ so that form of aggregate is determined by place of application.
49 cl, 35 ex, 11 tbl, 33 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to medicine, more specifically to biocompatible alginate systems with the delayed gelatinisation process. There are offered sets and compositions for making a self-gelatinised alginate gel containing sterile water-soluble alginate and particles of sterile water insoluble alginate with a gelling ion. There are offered methods for dosing self-gelatinised alginate dispersion for making the self-gelatinised alginate gel. The methods can include dosing the dispersion in an individual. There is offered the self-gelatinised alginate gel of the thickness more 5 mm and not containing one or more sulphates, citrates, phosphates, lactates, EDTA or lipids. There are offered implanted devices coated with the homogeneous alginate gel. There are offered methods for improving viability of pancreatic islets or other cell aggregate or tissue, after recovery and while stored and transported.
EFFECT: group of inventions provides creation of the alginate gelling system which contains alginate and the gelling ions with high biological compatibility; enables the gelatinisation process without pH variations, connected with the other systems, and requires minimum ingredients, thus provides variation of gelatinisation time and gel strength depending on the specific requirements.
62 cl, 11 dwg, 2 tbl, 27 ex
SUBSTANCE: invention relates to a method of producing biologically compatible gel which is thickened with cross-linked polymer by cross-linking a given amount of at least one biologically compatible natural polymer in a solution by adding a defined amount of cross-linking agent, an additional amount of polymer with molecular weight over 500000 dalton in a solution, in which the reaction mixture is diluted to reduce concentration of polymer in the solution, and the cross-linking reaction is stopped by removing the cross-linking agent.
EFFECT: gel and its use for separating, replacing or filling biological tissue or for increasing volume of such tissue, or supplementing or replacing biological fluid.
11 cl, 1 tbl, 4 ex
SUBSTANCE: invention concerns medicine. Particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives which are suitable for injection with gel particles having the size in a range from 1 to 5 mm at action of a physiological saline solution are described. An implant for increase of volume of the soft tissues, containing particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives where the basic volume of the specified particles represents the gel particles, suitable for injection and having the size in a range from 1 to 5 mm at action of a physiological saline solution, is described. The way of increase of volume of soft tissues at a mammal, including a human being, including subepidermal introduction in a place of a body of the specified mammal in which it is desirable to enlarge volume of soft tissues is described.
EFFECT: augmentation of volume of soft tissues at a mammal.
24 cl, 4 ex
SUBSTANCE: invention relates to field of medicine. Claimed is composition with hyaluronic acid (HA), which includes gel particles of bound water-insoluble hydrated HA. HA includes bindings, represented with the following structural formula: HK'-U-R2-U-TK'. Where each group HA' represents the same or other molecule of bound HA'; each U independently represents optionally substituted 0-acylisourea or N-acylurea; and R2 represents optionally substituted alkyl, alkenyl, alkinyl, alkoxy, cycloalkyl, cycloalkenyl, cycloalkinyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroaralkyl or heterocyclolalkyl. Also claimed is method of developing tissues in individual, including introduction of needle into individual in place where development of tissues is necessary, needle is connected to syringe filled with composition with HA, and applying force to syringe in order to supply composition with HA to individual. Method of obtaining composition with HA includes formation of water-insoluble dehydrated particles of bound HA, separating insoluble in water particles by their average diameter, selection of subset of particles by average diameter and hydration of subset of dehydrated particles by means of physiologically compatible water solution. Other method of obtaining composition with bound HA includes binding precursor of bound HA by means of bis-carbodiimide in presence of pH buffer and dehydration of bound HA. Also included is method of developing tissues in individual that needs tissue development. Method of stabilisation of bound HA includes hydration of water-insoluble dehydrated bound HA by means of physiologically compatible water solution which includes local anesthetic, so that value of elasticity module G' for stabilised composition constitutes not less than approximately 110% from value G' for non-stabilised composition.
EFFECT: claimed composition of hyaluronic acid and method of preparation and application of HA composition are efficient for development of tissue and/or drug delivery.
27 cl, 22 ex, 2 tbl, 7 dwg
SUBSTANCE: invention concerns medicine, namely to reconstructive surgery, traumatology-orthopedy, maxillofacial surgery, stomatology and can be applied at osteo-plastic operations. For delivery of medical products immediately in a zone of defect and their prolonged influence in the centre of a lesion medicinal preparations are dissolved in a normal saline solution in a dose providing local effect, collagen-containing component is added to a solution in the ratio 9-20 g: 100 ml of a solution also admix with the carrier from dispersed allotransplants in the ratio of 1:1-3.
EFFECT: method allows lowering a dose necessary for reception of medical effect in 10 times, and also allows accelerating reparative processes in a defect zone.
SUBSTANCE: method of antibiotics fixation within porous implants is described. Result of method application lies in possibility of reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound due to application of 10% gelatine solution as antibiotic carrier. Specified result is achieved by filling porous implants with antibiotic solution in liquid gel. For this purpose implant is dipped in solution by 3/4. Filling occurs under the influence of capillary forces. After solution cooled to form dense gel, antibiotic is fixed in implant pores and gradually released after installation to bone defect area.
EFFECT: reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound.
3 cl, 1 ex
SUBSTANCE: present invention refers to methods for coating depositions on surgical needles of a metal alloy. A needle is covered with a lower coating containing vinyl-functionalised organopolysiloxane; the lower coating is cured, and the needle is covered with an upper coating containing polydimethylsiloxane with the lower coating bound to the upper one. The lower and upper coatings can be deposited by means of nozzles. According to the versions of the invention, covering the needle of the metal alloy with the lower coating is followed by depositing an undercoating containing polyalkylsiloxane and tetraethyl silicate covalently bound to the metal alloy. Further, depositing the lower coating is combined by binding the lower coating with the undercoating.
EFFECT: coated surgical needles are durable and can be multiply deposited through a tissue with a minimum effort.
24 cl, 10 dwg, 6 tbl, 4 ex