Method for stimulating neurotisation using nanostructured matrix and genetic constructs
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.
The invention relates to medicine, pre-emptive scope of its application - neurosurgery and traumatology, the treatment of long peripheral nerve gaps.
During reconstructive surgery to overcome long gaps nerve used autonervous box, which involves filling the defect with a fragment of another, functionally less important nerve. To save the nerve-donor and not disable its function as an alternative autonervous insert actively developing nerve conduits, tabulirovanie patterns that guide and support the growth of regenerating nerve fibers.
The conduit nerve consists of walls and contents. Of materials for the walls of the conduit, the most promising are bionetwork polymers, which provide the resorption of the conduit after a successful nerve regeneration and, unlike conduits based binarystring materials, does not require re-operation for removal of the conduit. The wall of the conduit should have adequate mechanical properties (strength, elasticity), optimal timing of bierstone and characterized by high permeability to molecules of the microenvironment that supports neuroregeneration. These requirements adequately meets the wall of the conduit, comprising nanostrukturirovannogo method electrospinning biorastvorimym polymer poly(ε-caprolactone).
The contents of the conduit is a three - dimensional matrix that fills the potential space growth of nerve fibers and imitating the structure of the extracellular matrix of biological tissue. By analogy with the extracellular matrix of the contents of the conduit should have a hydrogel nature. The main requirement for hydrogel contents of the conduit is to maintain the survival, migration and proliferation of Schwann and endothelial cells, which is necessary for neovascularization and regeneration of nerve fibers. Criteria maintain the survival of cells in vitro and regeneration in vivo self-assembling nanostructured hydrogel-based oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™) is one of the most appropriate conduit for nerve.
Improving the efficiency of neuroregenerative for plastic nerves through conduits associated with the simultaneous involvement of gene and cell therapy. Direct gene therapy (gene therapy in vivo) involves the injection of a DNA-containing vectors in the area of damage to the nervous tissue. This method eliminates the possibility of malignant transformation of transplanted cells is also investigated for the purpose of delivery of therapeutic genes. From the past to stimulate neuroregeneration the most studied genes for neurotrophic factors. Promising stimulus is tori nerve regeneration are presented vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). These factors are both neurotrophic and angiogenic. As neurotrophic they support the survival of neurons and stimulate axonal growth, and as angiogenic stimulate the formation and growth of new blood vessels, which also contributes to neurodegeneration. For delivery to target cells of therapeutic genes used viral and non-viral vectors. Non-viral vectors, in particular plasmids, despite the lower transfectional activity considered to be more secure.
Known plasmid pBud-VEGF-FGF2, simultaneously expressing both genes VEGF and FGF2 (application for invention of the Russian Federation No. 2009133970 IPC C12Q 1/00 publ. 20.03.2011). The effects of local delivery of this plasmid in the area of traumatic spinal cord injury, we studied earlier, showed a pronounced stimulating effect on neuroregeneration (RF Patent No. 2459630, IPC A61K 48/00 A61P 25/28 C12N 15/79 publ. 27.08.2012 bull. No. 24). These data allow us to consider plasmid pBud-VEGF-FGF2 as an effective tool to stimulate post-traumatic nerve regeneration.
Of synthetic polymeric materials to create the walls of the conduit nerve the most promising are bionetwork polyesters such as polyglycolide, polylactide and poly(ε-caprolacton). To this end, apply their copolymers, as well as various mixtures of substances, give what they need physico-chemical properties, adequate to the requirements of implantable materials for the reconstruction of the damaged nerve. This material was obtained by different methods: immersion-enveloping (dip-coating), immersion deposition, injection molding, extrusion, weaving and electrospinning. Research in this direction has allowed to obtain a large number of conduits, which differ according to the method of obtaining, chemical structure and physical properties. The number of these samples is so great that it could not be analyzed in the foreseeable timeframe for comparison after implantation in the body of an experimental animal by the criterion of maximum efficiency nerve regeneration. This analysis is also complicated by the fact that already done in this direction by implantation of synthetic nerve conduits differ in experimental models (different nerves), the length of the diastasis (the gap between the proximal and distal segment of the nerve), the follow-up period, methods of evaluating the effectiveness of regeneration and other
The high permeability material is critical to create the walls of the conduit nerve. This condition is best suited material obtained by the method of electrospinning and consisting of micro - and nanofibers. In some works by the method of electrospinning made nanostructured what e poly(ε-caprolactone), used as a polymer or copolymer to create nerve conduits (Chew, S.Y., R. Mi, et al. "Aligned Protein-Polymer Composite Fibers Enhance Nerve Regeneration: A Potential Tissue-Engineering Platform." Adv Funct Mater 17(8). - 2007. - P.1288-1296) (Panseri, S., C. Cunha, et al. "Electrospun micro - and nanofiber tubes for functional nervous regeneration in sciatic nerve transections." BMC Biotechnol 8. - 2008. - P.39.) (Yu, W., W. Zhao, et al. "Sciatic nerve regeneration in rats by a promising electrospun collagen/poly(epsilon-caprolactone) nerve conduit with tailored degradation rate." BMC Neurosci 12. - 2011 - P.68). Obtained by the method of electrospinning of poly(ε-caprolactone) material, in addition to high permeability, has other positive properties, such as optimal for carrying out implantation of elasticity and the best ratio of surface area to volume.
The method involves the preparation of the starting solution of poly(ε-caprolactone) (80 kDa) in dichloromethane, HEXAFLUOROPROPANE or a mixture of chloroform/methanol (3/1) at a concentration of 5-15%; the location of the solution in the syringe with feed needle, the distance between the collector and the end of the needle 5-32 cm; the feed rate of the polymer of 0.05-0.1 ml/min, voltage, forming the field 8-34 kV, the speed of rotation of the axis of the collector 500-600 rpm
Potential space growth of nerve fibers inside the conduit shall be filled hydrogel matrix. The procedure of forming a biocompatible hydrogel should be simple and standard for the formation of reproducible patterns that mimic the matrix t is Ani. These conditions to the greatest extent satisfies the amphiphilic matrix of self-assembled peptide nanostructures (nanofibers and nanofilms) on the basis of oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™) (Ortinau, S., J. Schmich, et al. "Effect of 3D scaffold formation on the differentiation and survival in human neural progenitor cells." Biomed Eng Online, 9(1). - 2010. - P.70)authorized for use in the clinic. This matrix supports the survival and differentiation of cell types, including neural cells (Lampe, .J. and S.. Heilshorn. "Building stem cell niches from the molecule up through engineered peptide materials." Neurosci Lett 519(2). - 2012. - P.138-146). PuraMatrix™ better than other hydrogels supports the differentiation of neural stem cells (Thonhoff, J.R., D.I. Lou, et al. "Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro." Brain Res 1187. - 2008. - P.42-51). The use of hydrogel-based amino acids that perform the function of the extracellular matrix in vivo creates a three-dimensional structure for the migration of cells, stimulates the growth of nerve cells and blood vessels in the graft (McGrath, A.M., L.N. Novikova, et al. "BD PuraMatrix peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration." Brain Res Bull 83(5). - 2010. - P.207-213).
There is a method of stimulating nerve regeneration by direct injection into the site of injury therapeutic genes (transgenes) neurotrophic factors. Their delivery into the area of damage is considered to be one of the most promising approaches to stimulirovaniju neuroregenerative (Zhang, F. and W.. Lineaweaver. "Gene transfer with DNA strand technique and peripheral nerve injuries." J Long Term Eff Med Implants 12(2). - 2002. - P.85-96; Mason, M.R., M.R. Tannemaat, et al. "Gene therapy for the peripheral nervous system: a strategy to repair the injured nerve?" Curr Gene Ther 11(2). - 2011. - P.75-89). Works to promote post-traumatic nerve regeneration by direct gene therapy (Fu, C., G. Hong, et al. "Favorable effect of local VEGF gene injection on axonal regeneration in the rat sciatic nerve." J Huazhong Univ Sci Technolog Med Sci 27(2). - 2007. - P.186-189; Alrashdan, M.S., M.A. Sung, et al. "Effects of combining electrical stimulation with BDNF gene transfer on the regeneration of crushed rat sciatic nerve." Acta Neurochir (Wien) 153(10). - 2011. - P.2021-2029; Esaki, S., J. Kitoh, et al. "Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury." Gene Ther 18(11). - 2011. - P.1063-1069). Basically they are implemented with the use of viral vectors, which is not considered completely safe because of the possibility of insertional mutagenesis, expressed in inflammatory and immune responses and toxicity.
To model the bridging of nerve using biocompatible (but not biorastvorimym, as in the claimed invention) silicone conduit established that direct single injection into the injury area, two-cassette plasmid pBud-VEGF-FGF2, simultaneously expressing cloned genes vascular endothelial growth factor (vegf) and fibroblast growth factor 2 (fgf2) man, leads to the improvement of the functional parameters of regeneration, which corresponds to the increase in the number p is generating the myelinated fibers in the peripheral nerve segment (Nikolaev, SR, A.R. (and others, "Regeneration of the sciatic nerve of the rat in terms of local gene delivery of vegf and fgf2." Morphological table (2). - 2012 - P.45-50).
In terms of ' wall of the conduit" closest to the claimed technical solution is the way (Panseri, S., S. Cunha, et al. "Electrospun micro - and nanofiber tubes for functional nervous regeneration in sciatic nerve transections." BMC Biotechnol 8. - 2008. - P.39.). In this work the method of electrospinning received the wall of the conduit, consisting of 2 layers: the inner - poly(ε-caprolacton) (15%) and the exterior is a mixture of poly(ε-caprolactone) (5,5%) and polylactic-to-glycolide (4%). As the contents of the conduit was used saline.
In terms of the contents of the conduit" as equivalent the applicant proposes to consider the method (McGrath, A. M., L.N. Novikova, et al. "BD PuraMatrix peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration." Brain Res Bull 83(5). - 2010. - P.207-213). In this work, a nerve gap was overcome by means of conduit containing PuraMatrix™. Criteria elongation of regenerating axons, the number of surviving motoneurons and weight of the gastrocnemius muscle, this approach proved to be more effective than using similar conditions hydrogel based on alginate-fibronectin.
In terms of ' local direct delivery of therapeutic genes" the closest technical solution is the way (Masguts Russia, having got, I.I. of Salahutdinov, and others, "Stimulation of the settlement of the traumatic regeneration of the sciatic nerve in rats using plasmids, expressing vascular endothelial growth factor and basic fibroblast growth factor." Cell Transplantology and tissue engineering 6(3). - 2010. - Page 67-70). In this work to model the bridging of the sciatic nerve in rats using autonervous insert is shown that the direct introduction of plasmids pBud-VEGF-FGF2 in the proximal and distal segments of the nerve, as well as directly in autonervous box, stimulates revascularization and regeneration of nerve.
A method of manufacturing tabulirovanie conduit (Panseri, S., S. Cunha, et al. "Electrospun micro - and nanofiber tubes for functional nervous regeneration in sciatic nerve transections." BMC Biotechnol 8. - 2008. - P.39.) has the following disadvantages:
- mechanical properties of the conduit, although similar to our case the thickness of its component fibers were not sufficiently adequate that 40% of the samples led to their network and drastically reduced the size of the lumen, and therefore reduced the amount of potential space for the growth of regenerating nerve fibers;
- the presence in the conduit two layers and a significant total wall thickness (average 150 microns suggests a more severe restrictions for transport of metabolites;
- on the same experimental model, not a comparison of the effectiveness of implantation in the gap nerve developed by these authors conduit with conduits of other materials or of the same gender is(ε-caprolactone), but when other conditions electrospinning.
The way to bridge the gap nerve using conduit, proposed in the work (McGrath, A.M., L.N. Novikova, et al. "BD PuraMatrix peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration." Brain Res Bull 83(5). - 2010. - P.207-213), has the following disadvantages:
the wall of the conduit consisted of ultrafiltration membranes based on cellulose, which is restricted permeability of many molecules stimulants regeneration of the microenvironment inside the conduit with a mass more than 10 KD, which is especially critical for peptide molecules, neurotrophic factors, molecular weight exceeds this threshold, on average 2-3 times;
- applied the conduit presents not a solid tube, and was formed from a rectangular membrane and had a longitudinal seam, which significantly complicates the process of manufacturing the conduit, and, most significantly affects its mechanical properties, which indicators for pulp and without that are less satisfactory than for other synthetic polymers, such as polyesters, poly(ε-caprolactone), and others;
- not applied functional tests, the effectiveness of the method for restoring nerve function remains unclear.
not established as effective for neuroregenerative the conduit, a combination composed of biocompatible and biorastvorimy based material with staticheskogo polymer poly(ε-caprolactone) and amphiphilic hydrogel matrix of the self-assembled peptide nanostructures (nanofibers and nanofilms) on the basis of oligopeptides acetyl-(Arg-Ala-Asp-Ala) 4-CONH2(PuraMatrix™);
not received data on the effectiveness of local delivery of plasmid vector with therapeutic genes in terms of bridging the gap nerve using nanostructured conduit, in particular consisting of poly(ε-caprolactone) and PuraMatrix™;
- in the conditions of implantation of the conduits on the basis of synthetic biocompatible and biorastvorimym materials do not contain information about the simultaneous action of two therapeutic genes delivered into the area of damage.
The disadvantages of the method the bridging of nerve in the study (Masguts Russia, having got, I.I. of Salahutdinov, and others, "Stimulation of post-traumatic regeneration of sciatic nerve in rats with plasmids expressing vascular endothelial growth factor and basic fibroblast growth factor." Cell Transplantology and tissue engineering 6(3). - 2010. - Page 67-70) is all the shortcomings of using autonervous insert: off nerve-donor education painful neurom and other technical difficulties associated with the timely availability of transplantable material, according to its size, the area of implantation, etc.
The objective of the proposed method is to promote nerve regeneration by implantation of the conduit, the wall of which presents material from disordered oriented micro-and nanofibers biorastvorimym polymer poly(ε-caprolactone), and the contents of the formed self-assembled nanostructured hydrogel matrix on the basis of oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™), in complex with direct local delivery of genes vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), which will overcome the above mentioned drawbacks of the invention and to achieve new technical result.
Research on the direct impact on neuroregeneration biorastvorimym conduit with high permeability nanostructured walls and adequate mechanical properties, filled amphiphilic hydrogel matrix of the self-assembled peptide nanostructures, in conjunction with local delivery in the injury area, therapeutic gene promoters regeneration available to the applicant the information sources are not identified.
The problem is solved by a method of stimulating nerve regeneration by implantation of the conduit, the wall of which is made by a method of electrospinning and presents material from disordered oriented micro - and nanofibers biorastvorimym polymer poly(ε-caprolactone), and the content is formed by self-assembling nanostructured hydrogel-based oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™), in complex with the nternet local delivery of genes vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2).
Thus the wall of the conduit filament electrospinning of biorastvorimym polymer poly(ε-caprolactone), initial concentration is 6%, and the plasmid vector with two neurotrophic genes and simultaneously angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) in therapeutically effective dose is administered during surgery under sterile conditions once in the proximal and distal nerve segments after implantation of the conduit in the gap of the damaged nerve.
The inventive method operates on the known sequence of stages: manufacture, implantation of the conduit in the interruption of the nerve, the wall of the conduit presents material from disordered oriented micro - and nanofibers biorastvorimym polymer poly(ε-caprolactone) at a concentration of 6%, and the content is self-assembling nanostructured hydrogel-based oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™), in complex with direct local delivery of genes vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), which is injected into proximal and distal segments of the nerve, and the formed conduit implanted into the nerve gap and fix its ends epinephelinae seams.
Moreover, after receiving the conduit wall tableround what about the conduit is subjected to vacuum degassing for 10 min with subsequent sterilization by prolonged washing in a large volume of sterile distilled water, self-assembling nanostructured hydrogel matrix on the basis of oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™) form in a sterile environment, and neurotrophic genes and simultaneously angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and 15 μg of DNA in a volume of 7.5 µl of phosphate-saline buffer injected during surgery under sterile conditions once in the proximal and distal segments of the nerve near the suture line.
The inventive method of stimulating nerve regeneration using the new conduit in combination with gene therapy has been studied in laboratory rats and described in detail in the following examples:
Samples of polymer tubes for the manufacture of conduit were obtained by the method of electrospinning (Chew, S.Y., R. Mi, et al. "Aligned Protein-Polymer Composite Fibers Enhance Nerve Regeneration: A Potential Tissue-Engineering Platform." Adv Funct Mater 17(8). - 2007. - P.1288-1296; Panseri, S., C. Cunha, et al. "Electrospun micro - and nanofiber tubes for functional nervous regeneration in sciatic nerve transections." BMC Biotechnol 8. - 2008. - P.39; Yu, W., W. Zhao, et al. "Sciatic nerve regeneration in rats by a promising electrospun collagen/poly(epsilon-caprolactone) nerve conduit with tailored degradation rate." BMC Neurosci 12. - 2011 - P.68). As polymer selected biocompatible and biorastvorimy polymer poly(ε-caprolacton) 80 kDa (Sigma) in the initial concentration of 2.3% and 6% (w/w) in a mixture of chloroform/methanol = 3/1 (v/v) (Panseri, S., C. Cunha, et al. "Electrospun micro - and nanofiber tubes for functional nervous regeneration in sciatic nervetransections." BMC Biotechnol 8. - 2008. - P.39). The initial volume of the polymer solution of 10 ml. Thickness of the feeding needle to 1.3 mm, the Distance between the collector in the form of a rotating axis and the end of the needle 20 see the feed Rate of the polymer 0.2 ml/min. Voltage, forming a field of 24 kV. Electrospinning was carried out at room temperature. The study of the structure of the wall of the polymer tube was carried out by the method of scanning electron microscopy (Philips XL30ESEM) and optical light microscope (Axioscop Imager A1, Carl Zeiss).
Before filling tubes hydrogel mass of them was kept in vacuum for 10 min to remove traces of solvents of the polymer, followed by sterilization by incubation and washing in a large volume of sterile deionized water for 30 min (Heydarkhan-Hagvall, S., K. Schenke-Layland, et al. "Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering." Biomaterials 29(19). - 2008. - P.2907-2914) (Kim, UT, V.K. Haftel, et al. "The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps." Biomaterials 29(21). - 2008. - P.3117-3127).
Immediately before surgery on the nerve implantation of the conduit ex tempore in sterile conditions formed 0.5% PuraMatrix hydrogel™ (BD Biosciences) in accordance with the recommendation of the manufacturer. Created previously by the method of electrospinning samples of polymer tube of poly(ε-caprolactone) was filled in the formed hydrogel. Created a conduit implanted in the interruption of the nerve and recorded his con is s epinephelinae seams. In the proximal and distal segments of the nerve near the suture line was introduced plasmid pBud-VEGF-FGF2 with two neurotrophic genes and simultaneously angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and 15 μg of DNA in a volume of 7.5 µl of phosphate-saline buffer.
The experiments were carried out on outbred rats-males weighing 150-200 g in accordance with the requirements of the local ethics Committee of the GOU VPO "Kazan state medical University". Animals were kept in plastic cages at a temperature of 18-20°C with free access to food and water.
Surgical procedures were performed under urethane anesthesia (600 mg/kg, intraperitoneally). The anesthetized animal in the left sciatic nerve at the level of the mid thigh dissected fragment nerve and formed a gap between the proximal and distal segments of length 5 mm. Created conduits with an inner diameter of 2.2 mm was implanted into the nerve gap, the ends of which were fixed using four epinephrine joints with monofilament 8.0 with an atraumatic needle. It is established that the conduits on the basis of a 6% poly(ε-caprolactone), compared with the conduits on the basis of 2.3% polymer, characterized by a more acceptable mechanical properties and most effectively support neuroregeneration. Therefore, in the experiments with D. the rate of the genes were used conduits based on a 6% poly(ε-caprolactone).
The operated animals were divided into three groups (experimental group, control group 1 and control group 2). Additionally operated animals with bridging the gap by means of the conduit of biocompatible silicone containing hydrogel based on PuraMatrix™ (control group 3).
1) Animals of the experimental group (12 rats) immediately after the implantation of the conduit at the proximal and distal segments of the nerve at a distance of 2 mm from the weld line using a Hamilton syringe (Sigma) were injected with two-cassette plasmid pBud-VEGF-FGF2 expressing the genes for vascular endothelial growth factor (yegf) and fibroblast growth factor 2 (fgf2) in the amount of 15 μg in phosphate-buffered saline in a volume of 7.5 μl (30 μg each animal). The choice of dose plasmids based on extrapolation of protocols similar experiments on the stimulation of neuroregenerative (Masguts Russia, having got, I.I. of Salahutdinov, and others, "Stimulation of post-traumatic regeneration of sciatic nerve in rats with plasmids expressing vascular endothelial growth factor and basic fibroblast growth factor." Cell Transplantology and tissue engineering 6(3). - 2011. - Page 67-70).
2) Animals of the first control group (10 rats) in the same conditions and the same number were injected with plasmid pEGFP-N2 (Clontech), containing the gene for enhanced green fluorescent protein (egfp).
3) the Belly of the output of the second control group (4 rats) in the same conditions and in the same volume were injected with saline.
4) animals of the third control group (4 rats) nerve gap of the same length was overcome with a tube of biocompatible silicone (A-M Systems), filled prepared in a similar situation a hydrogel based on PuraMatrix™.
About the efficiency of regeneration was assessed by functional tests: test for motor function recovery of hind limb functional index of the sciatic nerve) (Bain, J.R., S.. Mackinnon, et al. "Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat." Plast Reconstr Surg 83(1). - 1989. - P.129-138; Inserra, M.M., D.A. Bloch, et al. "Functional indices for sciatic, peroneal, and posterior tibial nerve lesions in the mouse." Made 18(2). - 1998. - 119-124) and recovery test the sensitivity of the skin of the limb (pinch test) (Bajrovic, F., M. Bresjanac, et al. "Long-term effects of deprivation of cell support in the distal stump on peripheral nerve regeneration." J Neurosci Res 39(1). - 1994. - P.23-30). Functional tests were performed on 7, 10, 13, 15, 18, 21, 24, 27 and 30 days after surgery. To assess post-traumatic recovery at 30 days after surgery produced a fence material. To do this, under urethane anesthesia (600 mg/kg) after a laminectomy was isolated spinal ganglia L5-side operations were fixed in 10% neutral formalin, obezvozhivani and placed in paraffin according to standard procedures. At the same time took 5 mm fragment peripheral segment of the nerve distal to the injury site were fixed in a 2.5% solution of glutaraldehyde and 2% solution is mirehouse osmium and embedded in Epone. Half-sections of the sciatic nerve stained toluidine blue was used to count the number of regenerating myelinated fibers. Sections of the distal fragment of the peripheral segment of the nerve were made on the cryostat CRYO-STAR HM 560 and held peroxidase immunohistochemical reaction with monoclonal antibodies against S100 protein (Millipore). Counting the number of myelinated fibers and S100-immunopositive cells was performed on digitized images obtained by the microscope Leica DM 1000. The significance of differences between groups was evaluated using student's t.
1. Testing motor function using the functional assessment index of the sciatic nerve.
The highest values of the functional index of the sciatic nerve at all times of observations registered the experimental animals with nerve grafting using conduit, the wall of which is made by a method of electrospinning of poly(ε-caprolactone)containing hydrogel environment PuraMatrix™, with injection of plasmid pBud-VEGF-FGF2 with therapeutic genes.
The rate of recovery of motor nerve function in the experimental group rapidly increases, especially in the range of 13-18 days after surgery, and by day 30 exceeds the index test in the control group 1 (the introduction of the plasmid with the gene of green fluorescent protein pEGFP-N2 instead of plasma is water with therapeutic genes) 28.5% (P< 0,05). By this measure significant differences between experimental group and control group 2 (injection of saline instead of plasmids with therapeutic genes) were not registered.
According to the criterion averaged over all times of observations for a total of 30 daily postoperative period index functional index of the sciatic nerve in the experimental group registered an improvement of motor function by 24.1% (P<0.05) as compared to control group 1. By this measure significant differences between experimental group, on the one hand, and control group 2 or 3 were not registered.
2. Testing the sensitivity of the skin of the plantar surface of the foot of the hind limbs using the pinch test.
The surface area of the skin with restored sensitivity in the experimental group of animals is higher in animals of the control group 3 39.1% (P<0,05). Significant differences when comparing the experimental group with the control groups 1 and 2 is not registered.
3. Counting the number of regenerating myelinated fibers
The number of myelinated fibers in the peripheral part of the nerve in animals from the experimental group 4.4 times more than in animals of the control group 1, 7.6 times more than the animals of the control group 2 and 3.5 times higher than in control animals the group 3.
4. Counting the number of S100-immunopositive cells
The number of S100-immunopositive (Schwann) cells in the peripheral part of the nerve in the experimental group exceeds the number in control group 1, 2 and 3 respectively by 36.9% (P<0,05), 71,0% (P<0,05) and 30.1% (P<0,05). These cells producing neurotrophic factors, molecules of the extracellular matrix and adhesion molecules are key to maintain the process of neuroregeneration. Therefore, the number of these cells in the damaged area is considered as a reliable criterion for assessing the effectiveness of the flow of the entire process of nerve regeneration in General.
Unlike a method of stimulating nerve regeneration (Masguts, Russia, having got, I.I. of Salahutdinov, et al. "Stimulation of post-traumatic regeneration of sciatic nerve in rats with plasmids expressing vascular endothelial growth factor and basic fibroblast growth factor." Cell Transplantology and tissue engineering 6(3). - 2011. - Page 67-70) with overcoming similar along the length of the gap using autonervous insertion and introduction to the area of damage of the same plasmid vector with the same therapeutic genes that the claimed method was not shown to improve motor activity to the end of the first month after surgery according to the criterion of the total amplitude of the action potential of the gastrocnemius muscle. In Appl the reception method of improving motor function according to the criterion of registration of the functional index of the sciatic nerve registered in two weeks. In the works by stimulating nerve regeneration using a method electrospinning conduits based on poly(ε-caprolactone) no indication of the recovery of sensory function. The results of applying the proposed method indicate the possibility of efficient recovery of sensory function.
Thus, the obtained results of experimental studies indicate that the inventive method allows to effectively stimulate the regeneration of myelinated fibers, to improve recovery of motor and sensory nerve function when it breaks due to:
creation of adequate biocompatible and biorastvorimym nanostructured matrix on the inner surface of the high permeability of the porous wall tabulirovanie conduit, supports a population of Schwann cells, axonal growth and the process of remyelination;
creation of adequate biocompatible and biorastvorimym nanogel inside tabulirovanie conduit, filling the potential space of nerve regeneration, supporting the survival and differentiation of Schwann cells, the processes of growth and myelination of nerve fibers;
incentive effects are delivered in the area of nerve damage transgene - therapeutic gene expression of neurotrophic factors that support you is ivanie neurons and axonal growth, they both angiogenic factors, activating the recovery of microcirculation and blood supply of the regenerating nerve. The results achieved by the claimed solution is the improvement of regeneration after traumatic rupture of the nerve in the form of a more complete recovery of motor and sensory functions controlled by this nerve. This result was achieved by the claimed method of creating a conduit nerve of nanostructured biorastvorimym polymer poly(ε-caprolactone) in combination with self-assembling hydrogel matrix based Oligopeptide nanostructures, the bridging of nerve with this conduit, in combination with local Newspapers in the area of nerve damage therapeutic genes neurotrophic and at the same time angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) in the plasmid vector pBud-VEGF-FGF2. Using the proposed method of stimulating post-traumatic nerve regeneration method plasticity of nerve conduit based on poly(ε-caprolactone)filled with PuraMatrix™ with the introduction of therapeutic genes allows you to:
to improve the results of post-traumatic peripheral nerve regeneration in the form of a more complete recovery of motor and sensory functions, to strairway this nerve.
to overcome longer breaks nerve.
- reduce the time of stay of patients with peripheral nerve injury in the hospital and improve the quality of life of patients of this cohort.
1. Method of stimulating nerve regeneration by implantation of the conduit, the wall of which presents material from disordered oriented micro - and nanofibers biorastvorimym polymer poly(ε-caprolactone), and the content is self-assembling nanostructured hydrogel-based oligopeptides acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™), in complex with direct local delivery of genes vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), which is injected into proximal and distal segments of the nerve, and the formed conduit implanted into the nerve gap and fix its ends epinephelinae seams.
2. The method according to claim 1, characterized in that after receiving the conduit wall tabulirovanie conduit is subjected to vacuum degassing for 10 min with subsequent sterilization by prolonged washing in sterile distilled water.
3. The method according to claim 1, characterized in that the concentration of the original solution biorastvorimym polymer poly(ε-caprolactone) to create the walls of the conduit is 6%.
4. The method according to claim 1, characterized in that Prox is normal and distal segments of the nerve using a plasmid vector is introduced simultaneously with two therapeutic gene: the gene for vascular endothelial growth factor (VEGF) and gene fibroblast growth factor 2 (FGF2).
SUBSTANCE: in canal matrix in addition to plate of monocrystalline silicon of hole type with opened canals and deposited material on frontal surface of this plate intermediate dielectric layer of silicon dioxide is created and metal film is applied on the frontal surface of plate with opened canals which have specified diametrical dimension.
EFFECT: improvement of exploitation characteristics by introduction of electrodes and application of electrokinetic and electrophysiological control, which makes it possible to extend nomenclature of membrane equipment products based on biocompatible and highly technological silicon.
8 cl, 8 dwg
SUBSTANCE: invention relates to nanostructures with high thermoelectric properties. The one-dimensional (1D) or two-dimensional (2D) nanostructure is proposed, which is a nanowire of silicon, prepared by the method of non-electrolytic etching or grown by the method of VLS (vapour-liquid-solid). The nanostructure has a rough surface and comprises a doped or undoped semiconductor. The variants of the method of generation of electric power using the claimed nanostructures are proposed, as well as variants of devices for thermoelectric conversion using them.
EFFECT: proposed nanostructure can be located between two electrodes, and used effectively for thermoelectric power generation or for thermoelectric cooling.
48 cl, 9 dwg, 2 ex
SUBSTANCE: invention relates to the field of physical and colloidal chemistry and can be used in obtaining polymer compositions. Finely-disperse organic suspension of carbon metal-containing nanostructures is obtained by interaction of nanostructures and polyethylene polyamine. First, powder of carbon metal-containing nanostructures, representing nanoparticles of 3d-metal, such as copper, or cobalt, or nickel, stabilised in carbon nanostructures, are mechanically crushed, after which, mechanically ground together with introduced in portions polyethylene polyamine until content of nanostructures not higher than 1 g/ml is reached.
EFFECT: invention ensures reduction of energy consumption due to the fact that obtained finely-disperse organic suspension of carbon metal-containing nanostructures is capable of recovery as a result of simple mixing.
2 cl, 5 dwg, 2 ex
SUBSTANCE: invention relates to a method of immobilising protein molecules on the surface of magnetically controlled iron nanoparticles coated with a carbon coating. The method involves reacting powder with 4-carboxybenzene diazonium tosylate dissolved in water to form a covalent bond between organic functional groups and the surface of the powder of iron nanoparticles coated with a carbon coating. Carbodiimide activation is then carried out using the following systems: dicyclohexylcarbodiimide with N-hydroxysuccinimide in dimethyl sulphoxide (DCC/NHS in DMSO) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with N-hydroxysuccinimide in water (EDC/NHS in H2O) or a phosphate buffer solution. Covalent linking of protein molecules with the activated COOH group is then carried out in an aqueous or buffer medium.
EFFECT: invention enables to immobilise biomolecules on the surface of magnetically controlled iron nanoparticles coated with a carbon coating.
3 dwg, 8 ex
SUBSTANCE: invention relates to luminescent photovoltaic generator (1) and waveguide to be incorporated with said generator. Photovoltaic generator comprises photovoltaic element (4) and waveguide including transparent matrix (2) containing particles of inorganic luminescent material distributed therein and/or inorganic luminescent material located on at least one its side. Waveguide is associated with photovoltaic element (4) so that at using a portion of light emitted from luminescent materials is fed to photovoltaic element (4) to generated voltage therein. Note here that said inorganic luminescent material features, in at least one of UV, visible and IR bands, the absorption line width of 50 nm or larger, emission line width of 20 nm of smaller and Stokes shift of 50 nm or larger.
EFFECT: alternative to or perfection of known photovoltaic generators.
14 cl, 3 dwg, 1 tbl
SUBSTANCE: method to strengthen an asphalt road surface with a carbon nanomaterial includes usage of an asphalt concrete mix containing crushed stone, crushed stone siftings, sand and oil bitumen of "БНД" 90/130 grade. Besides, the oil bitumen is modified by carbon nanotubes in the amount of 0.005% from the bitumen mass, and modification of the oil bitumen with carbon nanotubes is modified under ultrasonic effect.
EFFECT: increased strength and elasticity of a produced asphalt surface, higher water resistance, heat resistance and frost resistance and expansion of temperature range of its laying in the field of negative temperatures.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to cosmetology and represents a complex cosmetic agent containing hyaluronic acid, colloidal silver, emulsion wax, water, carnosine, enfolin recovered from hydrogel of hyaluronic acid in the process of photochemical nanostructuring at wave length equal to 280 nm, while hyaluronic acid is nanostructured at a diameter of single chains up to 5 nm with the ingredients of the agent taken in certain ratio, wt %.
EFFECT: invention provides rejuvenating effect, anti-inflammatory action and lifting effect, besides it possess local immunostimulative action, extended storage period and high biological activity.
1 tbl, 3 dwg
SUBSTANCE: invention relates to ecologically clean and economically effective layered silicate polymeric superconcentrates and based on them composition materials and can be applied in creation of quality construction products in motor car construction, cable, building and other fields of industry. Superconcentrate contains cyclic oligomer of butyleneterephthalate, methylene chloride and layered silicate nanofiller, which represents preliminarily purified from ballast substances and modified by carbamide halloysite or montmorillonite. Application of claimed superconcentrate makes it possible to considerably simplify process of introducing nanosize nanofiller into polymeric matrix for obtaining compositional materials based on polypropylene or butyleneterephthalate.
EFFECT: obtained compositional materials possess required improved physical-chemical characteristics.
3 cl, 2 tbl, 2 ex
FIELD: measurement equipment.
SUBSTANCE: invention may be used to create miniature sensors for triaxial magnetometry. The magnetic field sensor comprises sensor units implemented using the Hall effect, which are made within a curvilinear shell with a system of layers. The system of layers includes functional and shaping layers that perceive the magnetic field. The latter provide for curvature of the shell and the possibility of orientation of cross-shaped Hall elements of sensor units in the space with arrangement of compliance between measured Hall voltages and orthogonal components of the external magnetic field vector. The method to manufacture a magnetic field sensor consists in the following. A multilayer film element/elements are formed on the substrate. At the same time they use materials, geometry and internal mechanical stresses providing for orientation of cross-shaped Hall elements of sensor units in the space, when there is compliance between the measured Hall voltages and orthogonal components of the external magnetic field vector. At the stage of formation of the film element they manufacture layers, shaping, mechanically stressed and functional, which perceive the magnetic field, with Hall contacts. The film element is separated from the substrate, transforming it under action of internal mechanical stresses into a shell with achievement of orientation of cross-shaped Hall elements in the space, when there is compliance between measured Hall voltages and orthogonal components of the external magnetic field vector.
EFFECT: solutions provide for achievement of accuracy and reliability of simultaneous measurements of orthogonal components of magnetic field, and also a component of magnetic field vector that is different from the one perpendicular to the sensor plane; increased reliability of a sensor and reproducibility of sensor parameters.
24 cl, 6 dwg, 5 ex
SUBSTANCE: in accordance with the invention claimed solar-powered generator (100) contains thermoelectric elements adjoining solar elements and located below solar elements. Concentrated flow of solar energy is provided. Heat sink (104), with changeable temperature and efficiency, contacts with cold soldered seam (108) of thermoelectric device (103). Thermal resistance is calculated with respect to energy flow, which results in creation in thermoelectrical device (103) of temperature gradient equal to several hundreds of Kelvin degrees. Solar element preferably contains semiconductor with large width of prohibited energy zone. Generator (100) preserves relatively suitable efficiency (efficiency factor) in some range of cold seam (108) temperature. System of hot water can serve as heat sink (104). High values of efficiency factor are obtained due to application of nanocomposite thermoelectrical materials. One-piece construction of solar element and thermoelectrical elements provides additional advantages.
EFFECT: uniformly but sparsely distributed thermoelectrical segments in matrix from material with high heat-insulating properties reduces quantity of material required for segments without impairment of working characteristics.
17 cl, 8 dwg
SUBSTANCE: osteosynthesis of long bones is followed by the hydropreparation through a Dufaut's needle; 0.9% sodium chloride 2 ml is introduced above an epineural space; epidural and epineural electrodes assisted by an electromyography and an electronic image converter are implanted and sutured on the skin. The postoperative period involves a complex course of the electrical stimulation through the epidural and epineural electrodes in a combination with a superficial cutaneous electrical stimulation of independent regions for 15-20 days.
EFFECT: method enables reducing the injuries accompanying the electrode placement and improving the motor activity of the extremities by the complex electrical stimulation.
2 dwg, 1 tbl, 1 ex
SUBSTANCE: invention refers to medicine, namely - to neurology. The method involves the integrated treatment. Azathioprine, dexamethasone and analgin are administered into the patient. Azathioprine 50 mg is administered three times a day, after meals for 21 days. Dexamethasone 4 mg is administered intramuscularly three times a day for 15 days. From the 16th to 18th day of treatment, dexamethasone 4 mg is administered 2 times a day - in the morning and afternoon. From the 19th to 20th day, the same is administered in a dose of 4 mg in the afternoon. Before bedtime, 50% analgin 2.0 ml is administered parenterally for 10 days. At 9 o'clock in the morning, patient's lumbar pain area is exposed to the magnetic laser treatment. The patient is exposed to infrared light of wave length 0.8-0.9 mcm, pulse power 5-8 Wt, pulse frequency 1000 Hz and magnetic strength 35 mT. The exposure is contact and stable, and covers the fields paravertebrally. The length of the one-field exposure is 2 minutes. The therapeutic course is 15 procedures. Trives spinal assistant is put on in the horizontal position of lying on back. The spinal assistant is taken off in the horizontal position before bedtime.
EFFECT: method reduces the length of treatment, prolongs a remission, including due to the developed modes and combinations of the various components of the exposure.
1 ex, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine, namely to therapy and neurology, and concerns using fatty acid/fatty acids Omega-3 as a part of an analgesic drug. That is ensured by administering an analgesic and/or preventative drug that contains the above acid/acids in the free or bound agent in the amount of more than 2 g. The administration is single oral or intravenous.
EFFECT: administering Omega-3 fatty acid/fatty acids in the above doses provides the analgesic effect regardless of causes of the pain.
10 cl, 3 ex
SUBSTANCE: invention refers to medicine, namely neurology, physiotherapy. The solution of proserine is introduced 30 minutes before conducting low intensity electrical stimulation. Amplitude of the electrical stimulation is 10-20 mA, frequency is 40-40 Hz, and length is 900 seconds.
EFFECT: method reduces the length of treatment, raises muscle tone, desensitizes the peripheral innervation region.
3 ex, 3 tbl
SUBSTANCE: invention relates to medicine, in particular to reconstructive surgery of peripheral nerves, and can be applied for substitution of peripheral nerve defects in case of their posttraumatic injury. Method includes intersection of neuromas and substitution of defect with autonervous insertions. Sampling of autologous fat tissue from patient is preliminarily realised by method of liposuction. Cells of stromal vascular fraction (SVF) are obtained from it after carrying out enzymatic processing. Obtained SVF cells are intraneurally transplanted into peripheral and central nerve segments, and into each insertion. Ends of nerve, as well as insertions, are covered with fibrin adhesive.
EFFECT: in conditions of clinical practice method makes it possible to restore functions of injured nerve with considerable reduction of treatment terms.
1 ex, 4 dwg
SUBSTANCE: invention refers to medicine. What is described is using the material for the purpose of neural dysfunction recovery with the above material containing a polysaccharide derivative hydrogel wherein 0.5 wt % of the aqueous solution contains a complex module in the amount of 1 to 1000 N/m2, while a loss factor makes 0.01 to 2.0 that is measured at angular velocity 10 rad/sec with using a dynamic viscoelasticity meter. The above material for neural dysfunction recovery may represent hydrogel injected with using a syringe and has an excellent body residence, and has a restorative effect on the damaged or degenerated nerve function.
EFFECT: preparing the material for neural dysfunction recovery.
19 cl, 6 dwg, 6 ex
SUBSTANCE: invention refers to medicine, namely to neurology, neurosurgery and rehabilitation, and may be used to recover the sensor-motor function of the central nervous system and peripheral nerves. The therapy is three-staged. At the first stage, at least two hollow electrodes made of biologically neutral conductive material in a proximal and distal direction from a nerve injury are implanted into the nerve. The second stage involves at least one course of a focused extracorporeal shock-wave therapy consisting in at least five sessions in a combination with intraneural electric stimulation and intraneural ionic medication aiming at stimulating the growth of peripheral nerve fibre axons and the regeneration of Schwann cells forming the medullary sheath. At the third stage, the implanted electrodes are removed, and at least one session of transcutaneous electric stimulation with one-step electric myoneurography is conducted to coordinate neural pulses passage through the newly formed peripheral nerve synapses.
EFFECT: method provides the high therapeutic effectiveness in case of injuring up to 90% of neural synapses, reduced length of rehabilitation up to the complete recovery in the patient with no contra-indications and a possibility of the outpatient care.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutics, namely a method for preparing and purifying monosialoganglioside GM1. The method for preparing pure monosialoganglioside GM1 in the form of its sodium salt involves (a) GM1 separation from Fucosyl GM1 in a lipid mixture containing monosialoganglioside GM1 as a main ganglioside component by a column ion-exchange chromatography using an eluent comprising potassium or cesium ions, (b) recovery of the dissolved substance from the eluted solution, (c) diafiltration of the aqueous solution of the recovered dissolved substance of the stage (b), (d) addition of sodium salt, and diafiltration of the prepared aqueous solution (d) recovery of GM1 in the form of its sodium salt. The method for purifying monosialoganglioside GM1 from Fucosyl GM1 in the lipid mixture, the column ion-exchange chromatography using the eluent comprising potassium or cesium ions. The preparation of monosialoganglioside GM1 has a purity of 99.0% or more, and contains less than 0.1% of Fucosyl GM1. The method of treating disorders and diseases of the central nervous system and the peripheral nervous system, comprising administering the preparation of monosialoganglioside GM1 to the patient in its effective amount. The use of the preparation of monosialoganglioside GM1 in preparing a pharmaceutical composition.
EFFECT: use the above preparation of monosialoganglioside GM1 in treating has the considerable advantages due to reducing side effects.
17 cl, 1 ex
SUBSTANCE: invention relates to medicine, namely to pediatric neurology, and can be used for rehabilitation of neurological disorders in children in case of neuroinfections. For this purpose, at the background of adequate complex and pathogenetic therapy, parenteral introduction of actovegin in acute period of disease additionally from the first days of disease cytoflavin is introduced intravenously by drop infusion in dose 0.6 ml/kg or 10 ml per day for 3-5 days, elcar perorally in dose 70-100 mg/kg of weight per day for 3-4 weeks. In the period of early reconvalescence pantogam is additionally introduced perorally in dose 50-70 mg/kg of weight per day for 4 weeks. In case if multifocal affection of brain substance is present, gliatilin is introduced intravenously by drop infusion in dose 1 ml per 5 kg of body weight per day in combination with intramuscular introduction of ipidacrine in dose 5-15 mg per day for 7-10 days, after that gliatilin perorally in dose 50 mg/kg of weight per day together with ipidacrine inside in dose 1 mg/kg per day for 4 weeks.
EFFECT: method makes it possible to improve disease outcome due to reduction of frequency of residual neurological deficiency formation with reduction of term of hospital treatment.
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed invention relates to field of pharmacology and clinical medicine and describes pharmaceutical compositions of 2-amino-2-[2-(4-octylphenyl)ethyl]propan-1,3-diole in free form and/or in form of pharmaceutically acceptable salt, where as auxiliary substances they contain lactose, starch and/or starch derivatives, selected from acetylated starch, sodium salt of starch carboxymethyl ether, pregelatinised starch, sodium starch glycolate, gelatin, binding agent, and lubricant with specified ratio.
EFFECT: invention makes it possible to extend possibility for cheaper in industrial manufacturing preparation for treatment of disseminated sclerosis.
7 cl, 16 ex
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