RU2745995C1 - Method for manufacturing a cellless hydrogel from warton's jelly made of human umbilical cord for intra-article application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method for manufacturing a cell-free hydrogel from Wharton's jelly of the human umbilical cord. A method of manufacturing a cell-free hydrogel from Wharton's jelly of the human umbilical cord for intra-articular use in order to restore damage to the articular cartilage, including obtaining a native umbilical cord, sterilization with a hydrogen peroxide solution, its preparation, fragmentation and homogenization, decellularization with sodium hydroxide followed by its neutralization with a solution of hydrochloric acid, washing and detached cells, neutralization with sodium hydroxide solution and re-centrifugation; lyophilization, then the resulting lyophilized matrix is solubilized by exposing the resulting matrix to pepsin in a solution of hydrochloric acid, after which the medium is neutralized by titration with sodium hydroxide to physiological pH values, under certain conditions.

EFFECT: above method allows to create a full-component matrix and hydrogel from the connective tissue of the human umbilical cord using the most physiological methods, which has regenerative properties to ensure the healing of cartilage and soft tissue injuries.

1 cl, 14 dwg

Description

The invention relates to the field of satisfying the vital needs of a person, namely to the field of medicine, biotechnology, regenerative medicine, pharmacology, in particular, to methods of treating traumatic injuries of the articular cartilage by creating a cell-free stored tissue-engineered product to replace cartilage defects with the provision of their regeneration and can be used , in addition to traumatology, in the restoration of soft tissues of the body.

There are the following technical solutions, known as such for their intended purpose, namely for the treatment of traumatic injuries of articular cartilage:

pharmaceutical - symptomatic drugs used in clinical practice, including for intra-articular administration (non-selective non-steroidal anti-inflammatory drugs, cyclooxygenase inhibitors; corticosteroids, hyaluronic acid, platelet-rich plasma);

surgical methods for the restoration of cartilaginous and osteochondral defects - abrasive mechanical chondroplasty, osteoperforation techniques (microfracturing, tunneling, reaming), the use of autologous osteochondral grafts (mosaic chondroplasty); methods using tissue engineering technologies (implantation of mesenchymal stem cells (hereinafter - MSC), chondrogenic stem cells, autologous chondrocytes, including matrix-induced ones). Each of these methods has separate advantages and disadvantages.

Long-term exposure to glucocorticosteroids potentiates gross cartilage damage due to toxic effects on chondrocytes [Wong M, Carter D. Articular cartilage functional histomorphology and mechanobiology: a research perspective. Bone. 2003; 33 (1): 1-13], contributing to the progression of post-traumatic osteoarthritis (OA). The effectiveness of corticosteroid injections decreases over time [Knutsen G., Drogset J., Engebretsen L., Grontvedt T., Ludvigsen T., Loken S., Solheim E., Strand T., Johansen O. A Randomized Multicenter Trial Comparing Autologous Chondrocyte Implantation with Microfracture. J. Bone Jt. Surg. 2016; 98: 1332-1339].

Intra-articular injections of hyaluronic acid are widely used in the treatment of osteoarthritis, but a meta-analysis of randomized clinical trials did not reveal a significant effect of intra-articular injections of hyaluronic acid compared with intra-articular injections of placebo [Jiang Y. Cell transplantation for articular cartilage defects: principles of past, present, and future practice ... CellTransplant. 2011; 20: 593-607].

The use of platelet-rich plasma by intra-articular injection for post-traumatic OA improves pain scores and functional outcomes. However, the method is not recommended by the OARSI (osteoarthritis research society international) for the treatment of post-traumatic OA due to the lack of convincing and reliable clinical data and long-term treatment results [Shahid M., Kundra R. Platelet-rich plasma (PRP) for knee disorders. EFORT OpenRev. 2017; 2: 28-34].

Surgical methods for osteoperforation of the subchondral bone (microfracturing, tunneling, reaming) are widely used because of their simplicity and low cost. However, this approach is effective only for small defects, provides a relatively short-term functional improvement due to the formation of fibrocartilage, and not native hyaline articular cartilage [Gobbi A., Whyte G. One-Stage Cartilage Repair Using a Hyaluronic Acid-Based Scaffold With Activated Bone Marrow- Derived Mesenchymal Stem Cells Compared With Microfracture: Five-Year Follow-up. Am. J. SportsMed. 2016; 44: 2846-2854]. Autologous osteochondral transplantation (mosaic chondroplasty) is the transplantation of osteochondral cylindrical grafts from the area of the unloaded articular surface into the area of the cartilage tissue defect. The disadvantages of the method include the limited availability of donor tissue and soreness of the donor site, which makes mosaic plasty only applicable to small defects. In addition, mosaic plasty is a surgically challenging task, since all implanted cylinders must be modeled in order to provide a flat cartilage surface [Gracitelli G.C., Meric G., Briggs D.T., Pulido P.A., McCauley J.C., Belloti J.C., Bugbee W.D. Fresh Osteochondral Allografts in the Knee. Am. J. Sports Med. 2015; 43: 885-891].

The main limitations of cell therapy are the high cost [Murray C, Vos T, Lozano R, Naghavi M, Flaxman A, Michaud C. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study. Lancet 2010; 380: 2197-2223], the need for at least two operations, the problem of fixing the cellular material in the area of the cartilaginous defect, and the formation of fibrocartilage, often due to dedifferentiation of chondrocytes during cell expansion [Goyal D., Goyal A., Keyhani S., Lee E ., Hui J. Evidence-Based Status of Second- and Third-Generation Autologous Chondrocyte Implantation Over First Generation: A Systematic Review of Level I and II Studies. Arthrosc. J. Arthrosc. Relat. Surg. 2013; 29: 1872-1878].

Currently, tissue engineering is being developed for the production and use of acellular scaffolds as natural scaffolds from various tissues, such as heart valves, blood vessels, skeletal muscles, placenta, and skin. Biomaterials for obtaining matrices from these tissues can be xenogenic and homologous donor tissues, as well as their individual components - biopolymers. Bioactivity, biodegradability, and the presence of surface ligands for cell adhesion are inherent in matrices made from natural materials. Tissue biomaterials from which cells have been removed are some of the best options for preparing biomimetic scaffolds. The decellularized matrix retains the architecture created by nature for a particular tissue to support cell growth and tissue organization, so the matrix is tissue-specific. The decellularization process, by removing the cells of the donor tissue, preserves the components of the native ECM with the growth factors fixed on it. Therefore, decellularized matrices are the most attractive design for creating a tissue-engineered graft on its basis.

The tissue-specificity of the matrix, its three-dimensional structure, composition, porosity are the necessary conditions for the colonization of cells, their distribution and differentiation into functioning elements. However, the receipt of intravital donor tissues for the creation of matrices is extremely limited due to their limited nature, and posthumous ones due to legal and ethical difficulties that require time to obtain the documentary consent of relatives [Federal Law of 23.06.2016 N 180-FZ "On Biomedical Cell Products " // "Russian newspaper". - 2016. - No. 139. - June 28].

A suitable source of biomaterial for creating tissue-engineered grafts is preferably limited on the basis of homologous origin. This is associated with the risk of immunological conflicts [Galili U. Acceleration of Wound Healing by α-gal Nanoparticles Interacting with the Natural Anti-Gal Antibody Journal of Immunology Research Volume 2015, Article ID 589648, 13 pages http://dx.doi.org/ 10.1155 / 2015/589648] and transmission of prion infections by xenogeneic matrices. Among homologous tissues, even matrix-specific donor tissues (both postmortem and lifetime) are not ideal, since the composition and structure of tissues have undergone changes during the donor's life. Malformations, past illnesses, age-related changes, the effects of stress, taking medications, the influence of harmful working conditions or the habitat can significantly change the components and architecture of tissues, which can be aggravated by decellularization procedures. As a result of these influences, matrices can adversely affect the attachment of recruited cells, their dispersal, maturation and functioning to create their own remodeled matrix in place of the degraded transplanted matrix.

However, the main biological reason for the non-optimal use of a donor biomaterial for the manufacture of matrices is its postnatal structure with a loss of regenerative potencies. It is known that the tissues of the embryo and provisional organs have special components that provide regenerative recovery processes at the stages of gestation, similar to the ontogenetic processes of fetal development. Unlike adult tissues, the extracellular matrix (ECM) of fetal tissues and provisional organs consists of more immature collagen with fewer cross-links, an abundance of high molecular weight hyaluronan, the presence of growth factors in the matrix, which contributes to more effective tissue remodeling [Badylak S.F. Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response. Ann. Biomed. Eng. 2014; 42: 1517-1524. https://doi.org/10.1007/s10439-013-0963-7]. Therefore, it can be assumed that ECM from extraembryonic tissues will provide more efficient and constructive tissue remodeling than ECM from tissues of adult donors. Being an extraembryonic tissue, it is free of age-related disadvantages, such as pathological ECM remodeling, fibrosis, oxidative stress and other negative changes [Koci Z. Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues. Tissue Engineering Part C-Methods. 2017; 23 (6): 333-345. https://doi.org/10.1089/ten.tec.2017.0089].

The umbilical cord, originating from the extraembryonic mesoderm, provides intrauterine development of the fetus by delivering substrates, oxygen and removing metabolites by the mother's blood. Wharton's umbilical cord jelly surrounds the vessels of the fetus and is believed to prevent compression, twisting and bending of the umbilical cord. Wharton jelly of the human umbilical cord is a hard mucous connective tissue of extraembryonic origin, consisting of ECM and mesenchymal cells. The Varton's jelly VKM is compositionally similar to the matrix of hyaline cartilage, the structure of which is represented by different types of collagens, glycoproteins, and proteoglycans. Moreover, each of the components separately is considered today as a potential therapeutic agent for regenerative medicine [Pearsall A, Madanagopal S, Tucker J. The Evaluation of Refrigerated and Frozen Osteochondral Allografts in the Knee. SurgicalScience. 2011; 2 (5): 232-241. https: //doi/org/10.4236/ss.2011.25052]. The strategic similarity of the processes of regeneration and embryogenesis suggests that morphogenetic signals of extraembryonic structures can be used in the development of tissue-engineered structures for tissue regeneration [Kalyuzhnaya LI, Kharkevich ON, Shmidt AA, Protasov OV. Regenerative properties of extraembryonic human organs in tissue engineering // Bulletin of the Russian Military Medical Academy. - 2018.- T. 64. - No. 4. - S. 192-198.]. It is known that fetal wounds, in contrast to postnatal wounds, quickly regenerate. This is facilitated by: 1) the dominance of the transforming growth factor tgf-β ₃ over tgf-β _1,2 , characteristic of the tissues of an adult organism, the abundance of other growth factors; 2) features of structural components (predominance of immature fetal collagen over mature, a small amount of cross-linked collagen; predominance of high molecular weight hyaluronan in the fetus over low molecular weight hyaluronan of postnatal tissues; 3) features of fetal cytokine regulation (anti-inflammatory IL-10 is more significantly expressed than in the fetus IL-6, IL-8); 4) features of the cellular composition of fetal tissues (the predominance of fibroblasts over myofibroblasts, a small number of mast cells and macrophages, inactivity and non-adhesiveness of platelets; the presence of MSCs). Scaffolds from tissues of provisional organs after the decellularization procedure retain signaling molecules and growth factors that promote regeneration in the area of matrix implantation [McAlindon T., Bannuru R., Sullivan M., Arden N., Berenbaum F., Bierma-Zeinstra S., Hawker G ., Henrotin Y., Hunter D., Kawaguchi H. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthr. Cartil. 2014; 22: 363-388].

Studies on the use of Varton's jelly VCM in experiments have shown that: 1) its biocompatibility and adhesive properties are comparable to those of cartilage matrices; 2) the high chondrogenic potential of the matrix is due to the similarity of the ECM microenvironment with native cartilage; 3) growth factors of ECM promote the production of collagen II and aggrecan by chondrocytes, which are specific for hyaline cartilage; 4) ECM, initially highly hydrated due to the abundance of hyaluronan, can be transformed into an injectable hydrogel, which polymerizes under physiological conditions inside the joint [Badylak S.F. Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response. Ann. Biomed. Eng. 2014, 42: 1517-1524. https://doi.org/10.1007/s10439-013-0963-7; McAlindon T., Bannuru R., Sullivan M., Arden N., Berenbaum F., Bierma-Zeinstra S., Hawker G., Henrotin Y., Hunter D., Kawaguchi H. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthr. Cartil. 2014, 22: 363-388; Shahid M., Kundra R. Platelet-rich plasma (PRP) for knee disorders. EFORT OpenRev. 2017; 2: 28-34].

The main goal of foreign researchers in the creation of cell-free matrices is to preserve the three-dimensional architecture of tissue-specific donor tissue. For reasons of its inaccessibility and suboptimality, our attention is directed to the tissues of provisional organs that have not undergone changes due to age, stress or disease, and, moreover, retain the fetal phenotype of tissues due to their extraembryonic origin. It has been proven that adhesion molecules and growth factors are fixed on the structural components of the matrix [Leung A. Fetal wound healing: implications for minimal scar formation. CurrOpinPediatr. 2015; 24 (3): 371-378]. The extracellular matrix (ECM) from these tissues and organs is a rich source of growth factors and biologically active molecules of cell proliferation, differentiation and migration [Taylor-Weiner N., Schwarzbauer J.E., Engler A.J. Defined Extracellular Matrix Components are Necessary for Definitive Endoderm Induction. 2013 doi: 10.1002 / stem.1453]. The applicants were interested in the component similarity of the mucous connective tissue of the umbilical cord with hyaline articular cartilage, in connection with which the idea arose to use this similarity to repair articular cartilage defects.

From the investigated prior art, the applicant identified an invention under the patent USA 2011/0256186 A1, 2011 [Perez J.F., Basterrecchea M.D.O., Feo M.B.C., Martinez A.I., Varona A.I.A., Casado T.P. Biomaterial based on Wharton's Jelly from the human umbilical cord], which describes a method for producing a biopolymer from the human umbilical cord, consisting exclusively of glycosaminoglycans (non-sulfated hyaluronic acid, sulfated dermatan sulfate and heparan sulfate). The essence of the invention consists in the manufacture of a hyaluronic hydrogel with greater than natural rigidity due to chemical cross-linking (crosslinking) using bimethylaminopropyl-carbodiimide (1-ethyl-3- (3-dimethylaminopropyl carbodiimide) structural glycosaminoglycan molecules, for its subsequent use either independently or in combination with cells for the reconstruction of soft tissues, treatment of scars, burns, ulcers, degeneration of intervertebral discs, osteoarthritis, etc. The material in solid (dried in vacuum) form, the authors of the invention propose to use in the treatment of burns, ulcers, skin defects, ophthalmic diseases, osteochondral defects, as a feeder during cell cultivation.At the same time, despite the positive results of cartilage restoration shown by the authors 4 weeks after resection of the anterior cruciate ligament of the knee joint of rabbits, there are the following disadvantages of this method:

First, in this patent, the hydrogel is based on only a certain part of the natural structural proteins of the valuable biological material of the human umbilical cord, namely glycosaminoglycans. All collagen structures, including collagens important for the subsequent remodeling of the basement membrane of the human umbilical cord, were removed by centrifugation. However, it is known that the ability to self-assemble and form a three-dimensional structure is possessed just by combinations of glycosaminoglycans and collagens [Koci Z. Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues. Tissue Engineering Part C-Methods. 2017; 23 (6): 333-345. https://doi.org/10.1089/ten.tec.2017.0089].

Secondly, the cited patent used papain peptidase, an enzyme that has abnormal activity, to remove cells and extract glycosaminoglycans. The patent does not indicate whether its activity was neutralized at the end of digestion, and does not describe how to eliminate it from the product. The ability of papain in the composition of dried hyaluronan, after transformation into a gel, to restore its activity at physiological pH and temperature has not been studied. This circumstance preserves the risk of restoring the peptidase activity of papain in vivo.

Thirdly, a feature of the hydrogel obtained in the patent from glycosaminoglycans is its low conformational rigidity. To obtain a tougher hydrogel that retains a given shape, the authors “crosslink” it using 1-ethyl-3- (3-dimethylaminopropyl carbodiimide, after this procedure the crosslinking agent must be washed out of the product. The patent does not indicate how the quality of product washing is controlled) from the agent.

Structural proteins of the native umbilical cord are distinguished by a natural feature consisting in the absence of cross-linking, due to which the wounds of the fetus heal with gentle elastic scarring or complete regeneration. The high mechanical strength of the hydrogel or matrix, created artificially by chemical agents or photoprocesses, potentiates the development of not regenerative, but cicatricial healing.

From the investigated prior art, the applicant identified scientific publications on the manufacture and use of acellular matrices and hydrogels to restore lost or damaged tissue [Herrero-Mendez A., Palomares T., Castro B. HR007: a family of biomaterials based on glycosaminoglycans for tissue repair. JTissueEngRegenMed. 2017; 11 (4): 989-1001; Koci Z. Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues. Tissue Engineering Part C-Methods. 2017; 23 (6): 333-345. https://doi.org/10.1089/ten.tec.2017.0089].

In the invention of the USA patent 2011/0256186 Al, 2011 [Perez J.F., Basterrecchea M.D.O., Feo M.B.C., Martinez A.I., Varona A.I.A., Casado T.P. Biomaterial based on Wharton's Jelly from the human umbilical cord] describes a method for producing a biopolymer from the human umbilical cord, consisting exclusively of glycosaminoglycans (non-sulfated hyaluronic acid, sulfated dermatan sulfate and heparan sulfate. The essence of the invention consists in the manufacture of a hyaluronic hydrogel with greater than natural rigidity chemical cross-linking (stitching) using dimethylaminopropyl carbodiimide (1-ethyl-3- (3-dimethylaminopropyl carbodiimide) structural glycosaminoglycan molecules, for its subsequent use either alone or in combination with cells for soft tissue reconstruction, treatment of scars, burns, ulcers, degeneration of intervertebral discs, osteoarthritis, etc. The material in solid (dried in vacuum) form, the authors of the invention propose to use in the treatment of burns, ulcers, skin defects, ophthalmic diseases, osteochondral defects, as a feeder for cell cultivation.

There are patent documents in which the umbilical cord is used to obtain mesenchymal stem cells, for this purpose, the processes of separation of Wharton's jelly, from which these cells are obtained, are carried out. For example, PCT document 98/17791 describes the isolation of pre-chondrocytes from the umbilical cord, which are subsequently used therapeutically for the production of cartilage. In document WO 2004/072273 A1, progenitor cells are extracted from Wharton's jelly surrounding the umbilical cord vessels and are used to repair human tissue.

However, there is no document that mentions a biomaterial, which is a natural mixture of linear polymers, devoid of cellular material and DNA, retaining in its composition most of the important molecules for subsequent colonization by patient-specific cells, which can form a hydrogel with the necessary viscosity properties for its use. in order to restore damaged / lost human tissue. Based on the above, the most promising direction in the creation of acellular tissue-engineered constructions can be considered the use of the umbilical cord biomaterial, the shape of such constructions can be three-dimensional or injection.

Based on the performed and stated analysis of the studied state of the art in scientific and technical literature and patent databases, as of the date of submission of the application materials, the applicant did not identify technical solutions for creating a cell-free full-component tissue-engineered construct and a hydrogel from it, using the solid mucous membrane of the umbilical cord as a biomaterial human (Varton's jelly). The closest to the claimed technical solution, selected by the applicant as a prototype, in terms of the coinciding set of features and purpose, is the USA patent 2011/0256186 A1, 2011: Perez J.F., Basterrecchea M.D.O., Feo M.B.C., Martinez A.I., Varona A.I.A., Casado T.P. Biomaterial based on Wharton's Jelly from the human umbilical cord. The biomaterial developed in this invention consists of human umbilical cord glycosaminoglycans, has a three-dimensional structure, which allows it to be used as a base matrix for tissue engineering. When applied alone or together with cells, it interferes with the regenerative process, exerting a noticeable effect on the cells of the tissue itself and providing a favorable environment for the activation of cellular processes

It should be noted that the product manufacturing method used in the prototype has the following disadvantages.

First, in this patent, the hydrogel is based on a certain part of the natural structural proteins of the valuable biological material of the human umbilical cord, namely glycosaminoglycans. All collagen structures, including collagens important for the subsequent remodeling of the basement membrane of the human umbilical cord, were removed by centrifugation. However, it is known that it is precisely combinations of glycosaminoglycans and collagens that are capable of self-assembly and the formation of a three-dimensional structure. Therefore, in the research of the applicant, a successful attempt was made to preserve all structural and functional components of a biomaterial with a high regenerative potential in a cell-free product.

Secondly, the cited patent used papain peptidase, an enzyme that has abnormal activity, to remove cells and extract glycosaminoglycans. The patent does not indicate whether its activity was neutralized at the end of digestion, and does not describe how to eliminate it from the product. The applicants conducted a series of experiments on the use of papain for similar purposes and found that under the conditions specified in the patent (60 ° C in an extraction buffer solution for 24 hours), papain continues to degrade matrix proteins in quantities four times higher than that used in the cited patent for four the next day, and at a temperature of 37 ° C. It can be expected that when a product made with papain is used, after its introduction into the cavities, its peptidase activity can be resumed. Therefore, to remove cells, applicants used 0.2 M NaOH, which at the end of the process was completely neutralized with a 0.1 N HCl solution. The resulting NaCl salt dissociates in water and can be easily removed by centrifugation and lyophilization. In addition, natural glycosaminoglycans exist in the umbilical cord biomaterial in the form of sodium salts. Thus, the decellularization reagent used by the applicants, being in itself non-toxic to structural proteins, allows not only to effectively detach cells, but also to be easily eliminated from the matrix.

Thirdly, a feature of the hydrogel obtained in the patent from glycosaminoglycans is its low conformational rigidity. To obtain a more rigid hydrogel that retains a given shape, the authors of the prototype "crosslink" it using 1-ethyl-3- (3-dimethylaminopropyl carbodiimide, after this procedure the crosslinking agent must be washed out of the product. The patent does not indicate how the quality of washing is controlled product from the agent.

From the point of view of maintaining the advantages of a biomaterial with high regenerative properties, including a spatial network of glycosaminoglycan fibers (and in the applicant's study also collagen), naturally free of crosslinks, the applicants avoided any artificial tightening of the matrix using crosslinking agents. Structural proteins of the native umbilical cord are distinguished by a natural feature consisting in the absence of cross-linking, due to which the wounds of the fetus heal with gentle elastic scarring or complete regeneration. Judging by the publications, it is possible to make a tissue-engineered product more mechanically strong using an additional component (silk fibroin, polylactide, chitosan). In this case, the main component of such a combination will have the fully preserved advantages of an extraembryonic source. Therefore, the applicants set themselves the task of preparing a full-component matrix from the umbilical cord, containing all structural as well as non-structural functionally significant molecules of the extracellular matrix, using the most physiological methods.

Based on the foregoing, according to the applicant, the most promising direction can be considered the creation of full-component uncrosslinked artificially hydrogels from the cell-free matrix of the human umbilical cord for the purpose of their injection into the cavities of defects.

The aim of the claimed technical solution is to create a more effective method of treating traumatic injuries of the articular cartilage by intra-articular injection of a full-component hydrogel from the cell-free matrix of the human umbilical cord by creating a full-component matrix and a hydrogel from the connective tissue of the human umbilical cord using the most physiological methods of matrix decellularization and its solubilization into injection with regenerative properties to ensure the healing of damage to cartilaginous and soft tissues.

The solution to this problem is provided by the fact that in the method of manufacturing a cell-free hydrogel from Wharton's jelly of the human umbilical cord for intra-articular use in order to restore damage to the articular cartilage, including obtaining a native umbilical cord, sterilization with 10% hydrogen peroxide solution, its preparation, fragmentation and homogenization, decellularization, washing from reagent and detached cells, neutralization with sodium hydroxide solution and repeated centrifugation, lyophilization, sodium hydroxide at a concentration of 0.2 M is used to remove cells from the matrix during decellularization, which is neutralized at the end of the process with a solution of hydrochloric acid 0.01 N, and the procedure for solubilization of the resulting lyophilized matrix produced by exposing the resulting matrix of pepsin in a solution of 0.01 N hydrochloric acid at a concentration of 2 mg / ml for 24 hours, after which the medium is neutralized to physiological pH values by titration with 0.01 N sodium hydroxide.

The goal is achieved by using the claimed method of manufacturing a cell-free matrix and hydrogel from Wharton's jelly of human umbilical cord using 0.2M sodium hydroxide solutions and 1 mg / ml pepsin solution in 0.01N hydrochloric acid.

The claimed method for stimulating the regeneration of hyaline cartilage is realized by three times injecting a cell-free hydrogel made from the human umbilical cord into the knee joint of a rabbit with a preliminary simulated defect with a diameter and depth of 3 mm.

A feature of the hydrogel made and used by the applicant is its basis - a full-component matrix composition, including hyaluronic acid, sulfated proteoglycans, various types of collagens, while the components are separately investigated as potential therapeutic agents for regenerative medicine. An additional justification for the biocompatibility of the hydrogel and the matrix of the umbilical cord with hyaline cartilage is the literature data on the preservation of growth factors in them that stimulate chondrocytes to produce collagen II and aggrecan.

A feature of the claimed invention is also the use of a reagent to remove cells from the matrix, which, upon completion of decellularization, is completely eliminated from the product. Sodium hydroxide in a minimum concentration (0.2M) is neutralized with a solution of hydrochloric acid (0.01N) with the formation of sodium chloride, which is removed with water during centrifugation.

Instead of obtaining a three-dimensional matrix of the umbilical cord as a regenerative material, we decided to make its liquid form for intra-articular injection with the assumption that the hydrogel has not only compositional but also biomimetic properties similar to the matrix. Our approach is distinguished by the use of suspension macromolecules of matrices instead of their three-dimensional structures. Hydrogels are an innovative way to work with matrix. Enzymically treated scaffolds contain nanovesicles with inductive properties, including growth and proliferation, survival, migration and differentiation of recruited recipient cells [Huleihel L., Hussey G.S., Naranjo J.D., Zhang L., Dziki J.L., Turner N.J., Stolz D.B., Badylz Matrix-bound nanovesicles within ECM bioscaffolds. Sci. Adv. 2016; 2: e1600502, 11p. http://advances.sciencemag.org]. The resulting liquid injectable suspension is able to fill the cavities of defects, subsequently colonized by cells [Eweida, A.M. Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells? / A.M. Eweida, M.K. Marei // Biomed. Res. Int. 2015; 9. https://doi.org/10.1155/2015/839694].

The experiments were carried out on 15 sexually mature chinchilla rabbits weighing 2.0-2.5 kg. Care, maintenance and work with animals was carried out in accordance with the requirements of the law "On Veterinary Medicine" (No. 243-FZ as amended on July 13, 2015) and the local ethics committee at the Military Medical Academy (No. 229 dated December 02, 2019, Saint Petersburg). The animals were kept in separate cages, they were provided with a standard daily regimen in accordance with zoohygienic and veterinary and sanitary requirements.

The claimed method is illustrated in FIG. 1-14, which show

- the stages of making a cell-free matrix (Fig. 1. a - native umbilical cord; b - removal of arteries and veins of the umbilical cord and isolation of Wharton's jelly; c - fragmentation, d - homogenized umbilical cord);

- the results of DNA detection by gel electrophoresis (Fig. 2. DNA in the native umbilical cord (E1, H1), cell-free matrix (F1) and hydrogel (G1, B2));

- histological confirmation of the effectiveness of umbilical cord decellularization (Fig. 3. Native umbilical cord, a - hematoxylin-eosin staining, many nuclei; b - DAPI staining, many nuclei; c - hydrogel, hematoxylin-eosin staining, no nuclei; d - matrix, DAPI staining , there are no cores);

- preservation of the main components in the matrix and hydrogel (Fig. 4. a - staining with alcian blue, the presence of glycosaminoglycans; b - van Gieson staining, the presence of collagens; c) orsein staining, reveals elastin fibers);

- a view of a lyophilized cell-free matrix of the umbilical cord (Fig. 5. A cell-free lyophilized three-dimensional matrix with a diameter of 50 mm, a height of 15 mm and a weight of 2.9942 g);

- scanning electron microscopy. Shown is a porous fibrillar structure (Fig. 6a) and a granular structure of the acellular matrix of the umbilical cord (Fig. 6b);

- injection hydrogel from the umbilical cord of a person (Fig. 7);

- solubilization with pepsin in hydrochloric acid solution allows to obtain the injectable form of the matrix in the form of a hydrogel (Figure 7);

- under physiological conditions, the hydrogel takes the form of a hydrogel both in vitro (Fig. 8) and in vivo (Fig. 9. After the introduction of an injection hydrogel stained with methylene blue into the knee joint of a rabbit with a preformed defect, the hydrogel turns into a hydrogel accumulating in the area damage);

- in Fig. 10 shows a defect (depth and diameter 3 mm) of the cartilage tissue of the medial condyle of the rabbit femur;

- in Fig. 11 - a view of an animal, which after the operation of modeling the intra-articular damage was put on a special collar to prevent the bandage from chewing;

- the defect zone in rabbits treated with the introduction of a hydrogel from the human umbilical cord, measured by magnetic resonance imaging (Fig. 12), decreases by the 60th day;

- on the 90th day after modeling the traumatic injury in rabbits treated with an injection hydrogel, the area of the defect is practically indistinguishable from the intact articular cartilage (Fig. 13);

- fig. 14 demonstrates the histological features of the regenerated hyaline cartilage in the defect area under the influence of the introduction of a cell-free hydrogel of the human umbilical cord.

The claimed method is performed according to a known sequence of steps and is described in detail in the following examples.

Example 1. A method of manufacturing a cell-free matrix of the human umbilical cord

From the umbilical cord obtained after childbirth with the informed consent of women after short-term sterilization with a solution of 10% hydrogen peroxide, arteries and veins were removed as structures containing an abundance of potentially immunogenic mature differentiated cells. Biomaterial in the form of hard connective mucous tissue (Warton's jelly) on the umbilical cord membrane is subjected to washing from blood with phosphate buffered saline (pH = 7.25) and homogenization (gentle MACS ™ Dissociator Milteniy Biotech, Germany) (Fig. 1).

Then the biomaterial was decellularized. The decellularization (cell removal) procedure is carried out using 0.2M NaOH solution for 3 hours in a flask on a shaker platform (Biosan, Latvia) at a speed of 140 rpm at room temperature. Removal of decellularizing solution and detached cells was carried out by filtering through a Schott funnel and three times washing with phosphate buffer and centrifugation (Beckman Avanti, USA) 3000 rpm for 5 min. each one. The efficiency of decellularization was checked by staining using light microscopy, staining the material with hematoxylin-eosin and DAPI (DAPI, 1: 1000 Invitrogen Paisley, UK), as well as quantitative determination of DNA using InstaGene matrix reagent kits (BioRad, USA) according to the manufacturer's instructions and on spectrophotometer Implen NanoPhotometer NP80 Touch (GmbH, Germany). Gel electrophoresis confirmed the absence of DNA in the decellularized matrix (track F1) and hydrogel (tracks G1, B2). (Fig. 2) and the presence of DNA in the native umbilical cord (tracks E1, H1).

The efficiency of decellularization determines the non-immunogenicity of a hydrogel made from a cell-free matrix, injected into the cavity of the injured joint; it is determined by the low content of DNA and cellular material in the product. The criteria for quality decellularization are considered to be DNA content less than 50 ng per mg of dry weight of ECM and DNA fragments less than 200 nucleotide pairs in size, as well as the absence of nuclear material during histological examination using fluorescent dyes. The method of cell removal we used made it possible to obtain a product that meets the above standards [Badylak S.F. Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response. Ann. Biomed. Eng. 2014; 42: 1517-1524. https://doi.org/10.1007/s10439-013-0963-7]. The average DNA content in the homogenized biomaterial of the native umbilical cord was 468.75 (453.65-475.15) ng / μL, after decellularization - 29.2 (27.8-30.7) ng / μL, the decrease is statistically significant, p < 0.001.

Upon completion of decellularization, the acellular matrix was washed from detached cells by centrifugation at 3000 rpm. and from a sodium hydroxide solution by titration with 0.01N HCl solution, after which it was re-centrifuged and lyophilized for 24 hours.

FIG. 3 shows histological preparations before (a, b) and after decellularization (c, d).

The matrix retains the main components of the umbilical cord connective tissue, detected using histological dyes (Fig. 4). Staining of samples with alcian blue (Fig. 4, a) demonstrates the presence of glycosaminoglycans, staining according to Van Gieson (Fig. 4, b) - the presence of structural collagens, orsein staining (Fig. 4, c) - elastin fibers. Thus, the decellularization procedure successfully removed cells, nuclei, debris. The procedure took about 5 hours (from the beginning of the preparation of the umbilical cord to complete neutralization of the pH to 7.5).

In addition to the effectiveness of decellularization, this method can be considered quite physiological, since it is possible to remove the reagent from the product using simple techniques by titration with 0.01 N hydrochloric acid solution, during which harmless products (sodium chloride and water) are formed in small quantities, which are removed by subsequent centrifugation:

NaOH + HCl = NaCl + H ₂ O.

The resulting biomaterial was freeze-dried for 24 hours (Zirbus VaCo5II, Germany) until completely dry (Fig. 5).

A series of images taken with a JEOL JSM-639 OLA scanning electron microscope (Japan) and ImageJ software (Rasband, WS, US National Institutes of Health; Bethesda, MD) shows that the lyophilized cell-free matrix from the human umbilical cord partially retained reticular collagen structure (Fig. 6a). A large magnification makes it possible to consider not only fibrillar, but also globular granular polymers on the pore walls with sizes corresponding to nanovesicles (Fig. 6b).

The pore sizes of the resulting product are in the ranges that allow cells to migrate in them, to occur the processes of movement of fluids, nutrient substrates and metabolites to ensure the vital activity of cells. It is known from scientific publications that the structure of the matrix includes important adhesive molecules and growth factors, as well as nanovesicles that direct the interaction and behavior of cells that populate the matrix. Therefore, a cell-free matrix of highly regenerative tissues is an independent product for its possible use in the treatment of deep damage to the skin and soft tissues.

Example 2. A method of producing an injectable hydrogel from a cell-free matrix of the human umbilical cord and studying its ability to form a three-dimensional hydrogel in vitro and in vivo.

Using our modified solubilization procedures (Freytes DO, Martin J., Velankar SS, Lee AS, Badylak SF Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials, 2008. - 29: 1630-1637. Doi: 10.1016 / j.biomaterials.2007.12.014), consisting in the action of pepsin in a solution of 0.01N hydrochloric acid at a concentration of 2 mg / ml, the matrix was transformed into a hydrogel with a viscosity that allows passage through the syringe needle 27 G (Fig. 7). After sterilization of the injection product with a solution of steptomycin-penicillin (Biolot), it was immediately frozen until it was used in the experiment.

Injectable hydrogel under physiological conditions (pH = 7.35-7.45 and t = 37 ° C) polymerizes in vitro into a three-dimensional structure of the hydrogel (Fig. 8).

The procedure for polymerizing a fluid (liquid) hydrogel into a three-dimensional hydrogel is possible due to the high content of high molecular weight hyaluronic acid in its composition. The obtained injection material with adjustable viscosity also polymerizes in vivo, filling the defects of the articular cartilage (Fig. 9).

Example 3. Simulation of damage to the hyaline cartilage of the knee joint of rabbits.

In a sterile operating room, all animals under combined anesthesia (propofol solution 1% - 2 ml, diethyl ether - 15 ml) through the medial parapatellar approach with a drill formed an osteochondral defect in the loaded zone of the medial condyle of the right femur with a diameter and depth of 3 mm (Fig. 10).

The operation ended with layer-by-layer wound closure and aseptic dressing. In the postoperative period, for the prevention of pyoinflammatory complications, the animals were injected intramuscularly with ceftriaxone 10 mg / kg once a day for the first 3 days. The operated limb was not immobilized. All rabbits wore a transparent plastic collar for 5 days after surgery to protect the dressing and suture from being chewed, which did not interfere with the feeding of the animals (Fig. 11).

During the observation of the animals for 10 days after modeling the traumatic injury of the articular cartilage (Fig. 8) and in the post-injection periods, no signs of wound infection were detected, synovitis of the operated knee joints in both groups, the rectal body temperature did not exceed 38 ° C.

Example 4. Study of the regenerative effect of a cell-free injection hydrogel on the restoration of defects in hyaline articular cartilage in the experiment.

Animals with a simulated articular cartilage defect were randomly divided into 2 groups. The first (control) group of animals (8 individuals) in the cavity of the operated joint was injected with 0.4 ml of 0.9% sodium chloride solution three times on the 14th, 21st, 28th days after the simulation of the defect. The animals of the second experimental group (7 individuals) were injected with 0.4 ml of hydrogel from the umbilical cord biomaterial at the same time.

60 days after the operation, all animals underwent magnetic resonance imaging (hereinafter - MRI) of the knee joints to measure the depth and diameter of the defect (Fig. 12a, b, c, d).

There was a statistically significant increase in the diameter of the lesion, illustrated sequentially in FIG. 12 a, b, c, d, in the control group (n = 8), in which 0.9% sodium chloride solution was used for treatment. After 60 days, the average diameter of the defect was 3.25 mm, p ¹ = 0.018. A decrease in the diameter of cartilage damage in the group of animals, which were injected intra-articularly with umbilical cord hydrogel to replace the cartilage defect, was statistically significant. After 60 days, the average diameter of the defect decreased to 2.69 mm; p ¹ = 0.028. Two-sided analysis of changes in the diameter and depth of the articular cartilage defect under the influence of the treatment showed a statistically significant difference in the characteristics of the control and experimental groups (p ² = 0.001, p ² = 0.004, respectively).

The animals were removed from the experiment after 90 days by intravenous injection of propofol (1% - 10 ml). Macroscopic preparations of the knee joints were examined visually for the presence in the tissues of the inflammatory process and degenerative-dystrophic changes (respectively, Fig. 13a, b and 13c, d).

Comparison of the results of microscopic examination 90 days after the formation of the defect showed that the predominant tissue in the area of the defect in animals in the experimental group was statistically significantly more often (p = 0.014) characterized by the presence of hyaline articular cartilage (n = 4) or incompletely differentiated mesenchyme (n = 3 ), while in the control group, in three animals, fibrous tissue or bone was present in the wound area, and in 5 animals, an incompletely differentiated mesenchyme. The indicators characterizing the surface, the integrity of the structure, the thickness of the cartilage in the defect zone were higher in the experimental group.

Analysis of the level of cell deficiency in the area of the defect showed that in the experimental group, normal cellularity was determined in 2 animals, in 4 more animals there was a slight deficiency, chondrons were more common. At the same time, the animals of the control group were more likely to have moderate hypocellularity (n = 5). Comparison of the cellularity indices of the two groups revealed a tendency to difference (p = 0.054).

The degree of functional activity of cartilage tissue cells can be judged by the content of glycosaminoglycans in the tissues. A qualitative reaction is the intensity of the alcian blue staining. A high intensity of staining of cartilage in the joints of animals treated with acellular umbilical hydrogel was revealed (Fig. 14 a, b, c, d, e, f).

In vivo studies have confirmed the validity of the initial hypothesis about the possibility of creating and using a full-component acellular matrix of Wharton's umbilical cord gel in the form of an injectable hydrogel for the restoration of traumatic injuries of the articular cartilage.

The discovered positive dynamics, consisting in a progressive decrease in the diameter and depth of damage to the medial femoral condyle in animals with intra-articular hydrogel injection, suggests that the umbilical cord is a suitable resource for creating a hydrogel as a favorable environment for the restoration of intra-articular cartilage.

The injectable, cell-free, full-component umbilical cord hydrogel obtained by the applicants for intra-articular administration meets the existing standards for DNA content, polymerizes in vitro and in vivo, is biocompatible and safe during administration. Intra-articular injection of a cell-free injectable hydrogel from the umbilical cord leads to a 60-day decrease in the size of traumatic damage to the articular cartilage of a rabbit, by 90 days - to a significant restoration of hyaline cartilage in the area of the articular surface defect.

Thus, based on the results of the study, it seems possible to conclude that the claimed method of making a cell-free hydrogel from Wharton's jelly of the human umbilical cord for intra-articular use in order to restore damage to the articular cartilage, which consists in injecting a hydrogel from the cell-free matrix of the umbilical cord, can effectively stimulate regenerative processes in the area of defects in hyaline cartilage. The research methods described above objectively indicate the positive effect of the applied action on the regeneration of the articular cartilage and ensure the restoration of motor functions in the damaged joints.

By applicants for the first time:

• A method of obtaining a cell-free matrix and hydrogel from the human umbilical cord using simple methods of removing cells from the tissues of the human umbilical cord has been applied; the effectiveness of the methods and reagents used is confirmed by modern technologies of DNA gel electrophoresis, light and scanning electron microscopy, spectrophotometry.

• The regenerative effect of intra-articular injection of a hydrogel from the acellular matrix of the umbilical cord on the restoration of post-traumatic damage to the hyaline cartilage of the knee joint, documented by modern highly informative research methods - magnetic resonance imaging, histological analysis with special staining.

The use of the claimed method for the manufacture of a hydrogel from the cell-free matrix of the human umbilical cord and its use for the treatment of intra-articular damage to the hyaline cartilage allows to improve its regeneration to restore the structure and function of the joint.

The claimed technical solution allows to ensure the implementation of the set goal, namely, a more complete and physiological restoration of the structure of hyaline cartilage at the site of the defect, which is responsible for the performance of motor functions and an improvement in the patient's quality of life, and an increase in the availability of a method for making a hydrogel and a method of treatment, which consists in the possibility of intra-articular administration of a hydrogel ...

The claimed technical solution meets the "novelty" criterion for inventions, since as of the date the applicant submitted the application materials from the analyzed sources of patent and non-patent information of the Russian Federation and foreign countries, the claimed set of features with features identical to the claimed technical solution was not revealed.

The proposed invention satisfies the criterion of "inventive step" for inventions, since the totality of the stated features provides for obtaining technical results that are not obvious to a specialist.

The claimed technical solution meets the criterion of "industrial applicability" for inventions, since was tested in practice in the research laboratory of the Military Medical Academy of the Ministry of Defense of the Russian Federation, as a result of which all the goals set in the declared technical solution were realized, namely, a more complete restoration of hyaline intra-articular cartilage was realized, which is necessary to restore the structure and function of the joint.

Claims (1)

A method of manufacturing a cell-free hydrogel from Wharton's jelly of the human umbilical cord for intra-articular use in order to restore damage to the articular cartilage, including obtaining a native umbilical cord, sterilization with 10% hydrogen peroxide solution, its preparation, fragmentation and homogenization, decellularization, washing from the reagent and detached cells, neutralization sodium and re-centrifugation; lyophilization, characterized in that sodium hydroxide at a concentration of 0.2 M is used to remove cells from the matrix during decellularization, which is neutralized at the end of the process with a solution of hydrochloric acid 0.01 N, and the procedure for solubilizing the resulting lyophilized matrix is performed by acting on the resulting matrix of pepsin in solution 0.01N hydrochloric acid at a concentration of 2 mg / ml for 24 hours, after which the medium is neutralized to physiological pH by titration with 0.01N sodium hydroxide.

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