Method for preparing biodegraded composite matrix of regenerated silk fibroin bombyx mori and its use

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine. The invention aims at creating biodegraded implants of the regenerated silk fibroin Bombyx mori. A method for preparing the biodegraded composite matrix of the regenerated silk fibroin Bombyx mori involves the stages: - dissolving the regenerated fibroin in a buffer containing CaCl2, C2H5OH, H2O in molar ratio 1:2:8. That is followed by dialysis of the prepared solution to the fibroin concentration min. 20 mg/ml. The fibroin solution of the concentration min. 20 mg/ml is mixed with dimethyl sulphoxide (DMSO). Gelatin or polylysine is added to the prepared mixture. The prepared mixture is frozen and thawed with an organic solvent added; what is also presented is the use of the specified biodegraded composite matrix as a base for the biodegraded implant.

EFFECT: group of inventions enables improving the biocompatibility, increasing the adhesive properties of matrix and cell proliferation in a mammalian body.

2 cl, 3 dwg, 3 ex, 1 tbl

 

The group of inventions relates to the field of creating biodegradable implants based on the regenerated fibroin of Bombyx mori silk.

Development of cellular technologies and the use of new methods of biochemistry and bioengineering opportunities for recovery of major defects of tissues and organs using established in vitro artificial fabrics. The basis of artificial fabrics are the holders of three-dimensional structure (called matrices or matrices), which serve as a substrate for adhesion of autologous cells, helping to maintain their proliferative activity, define the shape and physico-mechanical properties of the implant. Currently, preference is given to polymers of biological origin or composite materials based on them, as they possess high strength, no toxic effects, and, moreover, can serve as a highly effective biostimulants.

Of particular interest to researchers are attracted silk proteins. Biopolymers based silk cocoon silkworm well studied, because of the accessibility of the material and decades of experience of its use for medical purposes as a suture material. Source polymers usually are the cocoons of the silkworm Bombyx mori. The main polymer silk thread is fibroin, which can be the basis with the building of matrices for tissue engineering. Preparation of material for the synthesis of artificial matrices based on it include a step for purification from other substances that are included with silk thread, such as sericin, which are able to induce an immune response. Fibroin able to biodegradation under physiological conditions, do not have cytotoxic properties and is resistant to environmental conditions. In addition, the gut is very durable and elastic, so the products can be set firm fixed-form and used for the production of strong yet flexible structures. On the basis of a gut you can create composite matrices, the surface of which functionalities various biologically active substances that affect cell adhesion, proliferation and differentiation.

Fibroin is a protein consisting mainly of serine, glycine, alanine, and to a lesser extent - tyrosine, valine, aspartate and glutamic acid. Fibroin consists of light, which has a weight of 25 kDa, and heavy chains having a weight of 350 kDa, which are linked by disulfide bonds in the intracellular synthesis of membranes EPR and small glycated fragment P25, which has a weight of 25 kDa. Heavy, light chains and protein P25 usually associated total complex in the ratio 6:6:1 with a mass of 2.3 MDA [Altman GH et al. Silk-based biomaterials. Biomaterials 2003, 24(3), 401-416].

T is egernia the matrices of the porous structure are the basis for the maintenance of in vitro natural microenvironment for cell populations. On the basis of such matrices are designed bioisostere bodies specified shapes and physical characteristics. The ability to modify the surface of the material is biologically active molecules, such as components of the extracellular matrix, growth factors and differentiation, opens unlimited possibilities of control over the development of culture, allows to maintain the desired level of expression of certain genes and the functional activity of the cell population [Gellynck K, Verdonk P.C, Van Nimmen E., Almqvist K.F., Gheysens T., G. Schoukens, Van Langenhove L., Kiekens P., Mertens J., Verbruggen G. Silkworm and spider silk scaffolds for chondrocyte support. J. Mater. 2. Sci. Mater. Med.; 2008, 19(11): 3399-3409, Wang Y., Kirn H.J., Vunjak-Novakovic is G., Kaplan D.L. Stem cell-based tissue engineering with silk biomaterials.

Biomaterials; 2006, 24(36): 6064-6082.]. When developing a three-dimensional matrices are preferred polymers of natural origin (biopolymers) and various composite materials on their basis: alginates, collagen, gelatin, chitosan, hyaluronic acid, polyesters of bacterial origin [Volova T.G., Sevastianov V.I., shyshatskyy H. Polyoxyalkylated, 2nd edition. Krasnoyarsk: Platinum, 2006: s]. Such biopolymers possess high biocompatibility, ability to biodegradation (breakdown products are non-toxic to the body) and, often, the properties of biostimulants. Ability to biodegradation have not only the natural polymers ol the origin, but synthetic aliphatic polyesters, but, unlike the latter, the biopolymers in the decay do not form compounds with toxic effects. Decomposition products or derived from animal body or take an active part in physiological processes [Dawson E, Mapili G, Erickson K, Taqvi S, Roy K. Biomaterials for stem cell differentiation. Advanced drug delivery reviews; 2008, 60(2): 215-228, Ma, BC Biomimetic materials for tissue engineering. Advanced drug delivery reviews; 2008, 60(2): 184-198, E. Sachlos, Czemuszka J.T. Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. European cells and materials; 2003, 30(5): 29-39]. The ability to biodegradation of polymers have found application in the framework of another actively developing areas of bioengineering, engaged in the development of systems for drug delivery. The use of biodegradable polymers for drug delivery to the destination allows you to temporarily postpone active substances to the destruction of the polymer carrier in the target organ [Torchilin V. Multifunctional and stimuli-sensitive pharmaceutical nanocarriers. European Journal of Pharmaceutics and Biopharmaceutics; 2009, 71; 431-444].

The search for materials with a wide range of properties required for various tasks, has led to renewed interest in polymers, based on the silk. Sources of such polymers are the threads of the cocoon of the silkworm. For use in biology silk washed from accompanying impurities dissolved in a chemical solvent and precipitates in the form of regenerated silk fibroin. The design of this biopolymer can be used to create implants on the basis of its own cells. Such implants have a biological inertness, sufficient mechanical strength, ability to bioresorption and biodegradation, support the adhesion and proliferation of cells. Immunogenicity of silk fibroin extremely low [Panilaitis Century, Altman GH, Chen J., Jin HJ, Karageorgiou V, Kaplan D.L. Macrophage responses to silk. Biomaterials; 2003, 24(18): 3079-3085.] and implants on the basis of the regenerated silk fibroin dissolved with the formation of non-toxic products.

To improve the properties of the matrix to the fibroin add various polymers [Asconco, Alestalo, III. Biocompatible materials of regenerated silk for tissue engineering and drug therapy. Biotechnology, 2010, No. 1, p.9-16], in particular the addition of chitosan improves the mechanical properties of the material, but reduces its adhesive properties, compromising biocompatibility.

The creation of such materials generally include a serial multistage processing of cocoons of Bombyx mori [EN 2270209, 2006-02-20].

The prior art [Tamada Ya. New process to form a silk fibroin porous 3-D structure, Biomacromolecules. 2005 Nov-Dec; 6(6):3100-6] a simple and convenient method of obtaining material on the basis of the regenerated fibroin to create a biodegradable matrix, which includes stages of freezing and thawing. Also is this source describes the application of the obtained complex as the basis for the implant. This source of information is selected as a prototype.

Our proposed method of obtaining differs from the prototype in that we dissolve the regenerated fibroin in buffer containing CaCl2C2H5OHN2O in a molar ratio of 1:2:8, dilithium the resulting solution to a concentration of fibroin not lower than 20 mg/ml and mixing it with DMSO, and then implemented by inclusion in the composition of the additional matrix polymer (gelatin or polylysine).

In the claimed composite structures as the primary matrix material is used regenerated silk fibroin, and as an added - gelatin (the hydrolysis product of collagen - the main protein of the extracellular matrix that retains the biological properties similar to the properties of native collagen or polylysine. In the structure of gelatin exists adhesion peptide RGD, which increases the adhesion and proliferation of eukaryotic cells. Polylysin at neutral pH, positively charged, which also enhances the adhesion of eukaryotic cells.

Thus, the technical result of the claimed group of inventions is to improve the biocompatibility, significantly increased adhesion matrix and cell proliferation in the body of a mammal.

In addition, the use of the buffer soteriades the l 2C2H5OH, N2Oh, can significantly reduce the cost of getting the material on the basis of gut compared to the prototype.

BRIEF DESCRIPTION of DRAWINGS

1. Murine fibroblasts ZTZ on the surface of the matrix of regenerated silk fibroin. Scanning confocal microscopy, cell nuclei stained Sytox Green.

2. The porous structure of the matrix of regenerated silk fibroin obtained by freezing and thawing. Scanning electron microscopy, 400-fold increase.

3. Histological examination of cardiac matrices. Paint hematoxylin-eosin. 2 months after implantation displacing material of the matrix of connective tissue. Shows newly formed blood vessel.

EXAMPLES ILLUSTRATING PREDLAGAEMYE INVENTIONS

Materials and methods: instruments and reagents

Materials

Dry culture medium DMEM (Gibco Laboratories, Germany); culture plastic dishes (Nunc, Denmark and Costar, USA); all other reagents production Sigma-Aldrich Chemie GmbH (Steinheim, Germany).

Methods

The experiments were carried out in accordance with GOST R ISO 10993-19-2009 "Study of physico-chemical, morphological and topographical properties of materials".

Scanning electron microscopy

To study the structure of the matrices using the Ali methods scanning electron microscopy. Preparation of samples for electron microscopy were carried out by standard method comprising fixation with glutaraldehyde, postfixation 1% osmium tetroxide, dehydrated by increasing concentrations of ethanol followed by immersion in acetone. Further preparations were dried by a method of crossing a critical point in the device. Before viewing in the microscope the samples deposited layer of gold with a thickness of 20 nm in argon atmosphere at ion current of 6 mA and a pressure of 0.1 mm Hg on the instrument Ion Coater IB-3 (Eiko Engineering, MiTo, Japan). For scanning electron microscopy were used Camscan S2 (Cambridge Instruments, Cambridge, UK) in SEI mode. The resolution of the microscope 10 of them, the operating voltage of 20 kV. Images were obtained using the software MicroCapture (SMA, RF).

Laser confocal microscopy

For laser scanning confocal microscopy was used microscope Axiovert 200M LSM510 META (Carl Zeiss, Jena, Germany) with lenses of Plan-Neofluar 10×/0.3, and Plan-Neofluar 20×/0.5 and Plan-Neofluar 40×/1.3 To a Series of optical slices to obtain high resolution images received from one or synchronously with two channels installed according to the manufacturers instructions, the size of the pinhole (pinhole). The diameter of the pinhole was 1 airy disk (Airy unit), giving the optimal ratio of signal to noise. Set up set up the laser and analyzing filters according to Recomendar the Yam producers.

Cultivation of cells

Three-dimensional polymer matrix was used for culturing murine fibroblasts T. The samples were placed in a die for cell culture was added 300 μl of a suspension of murine fibroblasts T at a concentration of 24,000 cells in 1 ml culture medium DMEM (Sigma)containing 10% fetal bovine serum. Laundered not adherent to the substrate cells, and incubated matrix at 37°C in the presence of 6.1% CO2. Culture medium was changed every 2 days. For the quantitative analysis of cell adhesion used application functionality 3D for LSM Version 1.4.2 (Carl Zeiss, Jena, Germany) and Imaris 6.1.5 (Bitplane AG, Zurich, Switzerland). With their help got a high-contrast image, allowing a given color channel, clearly separated from the background voxels, corresponding to nuclei detected with a fluorescent dye. Counting the number of nuclei stained Sytox Green, three-dimensional images using 3D for LSM automatically: we determined the number of structures, uniform in color, corresponding to the volume of the nuclei of the studied cells. This calculation requires the use of high resolution images, with minimal autofluorescence background and the maximum ratio of signal to noise.

Implantation of polymer matrices under the skin of mice.

The studies were conducted in compliance and with GOST R ISO 10993-6-2009 "Study of local effects after implantation".

To study local effects after implantation and the ability to degradation, the matrices of the regenerated silk fibroin implanted subcutaneously laboratory mice of Balb/c four months of age. Prior to surgery the animals for 7 days were kept in vivarium for acclimatization. The keeping of animals and experimental conditions in vivo corresponded to GOST R ISO 10993-2-2009 Requirement "instinct" and GOST R ISO 10993-6-2009 "Study of local effects after implantation". Before implantation procedure removed the wool from the skin of mice depilatory cream. Conducted premedication with carprofen (carprofen): preparation Rimadyl (Pfizer Animal Health, USA) (with current substance carprofen) was injected at a rate of 5 mg per 1 kg, according to the manufacturer's recommendations.

The operation was conducted in sterile conditions under a General anaesthetic drug Zoletil 100 (Virbac Sante Animale, Kappoc, France) at a dose of 5 mg per 100 g Before implantation area of the skin at the site of incision was obezzarajivate 0.05% chlorhexidine solution (Biogen, RF), and the excess antiseptic was removed with sterile towels. For implantation did the cut length of 5 mm on the back of the animal, a subcutaneous pocket was formed by using a pointed trowel, separating the subcutaneous tissue from the muscle layer. The sample in the form of a disk with a diameter of 10 mm and thickness of 2 mm were placed in obrazovku the camping cavity using sterile tweezers. The incision was closed with non-absorbable polypropylene thread, imposing a simple knot surgical sutures. The wound was treated with antiseptic and covered with adhesive medical BF-6 (vertex ZAO, Russia). After waking up the animals were active and showed no signs of discomfort. After 2 months of implanted samples were removed for histological studies. Samples with adjacent tissue was removed, washed with phosphate-saline buffer and fixed in a mixture of Buena according to the standard method. Samples dehydrational in ethanol solutions of increasing concentration (ethanol solutions of 50%, 60%, 70%, 80%, 96%, 100%) for 2 hours each, removing the alcohol by incubation in a mixture of isopropanol and O-xylene (1:1) for 2 hours in two shifts pure O-xylene for 30 minutes. Dehydrated samples were prepared to fill in paraffin sequential incubation in the molten mixture Histomix® (BioVitrum, RF) and O-xylene (1:1) for 1 hour and, then, in three shifts molten Histomix® at 52°C for 30 minutes each. The prepared samples were placed in a mold for casting blocks and poured molten Histomix®.

Preparation of sections for histological studies

For the preparation of slices with a thickness of 5 μm of the prisoners in paraffin samples used microtome, Rotic-1 (Orion Medic, Russia). Sections were pasted on the surface of glass slides. Cuts to the guy who tological studies have deparaffinization sequential incubation in two shifts of O-xylene and 100% ethanol, were rinsed in 100% ethanol, two changes of 96% ethanol and two changes of distilled water.

Dewaxed sections were stained with a mixture of hematoxylin and eosin by standard procedures. Before staining, the sections were washed with distilled water. Next, the slice was incubated in a solution of alum of hematoxylin according to Ehrlich, washed in tap water, incubated in 1%aqueous solution of eosin and washed in tap water. After removal of excess water slices sequentially incubated in 96% ethanol and xylene, and then made into a balm.

Microscopic examination of the implant

Microscopic study of local effects after implantation was carried out according to GOST R ISO 10993-6-2009. Histological sections were examined using Zeiss microscope Imager Al (Zeiss) lenses Zeiss EC Plan-Neofluar 40×/0.75 In Ph2 and A-plan 20×70,45 Ph2. Images were obtained using camera AxioCam MRc 5 (Zeiss)and images were processed using the software AxioVision 4.7.2 (Zeiss). The sections were examined on a microscope Leica DM 1000 (Leica Microsystems, Inc., Bannockburn, USA) with Leica lenses Hi-Plan 40×/0,65 and 10×/0.25 and a Leica DFC 295 and the obtained images were processed using the software Leica Application Suite Version 3.4.0.

The implementation of the invention shown in examples

Examples

Example 1. The method of obtaining biodegradable HDMI is these matrix on the basis of the regenerated fibroin

Dissolve 150 mg of regenerated fibroin in previously prepared by stirring and heating the solution consisting of 513 mg l2, 388 μl of H2O, 420 μl of 96% With2H5HE (the molar ratio of the components: l2: C2H5HE: PLO 1:2:8). Vp-pa=1000-1050 µl. Fibroin=145 mg/ml of the resulting mixture was incubated in a water bath for 1 hour at 80°C until complete dissolution of the gut. Solution make in a dialysis bag. The dialysis is carried out against 500 ml of H2O for 80 minutes. Cialisovernight the fibroin solution with a concentration of not less than 20 mg/ml is mixed with DMSO (concentration of DMSO=1%)add gelatin or polylysine. The amount of gelatin or polylysine is from 10 to 90% of the total polymer. Then, the solution is stirred and poured into a mold. Then put in the refrigerator at - 18°C for a period of not less than 48 hours. The resulting matrix is taken out of the fridge, pour organic solvent, for example 96% ethanol and incubated for at least 60 minutes In the end you get a matrix with a pore size of from 10 to 200 μm (figure 1).

Example 2. Comparison of the adhesive properties of matrices

Carried out the preparation of the biodegradable composite matrix on the basis of the regenerated fibroin in the above manner.

Conducted a comparison of the matrices obtained by the method described in the prototype, and p is iegotovlenna according to the claimed method.

To determine the number attached to the matrices of cells studied samples by the method of confocal microscopy 24 hours after inoculation of murine fibroblasts T (Fig.2). Fibroblasts and secreted them components of the extracellular matrix are the basis of connective tissue that defines the key role of these cells in the regeneration of tissues of different organs. Samples were fixed in 4% formalin for 30 minutes and was identified cells SYTOX® Green nucleic acid stain (Invitrogen, Carlsbad, USA), nuclear dye with high affinity to nucleic acids. The matrix is incubated in the solution for 20 minutes with constant stirring environment to improve circulation and increase the availability of colour for the cells. After binding SYTOX Green with DNA observed an increase in its fluorescence 500 times. Method of confocal microscopy received image representing a series of horizontal optical sections of the matrix, allowing to observe cells and the structure of the matrix at a depth of up to 600 μm. Image used for counting cells. As can be seen from the table, adding in the composition of the matrix of gelatin or polylysine significantly increased the adhesion and proliferation of mouse fibroblasts T on the surface of the matrices.

We also were tested for adhesion and proliferation of mouse fibroblasts T on the surface m is trixul and other concentrations of gelatin or polylysine (in particular 50%, 90%), they also showed a significant increase in the adhesion and proliferation.

Example 3. Histological studies of implanted matrices

We obtained three-dimensional matrices were implanted under the skin of mice of BALB/c. Histological examination of samples obtained after extraction in two months, showed that inside the matrix is implemented connective tissue in the form of strands, consisting of fibroblast-like cells and their accompanying collagen fibers. Thus, there is a material degradation of the matrix and its replacement by young fibroblasts identified according to morphological characteristics, one of which is loose chromatin of the nucleus (euchromatin and 1-2 nucleoli). Histological study of the implants showed no phenotypic changes grow into the implant tissues, which could indicate violations of physiology in contact with the matrix cells. We also noted the high level of vascularization of tissues, replacing the matrix after implantation. Identified blood vessels lined by flat endothelial, and the newly formed vessels with high endothelium" (Fig.3). A large number of newly formed vessels testifies to the active processes of angiogenesis and canabrava occurring in the matrix 2 months after his impla is treated laboratory animals. The substitution matrix material vascularized tissue confirms the applicability of our matrices as the basis, including when designing bioisosteric bodies.

Comparison of the adhesion and proliferation of murine fibroblasts T on the surface of the matrices on the basis of the regenerated silk fibroin 24 hours after inoculation.
Sample matricesThe placeholder90% of the regenerated fibroin silk 10% gelatin90%of the regenerated fibroin silk 10% polylysin
The number of cells in 1 mm345±654±755±8

1. A method of obtaining a biodegradable composite matrix on the basis of the regenerated fibroin of Bombyx mori silk, which includes stages:
- dissolution of the regenerated fibroin in buffer containing l2With2H5HE, H2O in a molar ratio of 1:2:8,
- dialysis of the resulting solution to a concentration of fibroin not lower than 20 mg/ml;
mix the fibroin solution with a concentration of not less than 20 mg/ml with DMSO,
add gelatin or polylysine to receive the authorized mixture;
- freeze the mixture, followed by thawing in the presence of an organic solvent.

2. The use of biodegradable composite matrix obtained by the method according to claim 1, as a basis for biodegradable implant.



 

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