RU2824072C1 - Method of making medical artificial nerve stem implant containing silk fibroin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of implantable neuroprostheses for repair of defects of nerve trunks after their injury or gunshot wound, and concerns a method of manufacturing a medical artificial nerve stem implant. According to the proposed method, a polymer tube is filled with collagen gel and silk fibroin filaments, wherein the polymer tube is made from ε-caprolactone with an average molecular weight of 80 kDa, the thickness of the polymer tube is selected to be 50–100 mcm, and the length of the polymer tube is selected depending on the size of the nerve defect; native filaments of mulberry silk fibroin, the diameter of which is about 10 mcm, are introduced into the polymer tube, tubes with filaments are filled with a solution of collagen with concentration of 2 mg/ml, then the solution is polymerized into a gel inside the tube. All procedures are carried out under sterile conditions with pre-sterilized polymer tubes and silk fibroin filaments.

EFFECT: providing the possibility of nerve tissue regeneration, localization and direction of axon growth due to silk fibroin fibres embedded inside the polycaprolactone tube; higher efficiency of nerve tissue regeneration; achieving reproducibility in processes and results of tissue regeneration due to detailed description of implant manufacturing techniques.

1 cl, 2 dwg

Description

The invention relates to the field of implantable neuroprostheses for restoring defects of nerve trunks, namely to artificial nerves based on a tube made of polycaprolactone - a biodegradable polyester, collagen gel and silk fibroin. The invention can be used in neurosurgery, traumatology for injuries and gunshot wounds for the regeneration of damaged nerves over a large area in order to restore limb function.

A conductive polymer composite consisting of polycaprolactone fumarate and polypyrrole (an electrically conductive polymer) is known for nerve regeneration. When current was passed through the nerve in the presence of the said composite, restoration of the nerve fiber in the direction of the applied current was observed (US 2013331869 A1, A61F 2/04, 12.12.2013).

The limitation in the use of the described composite is that it cannot regenerate a damaged nerve, since there is no substrate along which axons can grow and it cannot be used as missing sections of the nerve that receive or transmit a signal to organs.

A medical artificial nerve trunk transplant is known, comprising a sheath or sheath with a construction fiber inserted into it, wherein the sheath contains a plurality of micropores, and the sheath and/or construction fiber contain silk fibroin (EP 1938774 A1, A61F 2/04, 02.07.2008).

The limitation in the use of the described transplant is the lack of biodegradation of its components, and there is no content inside the conduit.

The closest in design is an artificial implant, the tube of which consists of several layers. This can be polyglycolic acid, polylactic acid (L or DL), copolymer of glycolic acid and lactic acid, copolymer of lactic acid and ε-caprolactone, polydioxanone and copolymer of glycolic acid and trimethylene carbonate, which is able to maintain its shape in the body for a certain period of time. Inside the tube along the axis of the tube are fibers consisting of collagen, coated with laminin. Patent: US 6,589,257 B1 from 2003 (Tong, et al, "Sciatic Nerve Regeneration Navigated by Laminin-Fibronectin Double Coated Biodegradable Collagen Grafts in Rats," Brain Research, vol. 663, no. 1 (1994), pp. 155-162.)

The objective of the proposed invention is to create a harmless, flexible, wear-resistant artificial implant that can be an analogue of a nerve trunk.

The technical result of the proposed solution is:

- implementation of the possibility of regeneration of nervous tissue, localization and direction of axon growth due to silk fibroin fibers embedded inside a polycaprolactone tube;

- increasing the efficiency of regeneration of nervous tissue;

- achieving reproducibility in the processes and results of tissue regeneration through a detailed description of implant manufacturing technologies.

The invention is illustrated by Fig. 1, which shows a diagram of the formation of an artificial nerve, where 1 is a tube based on 50-100 μm PCL, 2 is a collagen gel 2 mg/ml, 3 are silk fibroin threads; Fig. 2 shows a photo of the external appearance of the artificial implant. a) a tube made of polycaprolactone with collagen gel; b) fibroin fibers - silk.

To create the artificial nerve, we used biodegradable components that have been well studied both in experiments and in practical application: polycaprolactone, collagen gel, and silk fibroin. The main difference between our artificial nerve and all other designs is that these components are used in the same way as they occur during nerve regeneration in the human body. Polycaprolactone is a prototype of the epineural sheath of the human nerve, collagen gel is a prototype of the internal epineurium, and silk fibroin is a prototype of Bügner's bands, along which the axons of regenerating nerves grow (Fig. 1).

Much attention is currently being paid to poly(ε-caprolactone) (PC). PC slowly degrades in the body by hydrolysis of ester bonds to form e-hydroxycaproic acid. The degradation products of PC are completely processed by the body's cells.

Polycaprolactone is a biodegradable polyester that is a component of the well-known monofilament suture Monocryl (Etchicon, Inc., Somerville, NJ) and has been widely used in several surgical fields for many years. Polycaprolactone is a strong structure and causes minimal reaction of surrounding tissues after implantation. In addition, polylactide (PLA) "Purasorb PL-10" from Corbion Purac (Netherlands) can also be used as a tube, and polypyrrole can be added to the polylactide tubes to improve electrical conductivity.

Collagen gel - collagen was isolated from the tendons of the tails of laboratory rats, which is a biodegradable medium in the form of an analogue of the extracellular matrix and is the main component of the extracellular matrix. Collagen gel has long been widely used in cosmetology and in the treatment of degenerative dystrophic diseases of the musculoskeletal system.

Silk fibroin is an insoluble protein present in silk produced by numerous insects, including various species of moths, silkworms, and spiders. Natural silk fibroin is the main component of fibroid fiber. Fibroin makes up 70 to 80% of the mass of silk. It is an important material used by humans, containing 18 amino acids. Our experimental studies have shown that it is related to biological tissues, non-toxic, non-polluting, and biodegradable. The artificial nerve trunk graft made from it is quite effective. The material of the proposed product is highly pure silk fibroin. It is a natural degradable material with good biocompatibility with the human body. The manufacturing technology of the product is such that it does not contain exogenous substances with toxic or other side effects.

The proposed design is a polymer tube filled with collagen gel and silk fibroin threads. The polymer tube was prepared using a previously patented technique (Nashchekina Yu.A., Yudintseva N.M., Nikonov P.O., Blinova M.I., Nashchekin A.V., Moskalyuk O.O., Yudin V.E., Mikhailova N.A. patent for invention "Method for producing a small-diameter bioresorbable vascular prosthesis" RU 2709621 C1 dated 12/19/2019). The polymer tube was prepared from poly (ε-caprolactone) with an average molecular weight of 80 kDa (Sigma, USA). The diameter of the polymer tube can be varied depending on the needs. The tube thickness was 50-100 μm. The length of the polymer tube can also be made depending on the size of the nerve defect. Native silkworm silk fibroin threads are introduced into the polymer tube. The diameter of the silk fibroin threads is about 10 μm (Nashchekina Yu.A., Nikonov P.O., Yudintseva N.M., Nashchekin A.V., Likhachev A.I., Moskalyuk O.A., Yudin V.E., Blinova M.I. “Interaction of bone marrow mesenchymal cells with native silk fibroin fibers”, Tsitologiya, 2016, Vol. 58, No. 11, pp. 843-849.). The tubes with threads were filled with a collagen solution with a concentration of 2 mg/ml. The solution polymerized into a gel inside the tube. All procedures were performed under sterile conditions with pre-sterilized polymer tubes and silk fibroin threads. The finished structure can be used either immediately after preparation or 1-3 months after preparation. During long-term storage, the collagen gel dries out, but when kept in a physiological solution, it partially swells again, which allows creating a favorable environment for axons to migrate (Fig. 2).

Claims (1)

A method for manufacturing a medical artificial nerve trunk implant containing silk fibroin, characterized in that the polymer tube is filled with a collagen gel and silk fibroin threads, wherein the polymer tube is made of ε-caprolactone with an average molecular weight of 80 kDa, the thickness of the polymer tube is selected to be 50-100 μm, and the length of the polymer tube is selected depending on the size of the nerve defect; native silkworm silk fibroin threads with a diameter of about 10 μm are introduced into the polymer tube, the tubes with threads are filled with a collagen solution with a concentration of 2 mg/ml, then the solution is polymerized into a gel inside the tube; all procedures are carried out under sterile conditions with pre-sterilized polymer tubes and silk fibroin threads.