RU2750021C1 - Method for restoration of diaphysis of long tubular bones using cellular technologies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: inventions group relates to medicine, namely to tissue engineering, regenerative medicine, traumatology and orthopedics, and can be used to restore bone tissue in the area of a bone defect. The method includes: installing a temporary insert-spacer corresponding to the size of the defect in the area of the bone defect; removal of the temporary spacer insert is performed 7-30 days after installation; installation of a perforated resorbable membrane with adhered cellular spheroids from the autologous periosteum deposited on its surface. In this case, the perforated membrane is folded in the form of a cylinder and is attached to the periosteum of the ends of the bone sawdust using surgical sutures. In special cases, the temporary spacer insert is made of polymethyl methacrylate, the resorbable membrane is made of a biodegradable material, the biodegradable material is selected from an allogeneic lyophilized periosteum. At the same time, when installing a perforated membrane with cellular spheroids, it is fixed at the edges with suture absorbable threads, the subject is a human, the bone defect is a defect in the tubular bone, a temporary spacer insert is installed in the form of a cylinder, and the cavity inside the membrane is filled with alginate gel. The resorbable membrane is collagen barrier membrane, holes of which have a diameter of 150-350 microns, the distance between the centers of the holes is 550-1000 microns and can be 10-50% of the total surface area of the entire membrane.

EFFECT: invention provides the ability to restore bone tissue in the area of a bone defect by growing bone regenerate in the area of the de novo de novo on a perforated resorbable membrane with adhered cell spheroids from the autologous periosteum deposited on its surface.

14 cl, 1 ex

Description

Technology area

The invention relates to medicine, namely to tissue engineering, regenerative medicine, experimental surgery, dentistry, plastic surgery, traumatology and orthopedics.

State of the art

Repair of a bone defect, including a critical size defect, remains one of the main clinical orthopedic problems. Bone defects of critical size are technically defined as those that do not heal spontaneously in a natural way. A critical defect can occur both in the case of an accidental event (trauma, osteomyelitis), forced surgery (tumor), and deliberately (experimental studies on laboratory animals). For the majority of bones, the sizes of critical defects have been determined; for each bone they are different, they differ both in volume and in configuration. In the presence of a critical defect, restoration of bone integrity by spontaneous regeneration is impossible without artificial stimulation.

Bone grafts are used in reconstructive surgery to restore the structural integrity of bones as components of the musculoskeletal system for the implementation of a private method of bone regeneration - transplant regeneration, as well as to increase the osteogenic potential of the regenerating connective tissue. The need for bone grafts arises for the reconstruction of the skeleton, for the restoration of the integrity of bones, during plastic surgery, restoration of the jaws in dentistry, when replacing defects at the site of bone tumors, revision arthroplasty, etc. In medicine, the need for bone transplantation has increased over the past decade. Bone transplantation is second only to blood transfusions in terms of demand.

Autogenous (autologous) bone grafting is a process in which bone material is collected from one site and transplanted to another site in the same patient. The method of autotransplantation of bone fragments is widely used in traumatology and orthopedics, maxillofacial surgery, plastic and reconstructive surgery, oncology, neurosurgery, etc. Currently it is considered the "gold standard" in bone restoration, due to the possibility of obtaining a predictable clinical result. However, the possibilities of obtaining bone autografts in sufficient quantities to replace extensive bone defects are very limited, therefore, the search for substitutes - bone-grafting materials and bio-artificial tissues that can provide a worthy alternative to autografts - continues. It is extremely important to preserve the viability of bone grafts, since during free transplantation their blood supply is disturbed, lysis of bone grafts often occurs, and this problematic side has not been sufficiently studied.

Known approach for protecting bone grafts from lysis. The so-called Masquelet technique, which involves the induction of a fibrous tissue membrane around the site of a bone defect using the body's foreign body response to the presence of a polymethyl methacrylate (PMMA) spacer. The aim of this study was to investigate the properties and characteristics of this induced membrane and its effectiveness when used in combination with an allograft or allograft / autograft mixture as a filler in a model of a critical size defect in sheep. The resulting induced membrane was found to be effective in holding the grafted materials in place. It has been demonstrated to be an organized pseudosynovial membrane that expresses bone morphogenic protein 2 (BMP2), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), von Willerbrand factor (vWF), interleukin 6 (IL-6) and interleukin 8 (IL-8). Although after 12 weeks more new bone growth was observed in the experimental groups compared to the control animals, the volumes were not statistically different and the defects did not completely overlap. Of the two graft groups, the allograft / autograft mixture has been shown to have a higher graft resorption rate than the allograft-only group. Although the Masquelet method was effective in producing a membrane covering the graft materials, its ability to aid in the healing of critical size segmental defects compared to blank controls remained inconclusive. Although after 12 weeks more new bone growth was observed in the experimental groups compared to the control animals, the volumes were not statistically different and the defects did not completely recover. Of the two graft groups, the allograft / autograft mixture has been shown to have a higher graft resorption rate than the allograft-only group (Christou C., Oliver RA, Yu Y., Walsh WR The Masquelet Technique for Membrane Induction and the Healing of Ovine Critical Sized Segmental Defects / PLoS One. 2014; 9 (12): e114122. Published online 2014 Dec 2.doi: 10.1371 / journal.pone.0114122).

To fill the bone defect, an osteoconductive and osteoinductive matrix of natural or artificial origin can be used, which, being a temporary framework (allogeneic, xenogenic cancellous or cortical bone, various osteoplastic materials), supports the ingrowth of bone regenerate from the living bone; can be a carrier of osteoinductive cytokines that stimulate and support the division of cambial cells of the connective tissue, their differentiation towards skeletal tissues, and, in general, bone differentiation of regenerated cells. Osteogenic cells (osteoblasts and their precursors) are capable of forming bone when placed in a suitable local regeneration environment.

It is important to note that an artificial material for replacing bone defects must have a set of certain properties, in particular, bioinertness. Bioinertness is the ability of a material to maintain the constancy of its composition and structure for a long time due to the absence of local and systemic interaction with the body or its minimally expressed chemical, electrochemical and surface-catalytic manifestation. Bioinert materials include metals, their alloys, polymers, corundum ceramics, and carbon. A fibrous capsule is often formed around bioinert materials, especially those with a smooth surface, through which the body is protected from a foreign body. The thickness and cellular composition of the capsule are a kind of measure of the biocompatibility of the material. It should be noted that any implants are capable of only temporary osseointegration and are inferior to living bone.

Numerous publications indicate that the problem of complete restoration of the anatomical integrity of the bone and the functional viability of bone tissue in the case of a defect, in particular, of a critical size, remains relevant.

Disclosure of invention

The object of the present invention is to develop a new method for the restoration (healing) of a bone defect, in particular, a defect that exceeds the critical size (critical bone defect).

The technical result of this invention is to develop a new and effective method for the restoration of bone and / or cartilage tissue in the area of a bone defect in a subject, in particular, a defect that exceeds the critical size (critical bone defect), allowing the growth of bone and / or cartilaginous regenerate in the area of the defect de novo on the induced pseudosynovial membrane using cell technologies. The method according to the invention is based on the use of cell spheroids deposited on the surface of a perforated membrane. Moreover, this method is characterized by a faster recovery period of bone and / or cartilaginous tissue and is less resource-intensive. In addition, spheroids from autologous cells of the growth zone of the periosteum, which is a source of bone regeneration, are used to restore bone. The periosteum may be removed from the subject in a simple outpatient operation under local anesthesia. At the site of the removed periosteum, its full regeneration is possible.

Achievement of the specified technical result is ensured when implementing a method for restoring bone and / or cartilage tissue in the area of a bone defect in a subject, which includes the following steps:

- installation of a temporary insert-spacer, corresponding to the size of the defect, in the area of the bone defect to form an induced pseudosynovial membrane;

- removal of the temporary insert-spacer 7-30 days after installation and formation of an induced pseudosynovial membrane around the insert-spacer;

- installation of a perforated resorbable membrane with deposited on its surface, adhered and partially spread out cell spheroids from the autologous periosteum, and the perforated membrane folds in the form of a cylinder and is attached to the periosteum of the ends of the bone sawdust using surgical sutures.

In particular embodiments of the invention, the resorbable membrane is made of a biodegradable material. In particular embodiments of the invention, the biodegradable material is selected from allogeneic, xenogenic, or autologous lyophilized periosteum.

In particular embodiments of the invention, the membrane is a collagen barrier membrane.

In particular embodiments of the invention, the holes of the perforated membrane have a diameter of 150-350 μm.

In particular embodiments of the invention, the distance between the centers of the holes is 550-1000 μm.

In particular embodiments of the invention, the holes in the membrane can represent 10-50% of the total surface area of the entire membrane.

In particular embodiments of the invention, when a perforated membrane with cell spheroids is installed, it is fixed at the edges with absorbable sutures.

In particular embodiments, the perichondrium cell spheroids are 250-450 μm in size.

In particular embodiments of the invention, the subject is a human.

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

In particular embodiments of the invention, the temporary spacer insert is installed in the form of a cylinder, which prevents the formation of end plates.

In particular embodiments of the invention, the cavity inside the membrane is filled with an alginate gel.

A temporary spacer insert is inserted into the defect, repeating the shapes and sizes of the removed bone. The spacer insert is compressed using an external fixation device with the edges of the bone sawdust around the open bone marrow cavities. The insert can be in the form of a cylinder, in particular, of bone cement (polymethyl methacrylate - PMMA), hydroxyapatite, silicone, or in the form of a silicone hollow tube sutured to the periosteum.

Operation to remove the spacer insert. In a sterile operating room, surgical access is performed, and the spacer insert is removed, in particular from bone cement, under general anesthesia. The induced pseudosynovial membrane is dissected from the side of the operative approach to remove the spacer insert in the longitudinal direction, as gently as possible.

The aim of the present inventors is to use this induced membrane, the main properties of which are to act as a biological chamber that prevents resorption of the graft and secretes growth factors as a recipient surface for cell transplantation. The induced pseudomembrane not only protects, but also serves as a source of regenerating vessels and maintaining a sufficient partial pressure of oxygen for the bone graft. It has been demonstrated that the induced pseudomembrane is abundantly vascularized by numerous capillaries in all layers. While high concentrations of vascular endothelial growth factor (VEGF) and transforming growth factor B (TGF-B) were obtained in the second week, the concentration of morphogenetic protein-2 (BMP-2) in the membrane was increased to its highest level in the fourth week. week.

The perforated membrane with spheroids is folded into a tube corresponding to the internal dimensions of the induced pseudosynovial membrane formed around the spacer insert. The surface with the transplanted spheroids should fit snugly against the induced pseudosynovial membrane. The perforated membrane along the edges is sutured with absorbable surgical sutures to the periosteum of the bone sawdust. The longitudinally perforated membrane is sutured along with an absorbable suture. Layer-by-layer suturing of tissues is carried out. A gel is injected into the cavity lined with a perforated membrane under pressure using a syringe needle, which ensures tight adherence of the in vitro perforated membrane with spheroids to the induced pseudosynovial membrane formed in vivo around the spacer insert in the defect area. The stage of formation and growth of bone regenerate is carried out in conditions of external hardware fixation of bone fragments.

Definition and terms

Various terms related to the objects of the present invention are used above and also in the description and in the claims. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing the present invention refer to well known techniques, including modifying these techniques and replacing them with equivalent techniques known to those skilled in the art.

In the description of this invention, the terms "includes" and "including" are interpreted as meaning "includes, among other things". These terms are not intended to be construed as “consists only of”.

In the description of this invention, the term "tissue" refers to a system of cells and non-cellular structures that have a common structure, in some cases a common origin, and specialized in performing certain functions.

As used herein, the term "defect" refers to bone tissue if it is absent, reduced in quantity, or otherwise damaged. A bone defect can be the result of illness, treatment for a disease, or injury. "Critical bone defect" is a bone injury in which bone fusion is impossible spontaneously, that is, in which the bone is organotypically organotypically restored in a natural way with the restoration of its structure and anatomical integrity, and the open bone marrow canal of bone fragments is often closed with end plates.

Critical-sized bone defects (CSBDs) are defined as "the smallest bone defect in a particular bone and animal species that does not heal spontaneously during the life of the animal."

Transplant regeneration - regeneration by graft, stimulation of the regeneration process or providing the very possibility of recovery through tissue transplantation, which are further subject to destruction and resorption, create a model (frame) that allows local tissues to regenerate. The transplantation method is used to stimulate the regeneration process or to provide the very possibility of recovery.

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 (cellular) spheroids have a tissue-like structure - often called three-dimensional micro-tissues, in which intercellular interactions are maximized during their production.

Biofabrication is the process of artificially constructing and growing outside the body of living, functional tissues or organs for subsequent transplantation to a patient in order to replace or stimulate the regeneration of damaged organs or tissues, most often using bioprinting technology.

An induced pseudosynovial membrane is a membrane induced by a foreign body (formed around a spacer insert at the site of a bone defect, represented by a connective tissue regenerate rich in vessels), which has the ability to consolidate fragments of a conventional cancellous bone autograft transplanted to repair long bone defects. The main properties of the membrane are to prevent resorption of the bone graft and to secrete growth factors. The induced membrane forms a kind of biological chamber that allows the creation of numerous experimental models of bone reconstruction.

The alginate gel herein, in particular, can be obtained by the method described in Stevens MM, Marini RP, Schaefer D. et al./ In vivo engineering of organs: the bone bioreactor / Proc Natl Acad Sci US A. 2005 Aug 9; 102 (32): 11450-5. In particular, gelation can be initiated by mixing 2% (w / v) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl with an equal volume of a solution containing 75 mM CaCl ₂ in 10 mM Hepes containing 150 mM NaCl and 10 mM KCl using a sterile Y-shaped mixer. The gel cures within 1 min and has a Young's modulus of 0.17 MPa. Growth factors (TGF-β1 and FGF-2, R & D Systems) can be added to the gel, included in the composition at a concentration of 10 ng / ml.

Detailed disclosure of the invention

An example of implementation of the invention

Under general anesthesia in a sterile operating room, surgical access to the rabbit tibia is made. With a surgical wire saw Gigli, a part of the shaft of the bone is cut out to form a bone defect of a critical size - 3 cm. The cut bone in a sterile F12 medium is transferred to the cell technology laboratory.

An external fixation device (such as the Ilizarov apparatus) is applied. A temporary insert-spacer made of bone cement of a cylindrical shape is inserted into the area of the bone defect, repeating the size and shape of the cut out part of the bone. With the help of an external fixation device, the spacer insert is compressed by means of bone sawdust on both sides. Layer-by-layer wound closure with the imposition of an aseptic dressing is performed. The animal is removed from anesthesia and kept in a vivarium.

An autologous periosteum is harvested from the extracted part of the bone in the laboratory. Sterile saline or F12 culture medium is injected under the periosteum using a syringe. In the form of a film, the periosteum is removed from the bone. The inner layer of the periosteum is carefully scraped from the inner layer with a sterile scalpel inside the Petri dish filled with F12. Fragments of the periosteum are placed in a collagenase solution.

To obtain a primary culture, cells isolated from the periosteum are seeded in plastic flasks with a culture medium of the following composition: DMEM (or DMEM / F 12) 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 U / ml, streptomycin 100 μg / ml, at 100% humidity, 37 ° C, 5% CO ₂ (this environment and conditions are used at all stages of subsequent cultivation). The cultivation is carried out for 24 days.

After the third passage, cells are removed from plastic vials with trypsin / EDTA. The resulting cell suspension is washed from trypsin / EDTA, including using DMEM medium with 10% serum, followed by centrifugation (10 minutes at 200g) and transferred to 81-well agarose plates with a well diameter of 800 μm at a concentration of up to 1.6 million cells on the plate, where spheroids begin to form in the wells from the cultured cells (the grown periosteal cells in the form of a suspension are transferred into 3D Petri Dish® from agarose (agarose microwells) (MicroTissues Inc.)).

Spheroids stay in the wells for up to 7 days with a change in the nutrient culture medium every 2-3 days.

To place the spheroids on a perforated membrane, they are collected from the wells and transferred to a test tube, where they settle to the bottom without additional centrifugation.

The spheroids are then placed in a syringe and transferred to a perforated membrane that is in the culture medium inside the culture flasks (cell culture mattresses) with a resealable side cap. All vials have a non-adhesive (untreated) bottom surface of the vial. Spheroids are manually placed under the binoculars over each perforated hole in the membrane. The vials are filled with a nutrient medium and placed in a CO ₂ incubator. Within 3-5 days, cell spheroids adhere and partially spread out on this membrane. In fact, a tissue-engineered structure is grown - a bio-artificial periosteum or a living equivalent of the periosteum.

The perforated membrane can be made of decellularized allogeneic lyophilized periosteum or OSSIX® Plus material - a resorbable cross-linked collagen barrier membrane for guided bone (GBR) and tissue regeneration (GTR), laser piercing of the holes is carried out over the entire area of the membrane due to the sublimation of the material.

When the living equivalent of the periosteum (perforated membrane with precipitated spheroids) is ready for transplantation, the spacer insert is removed. In a sterile operating room, surgical access is performed, and the bone cement insert is removed under general anesthesia. The external fixation device is loosened. The induced pseudosynovial membrane is dissected from the side of the operative access in the longitudinal direction as carefully as possible, and the spacer insert is removed as carefully as possible from the surrounding connective tissue capsule - the induced pseudosynovial membrane.

The perforated membrane with spheroids rolls up into a tube corresponding to the outer dimensions of the removed spacer insert. The surface of the perforated membrane with transplanted spheroids should adhere tightly to the induced pseudosynovial membrane. The perforated membrane along the edges is sutured with absorbable surgical sutures to the periosteum of the bone sawdust. The longitudinally perforated membrane, together with the dissected pseudosynovial membrane, is sutured along with an absorbable suture. Layer-by-layer suturing of tissues is carried out. A gel, in particular an alginate gel, is injected into a cavity lined with a perforated membrane under pressure using a syringe needle, which ensures tight adherence of the perforated membrane with spheroids grown in vitro to the induced pseudosynovial membrane formed in vivo around the spacer insert. The specified gel promotes osteogenesis and temporarily fulfills the function of filling the free space.

The external fixation device remains for the entire period of observation of the formation and growth of bone regenerate.

Within 2 months, the formation of reparative regenerates of skeletal tissues (hypertrophic cartilage, reticulofibrous mineralized (coarse-fibrous) bone tissue) in the area of bone defects is detected.

Cell spheroids

Spheroids according to the present invention refers to spherical cell aggregates formed from living cells 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 extracellular matrix. As a source of skeletal connective tissue cells for the production of spheroids according to the present invention, cells of the inner layer of the periosteum are used, which are the cambial zone for skeletal connective tissues, supplying progenitor and stem cells for physiological and reparative regeneration, therefore, containing a greater number of progenitor cells. In particular embodiments of the invention, the size of such spheroids is 200-450 μm. The method for preparing such spheroids is given below.

The collection of periosteal cells is carried out using a minimally invasive method. Local anesthesia of the skin, subcutaneous tissue and periosteum is performed. Through a small incision, we reach the periosteum, using a needle with saline, we separate the periosteum from the rib, fill the space between the compact bone of the rib and its periosteum, and then carefully dissect the flap - the periosteum. Isolation of cells from the periosteum was performed by preliminary mechanical scraping of the inner layer of the periosteum, followed by enzymatic dissociation of tissue pieces of this layer to single cells.

To obtain a primary culture, cells isolated from the periosteum are seeded in plastic flasks with a culture medium of the following composition: DMEM (or DMEM / F12) 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 U / ml, streptomycin 100 μg / ml, at 100% humidity, temperature 37 ⁰ С, 5% СО ₂ (this medium and conditions are used at all stages of subsequent cultivation).

Subcultivation of a monolayer culture of progenitor cells of skeletal connective tissues is carried out until the third - fourth passage with a change of medium every 2-3 days.

After the last passage, cells are removed from plastic vials with trypsin / EDTA. The resulting cell suspension is washed from trypsin / EDTA, including using DMEM medium with 10% serum, followed by centrifugation (10 minutes at 200g) and transferred to 81-well agarose plates with a well diameter of 800 μm at a concentration of up to 1.4 million cells onto the plate, where spheroids begin to form from the cultured cells in the wells.

Spheroids are formed in the holes for up to 7 days with a change in the nutrient medium every 2-3 days.

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 (17)

1. A method for restoring bone tissue in the area of a bone defect in a subject, comprising the following steps: - installation of a temporary insert-spacer, corresponding to the size of the defect, in the area of the bone defect; - removal of the temporary spacer insert 7-30 days after installation; - installation of a perforated resorbable membrane with adhered cellular spheroids from the autologous periosteum deposited on its surface, and the perforated membrane is folded in the form of a cylinder and is attached to the periosteum of the ends of the bone sawdust using surgical sutures. 2. The method of claim 1, wherein the temporary spacer is polymethyl methacrylate. 3. The method of claim 1, wherein the resorbable membrane is made from a biodegradable material. 4. The method of claim 3, wherein the biodegradable material is selected from allogeneic lyophilized periosteum. 5. The method of claim 3, wherein the membrane is a collagen barrier membrane. 6. The method of claim 1, wherein the holes in the perforated membrane have a diameter of 150-350 microns. 7. The method according to claim 6, wherein the distance between the centers of the holes is 550-1000 microns. 8. The method of claim 1, wherein the holes in the membrane can represent 10-50% of the total surface area of the entire membrane. 9. The method according to claim 1, wherein when the perforated membrane with cell spheroids is installed, it is fixed at the edges with absorbable suture sutures. 10. The method of claim 1, wherein the subject is a human. 11. The method of claim 1, wherein the bone defect is a tubular bone defect. 12. The method of claim. 1, wherein the temporary spacer is mounted in a cylindrical shape to prevent endplate formation. 13. The method of claim 1, wherein the cavity within the membrane is filled with an alginate gel. 14. Perforated resorbable membrane with adhered cell spheroids from the autologous periosteum deposited on its surface to restore bone tissue in the area of the bone defect of the subject by the method according to claim 1, characterized in that the resorbable membrane is made of biodegradable material, the holes of the perforated membrane have a diameter of 150-350 μm, the distance between the centers of the holes is 550-1000 μm, the holes in the membrane are 10-50% of the total surface area of the entire membrane.