RU2748544C1 - Method for targeted minimally invasive access for cell transplantation into bone regenerate - Google Patents

Abstract

FIELD: medicine and medical technology.

SUBSTANCE: inventions group relates to the field of medicine and medical technology. The system for minimally invasive introduction of the graft into the connective tissue regenerate at the site of the bone defect of the subject for the plate of bone osteosynthesis includes a port system, which is a device for inserting a cell graft under the skin, which consists of a main body, a silicone membrane and a titanium reservoir with a cannula, a silicone tube, a plate for osteosynthesis, having at least two threaded holes for fastening by means of screws to the bone in the area of the defect, and at least two screws for fixing the plate for osteosynthesis, intended for screwing through the above-mentioned threaded holes. The plate includes a needle for supplying a cell graft, which is fixedly connected to the plate and has a cannula. The proximal end of the silicone tube is attached to a titanium reservoir with a cannula. The distal end of the silicone tube is positioned in the osteosynthesis plate up to the cannula of the needle for supplying the cell graft. A method of introducing a graft into a connective tissue regenerate to restore bone defects in a subject, which consists in using the above system, before injection, the surface of the skin above the port is treated with a skin antiseptic, the graft is infused with a needle, which pierces the skin, partly the subcutaneous tissue and then the silicone membrane port.

EFFECT: provides ability to perform multiple transplants with reduced trauma and pain in the subject.

11 cl, 3 dwg

Description

Technology area

The invention relates to medicine and medical technology, namely to experimental traumatology and orthopedics, regenerative medicine, transplantology. The invention can be used in traumatology for fusion of bones without fusion of long bones after fractures and for restoration of the anatomical integrity of bones after traumatic and other defects exceeding critical dimensions in vivo.

State of the art

Bone restoration at the site of a defect exceeding the critical size remains the main clinical problem in traumatology and orthopedics. Critical bone defects are technically defined as those that do not heal spontaneously, naturally during the patient's life. A critical defect can occur both in the case of an accidental event (trauma), and deliberately (experimental studies on laboratory animals). The abilities of humans and mammals, in general, to restore bone as an organ by reparative regeneration are limited, therefore, for bones there is a concept of defects exceeding critical sizes, but for each bone they have their own parameters and differ both in the volume of lost tissues and in configuration.

In experiments on animal models with artificial critical bone defects, it was shown (Mironov S.P., Omelyanenko N.P., Ilyina V.K., Karpov I.N., Dorokhin A.I., Kozhevnikov O.V. CELLULAR TECHNOLOGIES FOR MANAGING REPARATIVE OSTEOGENESIS // Kremlin Medicine. Clinical Bulletin. - 2007. - No. 1. - P. 48-52) that in place of the lost supporting tissues - between the sawdust of the bones, a supra-tissue temporary structure is spontaneously formed - "granulation tissue", rich newly formed vessels. With further natural regeneration in this young connective tissue, a reduction of the vascular bed occurs, a coarse-fibrous structure, atypical for this localization, is formed - a connective tissue scar. Open medullary canals are closed with endplates, the bone is not restored.

Bone also regenerates through dynamically changing connective tissue regeneration, since bone tissue is a type of skeletal connective tissue. Secondary bone fusion sequentially goes through five phases of morphological reorganization of bone regenerate replacing each other: 1) formation of provisional (osteogenic) callus; 2) differentiation of provisory callus cells into cartilage and coarse-fibrous bone tissue; 3) the formation of primary bone marrow cavities with bone marrow; 4) destruction of cartilage and the formation of an enchondral bone in its place, which has a spongy structure; 5) replacement of coarse fibrous enchondral bone parallel-fibrous (Babaeva, Anna Georgievna Regeneration: facts and prospects / A.G. Babaeva; Russian Acad. Of Medical Sciences, State Research Institute of Human Morphology of the Russian Academy of Medical Sciences. - Moscow: Publishing House of the Russian Academy of Medical Sciences, 2009. - 333, [1] pp. 182-183).

The bone regenerate is rebuilt for a long time: first, reparative remodeling occurs, and then, already at the final stage, under the influence of a gradually increasing mechanical load, adaptive-functional remodeling is carried out. After these rather long-term rearrangements, the bone completely restores its functions as an organ: the bone regenerate is completely consolidated with bone fragments or the edges of the defect and does not differ from the neighboring intact bone tissue.

With defects exceeding the critical size, the bone regenerates, but the consolidation of the fracture is impossible, - domes of outgrowths are formed on the fragments (sawdust) of the bones, which do not interact sufficiently with each other, since their length is not enough for contact with the bone regenerate on the opposite side. Osteogenic structures independently limitedly penetrate into the granulation tissue. Open bone marrow canals are closed with end plates, and bone regeneration is completed at this point.

Thus, natural reparative regeneration is not enough to overcome the distance of the defect by the newly formed bone tissue; therefore, such a defect is called “a defect exceeding the critical size”. The bone fragments remain unconnected, the regenerate (granulation tissue) between the bone fragments turns into a normal scar, an important supporting function is not restored, the person remains disabled.

Various methods are known for replenishing a bone defect, for example, with directed cell transplantation using matrices made of biomedical materials (implants) into the defect area. There is a known method of restoring a 2.1 cm femur defect in dogs using a hollow porous matrix populated with allogeneic mesenchymal stem cells of the bone marrow (Arinzeh TL, Peter SJ, Archambault MP et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect // J Bone Joint Surg Am 2003. Vol. 85-A (10) P: 1927-35). To populate the matrices with cells, the method of vacuum loading of cellular material was used. For this purpose, the empty matrix was placed into a syringe, with the help of which the culture medium with the cell material was collected. One matrix was layered with 37.5 million cells. After 16 weeks, bone formation was observed at the site of the defect.

The disadvantage of this method is that a large amount of cellular material is required for the formation of bone tissue, in addition, not all of the matrix is replaced by bone tissue, and after 16 weeks it is detected in a sufficiently large amount.

Thus, there is a need to continuously administer osteogenic cells in multiples. In this case, natural osteogenic insufficiency is overcome, the replacement of the space of the bone defect by fibroblasts of the paraosseous tissues does not occur, and osteogenesis is stimulated.

A well-known document (S.P. Mironov, N.P. Omelyanenko, O.V. Kozhevnikov et al. Possibilities of cellular technologies in the treatment of congenital pseudoarthrosis of the lower leg bones in children. // KTTI - VII (2) - 2012 - P. 38- 39), which discloses a method of treating various pathological conditions associated with damage to connective tissue or impairment of its regeneration. In this method, autologous connective tissue cells (CCT) of the bone marrow are injected into the resection area of the pseudarthrosis, which leads to the formation of a full-fledged bone regenerate between two parts of one long tubular bone.

Limitations of cell transplantation are known that reduce their clinical efficacy due to the short life span of transplanted cells in the recipient's body, which is associated with the following circumstances:

1. Cells are usually transplanted as single monodisperse cells in suspension, most often by local injection. This strategy has met with limited success due to rapid cell death when delivered to a harsh microenvironment, insufficient retention in place, reducing their intended effects to a few days.

2. Loss of viability occurs due to limited transmission of signals from cell to cell (intercellular interaction is disrupted) and the cellular matrix (cell-matrix interaction is disrupted) and an uncontrolled local regeneration environment.

3. It is important to overcome with the help of cell transplantation the minimum threshold for the number of cells required for the implementation of reparative osteogenesis. It is often necessary to maintain a special artificial substrate in which the transplanted cells can attach and proliferate for a sufficiently long period to provide the necessary direction of differentiation and reparative osteogenesis.

Two new concepts for improving methods of stimulating bone regeneration - the concept of medicinal signal cells and the biological chamber of the "Diamond Concept" are based on the multiple supply of various products and living cells to the area of bone regeneration.

Delivery by the usual injection method is difficult, since it implies repeated extended damage to the soft tissues and regenerate by the needle and the creation of wound channels that directly connect the local regeneration environment with the external environment. It is important to correctly inject the necessary products into the area of regeneration, which means it is necessary to “grope” with the tip of the needle “blindly” the required insertion site, or to use medical imaging devices and actively move the needle in the living innervated tissues of the patient. Each introduction turns into a surgical operation requiring general or local anesthesia and sedation.

Accordingly, the relevance of the search for alternative methods of introducing cellular material is increasing, in which it is possible both to create and maintain an osteogenic environment for bone regeneration, and to carry out multiple cell transplantations, facilitating the connection of bone fragments through the unifying connective tissue (bone) regenerate into a single bone (by restitution), and also eliminating factors that interfere with osteogenesis.

Disclosure of invention

The objective of the present invention is to reduce the trauma of patients with long-term periodic introduction of a cell graft locally into the bone regeneration zone, to provide quick access of nutrient and biologically active substances to the defect area at any time during a long period of restoration of bone integrity.

The technical result of this invention consists in the development of a new system for the introduction of a cell graft, a method for introducing a cell graft into the area of bone regeneration, as well as biologically active substances, nutrient media, differentiation factors to create a favorable local regeneration environment that supports reparative osteogenesis, namely, in the development of a new the system, with the help of which the specified method is carried out, for the restoration (healing) of a bone defect exceeding the critical size (critical bone defect) or long-term non-healing fractures. This system allows for multiple transplants and is characterized by reduced trauma and pain in the subject.

The use of the new system for the introduction of the graft will allow to obtain effective and permanent access to the area of bone regeneration to feed it with cellular material (cell grafts, tissue spheroids) and bioactive substances in certain modes in order to restore (heal) a bone defect exceeding the critical size (critical bone defect). ).

The technical result and the technical problem are provided due to the fact that the proposed system for minimally invasive introduction of the graft into the connective tissue regenerate at the site of the bone defect includes:

- a port system, which is a device for inserting a cell graft under the skin, which consists of a main body, a silicone membrane and a titanium reservoir, which is connected to a needle for supplying a cell graft on an osteosynthesis plate, which is connected to the port system through a silicone tube with a cannula;

- plate for osteosynthesis, having at least two threaded holes for fastening by means of screws to the bone in the area of the defect, and the plate includes a needle for supplying a cell graft, fixedly connected to the plate, which is connected to the port system by means of a silicone tube of the port system when using a cannula, wherein the distal end of the silicone tube is located in the plate for osteosynthesis, the proximal end of the silicone tube is attached to the titanium reservoir with a cannula;

- at least two screws for fixing the osteosynthesis plate, intended for screwing through the above-mentioned threaded holes.

In particular embodiments of the invention, the screw is a conical frusto-conical screw.

In particular embodiments of the invention, the plate is made of titanium or stainless steel.

In particular embodiments of the invention, the graft is a suspension based on osteogenic cells, living cell cultures of autologous cells of the periosteum, perichondrium, bone marrow stromal cells, a suspension based on tissue or biocomposite spheroids, or their various combinations.

In particular embodiments, the spheroids are spheroids based on autologous perichondrium cells, spheroids based on autologous periosteal cells, spheroids based on autologous and allogeneic multipotent mesenchymal bone marrow stromal cells and ground partially demineralized allogeneic bone, or a mixture thereof.

In particular embodiments of the invention, the width and depth of the channel through which the silicone tube passes in the osteosynthesis plate is 1.5-3 mm.

In particular embodiments of the invention, the housing of the port system is made of a biocompatible inert material.

In particular embodiments of the invention, the biocompatible inert material is an epoxy resin or polysulfone.

In addition, the technical result is provided by implementing a method for introducing a graft into a connective tissue regenerate to restore bone in a subject with defects whose dimensions exceed the critical size by means of a system for minimally invasive graft insertion according to the invention.

In particular embodiments of the invention, the graft is administered repeatedly as the connective tissue regenerate develops periodically.

In particular embodiments of the invention, cytokines, osteoinducers, differentiation inducers, hydroxyapatite granules, calcium phosphate granules, a nutrient medium, biologically active substances and / or a suspension of crushed demineralized bone are additionally periodically administered.

In particular embodiments of the invention, the bone defect is a tubular bone defect.

The method according to the invention is based on the fact that in the area of the traumatic defect a temporary supra-tissue structure - granulation tissue - natural cell matrix (hereinafter ECM) is formed spontaneously, naturally and, in part, in response to the stimulating effect of the transplanted cellular material on angiogenesis and inhibition of vascular reduction due to paracrine effects. The specified EKM is a connective tissue regenerate mainly from paraosseous tissues and is saturated from the outside artificially through the proposed device with osteogenic cells, cytokines, inducers of bone differentiation - everything that is necessary to stimulate reparative osteogenesis. Further, the ECM with transplanted osteogenic cells or spheroids is gradually replaced by bone callus (partly by replacement by the type of transplant regeneration), which connects bone fragments, which, during the remodeling of the regenerate, is already naturally under the influence of mechanical stress during long-term remodeling with the formation of a full-fledged bone at the site of the defect.

The use of the present invention allows:

1) carry out multiple transplants with reduced trauma and pain in the subject;

2) precisely penetrate inside to bone regenerates and EKM, saturate EKM with osteogenic cells, cytokines and differentiation inducers, as well as nutrients;

3) prevent the reduction of vessels in the granulation tissue, the completion of the regeneration phase and the formation of an atypical regenerate - a connective tissue scar, which prevents reparative osteogenesis;

4) use EKM as a natural matrix well saturated with oxygen and nutrients, which is artificially saturated with osteogenic material, changes its properties and structure, as a result of which the bone effectively regenerates.

The system and method according to the present invention allows to create and maintain a local osteogenic environment favorable for bone regeneration, facilitating the union of bone fragments through unifying bone regenerate into a single bone, as well as eliminating factors that interfere with osteogenesis.

Brief Description of Drawings

The accompanying drawings, which are incorporated and form part of the present specification, illustrate embodiments of the invention and, together with the foregoing general description of the invention and the following detailed description of the embodiments, serve to explain the principles of the present invention.

FIG. 1 shows a schematic diagram of the system according to the invention, the numbers indicate the following positions:

10 - plate for osteosynthesis;

20 - port system;

30 - needle for supplying a cell graft;

40 - a tube of the port system;

50 - bolts (screws) for fixing the plate;

60 - bone;

70 - holes for screws (bolts) fastening the plate to the bone.

FIG. 2 shows the port system, the numbers indicate the following positions:

1 silicone membrane;

2 outer case;

3 titanium tank;

4 connecting sleeve;

5 catheter;

6 cannula port.

FIG. 3 shows a diagram of the system according to the invention:

10 - plate for osteosynthesis;

20 - port system;

30 - needle for supplying a cell graft;

400 - critical bone defect;

50 - plate fastening screw;

600 - channel connecting the needle for supplying the cell graft with the port system.

Terms and Definitions

For a better understanding of the present invention, below are some of the terms used in the present description of the invention. Unless otherwise specified, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

In the present description and in the claims, the terms "includes", "including" and "includes", "having", "equipped", "containing" and their other grammatical forms are not intended to be construed in an exclusive sense, but, on the contrary, are used in a non-exclusive sense (ie, in the sense of "including"). Only expressions of the type “consisting of” should be considered as an exhaustive list.

In the materials of the present application, the term "connective tissue regenerate" is understood to mean connective tissue newly formed as a result of reparative regeneration.

In the materials of this application, the term "granulation tissue" is understood as a temporary supra-tissue structure, a young connective tissue formed during the healing processes of defects in various tissues and organs. The development of granulations is an adaptive process that promotes the healing of wounds and ulcers, the organization and elimination of foreign bodies and non-viable tissues.

In the materials of this application, the term "bone regenerate", "regenerate" means: 1 - a set of immature proliferating cells in the damaged area; 2 - tissue newly formed as a result of regeneration, in this case bone.

In the materials of this application, the term "regeneration area" is the place of reparative regeneration in a living organism.

In the materials of this application, the term "traumatic defect" is any bodily disability resulting from an injury.

In the materials of this application, the term "fracture", "bone fracture" is understood to mean a complete or partial violation of the integrity of the bone under a load exceeding the strength of the injured area of the skeleton. Fractures can occur both as a result of trauma and as a result of various diseases, accompanied by changes in the strength characteristics of bone tissue.

In the materials of this application, the terms "osteogenic structures", "osteogenic cells" are synonymous, they represent the cells of the stage of osteoblastic differentiation following the stromal stem cells, are partially committed, cambial in the osteoblastic cell differentiation. The population of osteogenic cells is heterogeneous, it includes at least two types of cells - determined osteogenic progenitor cells (they do not need any induction to realize their osteogenic potential, but for osteoblastic differentiation they need close intercellular contacts with microenvironmental cells) and inducible osteogenic cells - precursors (exhibit osteogenic properties only after the action of certain inducers of osteogenesis; adventitious cells accompanying blood vessels, cells in the periosteum and in extraskeletal organs are inducible to osteogenesis).

In the materials of this application, the terms "inducers of osteogenesis", "factors or inducers of bone differentiation", "osteoinducers" mean a substance that can stimulate the differentiation of stem cells and progenitor cells in the direction of osteoblastic diferon - osteoblasts, the formation of bone tissue.

A "differentiation inducer" is a substance that can stimulate stem and progenitor cells to differentiate in a specific direction.

"Induced differentiation" is the differentiation of cells that occurs as a result of exposure to certain factors of a biological or chemical nature.

In the materials of this application, the term "induction factors and stimulants that support osteogenesis without removing the existing necessary biologically active substances that support regeneration" mean various chemicals with biological activity that are present in the tissue fluid in the region of regenerating structures. They are necessary for the implementation of processes aimed at restoring biological structures, in this case, bones.

In the materials of this application, the term "osteogenic environment for bone regeneration" means a local artificially maintained tissue environment for directed differentiation of stem and progenitor cells in the direction of the osteoblastic line of differentiation, favorable for bone regeneration, containing inducers of bone regeneration, including 0.1 μM dexamethasone , 0.05 mM ascorbic acid, 10 mM glycerol-2-phosphate.

In the materials of this application, the term "nutrients" (in the context of cellular technologies) means all substances in an easily digestible form necessary to meet the nutritional and energy requirements of living cells.

Used in this application, the term "nutrient medium" is a mixture of inorganic salts and other nutrient compounds designed to ensure the survival, maintenance of growth and / or proliferation of cells outside the body in vitro, usually a basic medium with additives, such as serum or factors growth.

In the materials of this application, the term "biologically active substances" are chemical substances that, at low concentrations, have high physiological activity in relation to certain groups of living organisms or to certain groups of their cells.

Used in this application, the term "spheroids" refers to tightly packed ball-shaped cell aggregates formed by three-dimensional cultivation. Their important property is the ability for mutual adhesion and subsequent tissue fusion, as well as for adhesion to the elements of the recipient's extracellular matrix. Tissue spheroids have a tissue-like structure - often called three-dimensional micro-tissues, which were created in artificial conditions to maximize intercellular interactions.

In the materials of this application, the term "cellular material", "cell transplant" means osteogenic cells, living cell cultures of autologous cells of the periosteum, perichondrium, bone marrow stromal cells, multipotent mesenchymal stromal cells of bone marrow or adipose tissue, as well as cellular (tissue) spheroids obtained from these cells, biocomposite spheroids, their various combinations.

In the materials of this application, the term "graft" means a suspension based on osteogenic cells, living cell cultures of autologous cells of the periosteum, perichondrium, bone marrow stromal cells, a suspension based on cellular (tissue) or biocomposite spheroids, or their various combinations. In this case, spheroids are spheroids based on autologous cells of the perichondrium, spheroids based on autologous cells of the periosteum, spheroids based on autologous and allogeneic multipotent mesenchymal bone marrow stromal cells and crushed partially demineralized allogeneic bone, or a mixture thereof.

Long bone non-union continues was defined as “failure to achieve bone union for 9 months after injury and there was no sign of recovery for 3 months” or for 6 months if there is no radiographic evidence of healing fracture.

The term "connected" means functionally connected, and any number or combination of intermediate elements between the connected components (including the absence of intermediate elements) can be used.

In addition, the terms "first", "second", "third", etc. are used simply as conditional markers, without imposing any numerical or other restrictions on the enumerated objects.

As a port system in the present invention, in particular, the Selsite port system, which is surgically implanted subcutaneously, with special Surekan needles for supplying a cell graft with a special shape of the tip, which does not damage the port for filling the port system, can be used (B. Braun Melsungen AG), the silicone catheter of this port system is replaced with a longer silicone tube of the same inner hole diameter, but with a thicker wall + 30% (device port system tube), connecting it to the cell graft delivery needle in the osteosynthesis plate. The connection of the outlet cannula of the port and the tube of the port-system is fixed with a special fixing sleeve, similar to that supplied with this port-system.

Detailed disclosure of the invention

In general, the present invention relates to a system for introducing a graft into a defect area, in particular for transplanting a suspension of cells or spheroids, and a method for introducing a graft into a connective tissue regenerate using the system.

The system for transplanting cellular material includes at least one port-system, an osteosynthesis plate, an elastic tube, screws for fixing the osteosynthesis plate, intended for screwing through threaded holes in the plate (Fig. 1). The plate and the port system are connected by means of a silicone tube, from the cannula of the port system to the cannula of the needle for supplying the cell graft. In the plate, a groove is selected with a cutter, into which a silicone tube is placed, the groove is made both from the inner and outer sides of the plate for osteosynthesis.

When transplanting cell material, a suspension with living material is first fed into the patient's body with a special needle for feeding the cell graft through the skin and a silicone membrane directly into the chamber of the port system, and then through the catheter, they enter a silicone tube (inner diameter 1 mm, wall thickness 0.5 mm). The periods of administration of cellular material and other substances are determined by specific treatment protocols. The introduction of the graft is described in more detail below.

Through the port systems, cellular material is periodically supplied to the connective tissue regenerate: a suspension of living cell cultures, in particular autologous cells of the periosteum, perichondrium, bone marrow stromal cells, cellular (tissue) spheroids obtained from these cells, biocomposite spheroids, and their various combinations.

Periodic introduction of cellular material is necessary so that one portion of the cellular material has time to take root, or to implement the scenario of medical signaling cells - the release of cytokines during temporary engraftment of cells, before the next portion is fed, which forms the next volume of the engrafted graft. The frequency depends on the treatment protocol. The introduction of the cellular material is multiple, one-step, repeated at intervals from 12 hours to several days.

The infusion port itself is installed under the skin during minor surgery (Fig. 2). The place and method of setting up the port system is determined by the general condition of the patient and the planned type of treatment. The infusion of drugs is carried out using special Huber-type needles, which, by puncturing the silicone membrane of the port, allow the infusion solution to be injected into the reservoir without damaging the silicone membrane. Further, the drug enters through the catheter into the silicone tube and then into the area of the defect (depending on the type of port - system).

Thus, large bone volumes can be designed in a predictable manner using new cell transplantation protocols, allowing frequent and painless introduction of cellular material for regeneration.

Example

The device is implanted under general anesthesia in aseptic conditions in a general operating room equipped with an X-ray surgical unit to control the position of the device.

The operating field is prepared for the rabbit over the tibia. A soft tissue incision is made, surgical access to the tibia. With the help of a twisted wire saw Jigli, a piece of tibia 3 cm long is cut out from the central part of the diaphysis. Hemostasis is performed.

Next, a specially selected plate for bone osteosynthesis is applied to two parts of the tibia, which is fixedly connected to a needle for supplying a cell graft. The plate allows you to reliably fix parts of the bone from the lateral surface of the bone due to blocking in the holes of the heads of the screws that are inserted into the bone fragments (Fig. 3). Two parts of the bone are fixed in the correct anatomical and topographic position, with open medullary canals opposite each other. Between the fragments there is a free space corresponding to the length and volume of the removed central part of the bone.

The port system is surgically implanted subcutaneously into a subcutaneous pocket, which is performed from the outer part of the superior thigh of the same hind limb of the rabbit. The subcutaneous pocket is made through a surgical approach to the tibia. The soft tissues are moved apart in a blunt way; a silicone tube of the device passes along the wound channel. The distal end of the silicone tube is positioned in the osteosynthesis plate, the proximal end of the silicone tube is attached to a titanium reservoir with a cannula. The titanium tank is sealed in a special case made of biocompatible inert materials (special epoxy resin or plastic - polysulfone). This coating facilitates the implantation of the port under the skin, and the absence of pronounced reactions to a foreign body. The central part of the chamber of the port system consists of a very dense silicone membrane. The connection of the outlet cannula of the port and the silicone tube of the device is secured with a special fixing sleeve. The titanium reservoir is sutured with surgical sutures to the adjacent superficial fascia and hypodermis. The silicone tube travels freely.

Before injection, the surface of the skin above the port is treated with a skin antiseptic. Infusion of the graft, in particular of cell suspensions and solutions, is performed using special needles - Surekan, which pierce the skin and partially the subcutaneous tissue, then the silicone membrane of the port, allow the introduction of aqueous solutions or suspensions into the reservoir without damaging the silicone membrane. Next, the cell suspension is fed into the bone regeneration zone through a silicone tube into a needle for supplying a cell graft, which is inserted into the cavity at the site of the bone defect.

Layer-by-layer wound closure is performed.

The implanted port system does not cause unpleasant sensations in the subject, in particular in the animal model, makes it possible to dispense with the introduction of the animal into a state of anesthesia, and makes the infusion procedure practically painless.

The service life of the system according to the invention depends on the duration of the treatment and can be several months. Combining the main advantages of the port system with the possibility of a controlled introduction of living cells and biologically active substances into the regeneration area.

Thus, the system proposed according to the present invention allows the use of other protocols of cell transplantation (more often to inject cells and substances, to maintain the in vivo mode of the bioreactor - the supply of substances that support bone regeneration). At the same time, the system allows all manipulations to be carried out minimally invasive, almost painless, does not require additional anesthesia and local anesthesia during transplants and injections.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are provided for the purpose of illustrating the present invention only and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims (11)

1. A system for minimally invasive introduction of a graft into a connective tissue regenerate at the site of a bone defect of a subject for a plate of bone osteosynthesis, including: a port-system, which is a device for inserting a cell graft under the skin, which consists of a main body, a silicone membrane and a titanium reservoir with a cannula, silicone tube, plate for osteosynthesis, having at least two threaded holes for fastening by means of screws to the bone in the defect area, and at least two screws for fixing the plate for osteosynthesis, intended for screwing through the above threaded holes, and the plate includes a needle for feeding cell graft, fixedly connected to the plate, and having a cannula, the proximal end of the silicone tube is attached to the titanium reservoir with a cannula, and the distal end of the silicone tube is located in the plate for osteosynthesis up to the cannula of the needle for feeding the cell transplant ntata. 2. The system of claim. 1, wherein the screw is a conical frusto-conical screw. 3. The system of claim. 1, wherein the plate is made of titanium or stainless steel. 4. The system according to claim 1, in which the graft is a suspension based on osteogenic cells, living cell cultures of autologous cells of the periosteum, perichondrium, bone marrow stromal cells, a suspension based on tissue or biocomposite spheroids, or their various combinations. 5. The system of claim 4, wherein the spheroids are spheroids based on autologous perichondrium cells, spheroids based on autologous periosteal cells, spheroids based on autologous and allogeneic multipotent mesenchymal bone marrow stromal cells and ground partially demineralized allogeneic bone, or a mixture thereof. 6. The system of claim. 1, in which the housing of the port system is made of a biocompatible inert material. 7. The system of claim 6, wherein the biocompatible inert material is an epoxy resin or polysulfone. 8. A method of introducing a graft into a connective tissue regenerate to restore bone defects in a subject, which consists in using the system according to claim 1, before injection, the surface of the skin above the port is treated with a skin antiseptic, the graft is infused using a needle that pierces the skin, partly subcutaneous cellulose and then the silicone membrane of the port. 9. The method according to claim 8, wherein the graft is administered periodically as the connective tissue regenerate develops. 10. The method according to claim 8, in which cytokines, osteoinducers, differentiation inducers, hydroxyapatite granules, calcium phosphate salts in the form of granules, a nutrient medium, biologically active substances and / or a suspension of crushed demineralized bone are additionally periodically administered. 11. The method of claim 8, wherein the bone defect is a tubular bone defect.

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