Joint structure reconstruction inducer and use method and application thereof

Abstract

The invention belongs to an in-situ regeneration induction part in the field of regenerative medicine, and particularly relates to reconstruction of joint-like structures of mammals.

Description

Technical Field

[0001] The present invention belongs to the in-situ regeneration induction part in the field of regenerative medicine, particularly to the reconstruction of joint-like structures in mammals. The present invention includes the joint-like structures regenerated in the toes of adult mice, which is the regeneration of mammals. Background Art

[0002] Lower organisms have strong regenerative abilities. For example, the forelimbs and hindlimbs of amphibians such as lizards, salamanders, and frogs can be completely regenerated after amputation. However, the regenerative ability of mammals is weak. For example, the distal phalanges (Phalange 3, P3) of humans and mice have only incomplete regenerative abilities (i.e., they can regenerate after distal resection, but not after proximal resection), and the remaining phalanges such as the middle phalanges (Phalange 2, P2) and the upper and lower limbs do not have regenerative abilities.

[0003] Arthroexesis is an injury to the joint structure and an abnormal joint with a defect in the joint component. Arthroexesis is different from ordinary joint injuries. In ordinary joint injuries, the joint structure is intact without the absence of joint components, such as osteoarthritis and microdrilling on the joint. The repair of joint structure injury is difficult and is an unsolved problem. Mammals cannot regenerate after arthroexesis, and the research on arthroexesis regeneration is generally carried out in regenerable amphibians [Tsutsumi, Rio et al. “Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.” Regeneration (Oxford, England) vol.3, 126 - 38.6 Jan. (2016), doi:10.1002 / reg2.49; Tsutsumi, Rio et al. “Reintegration of the regenerated and the remaining tissues during joint regeneration in the newt Cynops pyrrhogaster.” Regeneration (Oxford, England) vol.2, 126 - 36.8 Apr. 2015, doi:10.1002 / reg2.28]. So far, only one report has been seen on the regeneration research of completely absent joint components in mammals, which is the regeneration of mouse toe joints induced by BMP9: the mouse toe is truncated at P2, and P3 and sesamoid bones in the terminal joint of the mouse toe are completely absent, and the P2 component is partially absent. BMP9 can induce a joint-like structure at the P2 stump [Yu, Ling et al. “BMP9 stimulates joint regeneration at digit amputation wounds in mice.” Nature communications vol.10, 1424.5 Feb. (2019), doi:10.1038 / s41467 - 018 - 08278 - 412].

[0004] Therefore, there is an unmet need in the art for an inducer for joint structure reconstruction, especially for joint structure reconstruction in the case of joint component defects. This application is dedicated to solving this technical problem. Summary of the Invention

[0005] The cartilage of the mouse toe joint is hyaline cartilage, which is avascular connective tissue and its main component is cartilage matrix. The texture of the cartilage matrix is dense and can bear pressure; there are chondrocytes in the cartilage matrix. The chondrocytes of hyaline cartilage are generally round or biconvex lens-shaped, and the dark-colored cell nucleus is in sharp contrast with the white outside the cell nucleus, like an eyeball, which is very easy to identify; especially the hypertrophic chondrocytes with enlarged volume are even easier to identify. Cartilage can differentiate into bone through endochondral ossification.

[0006] In our study on P2 toe amputation and regeneration, we first used the P2 toe amputation model with soft tissues retained ( Figure 1 . Normal mouse toe): that is, the mouse toe was amputated at P2, the distal part of P2 and P3 were removed, and soft tissues were retained as much as possible. We have found in this model that the extracellular matrix (ECM, extracellular matrix) has a promoting effect on the bone regeneration of P2 (Patent Application PCT / CN2017 / 102270, Bone and Soft Tissue Synchronous Regeneration Inducer and Its Preparation Method and Use). The P2 toe amputation model was initially used as a bone regeneration model, but from the perspective of joint regeneration, this model is also a mouse toe end joint resection model: the mouse toe end joint consists of three joint components, P2, sesamoid bone and P3; in the P2 toe amputation model with soft tissues retained, the P3 component in this joint is completely missing, the P2 component is partially missing / partially retained, and the sesamoid bone is retained.

[0007] We unexpectedly found in the ECM-induced P2 toe amputation and regeneration that free bones appeared in the ECM group, while no free bones appeared in the control group ( Figure 2 ). In addition, it was also found that the retained sesamoid bone could form a joint-like structure with P2 in both the control group and the ECM group; the free bones in the ECM group could also form a joint-like structure with P2. Therefore, the present inventor unexpectedly found that: (1) ECM can induce free bones; (2) the sesamoid bone can induce a joint-like structure at the P2 stump; (3) the free bones can merge with the sesamoid bone, and the merged bone does not affect the formation and maintenance of the joint-like structure. The ECM in the above studies was implanted into the mouse toe at the same time as the toe amputation. Further research found that if the implantation of ECM was carried out after the wound healing of the toe amputation, the probability of the appearance of free bones could be increased ( Figures 3-7 ).

[0008] The present inventor also found that in the case of joint component defects, the remaining joint components can reform a joint-like structure with the stump of the partially missing joint components. That is, in the P2 toe amputation model, the sesamoid bone can reform a joint-like structure with P2 (among the three components of the mouse toe end joint, the P3 component is completely missing, the sesamoid bone component is retained, and the P2 component is partially missing). The inventor also found that in the case of joint component defects, ECM particles can form free bone at the injury site, and the free bone can form a joint-like structure with the remaining joint components (sesamoid bone), or can also form a joint-like structure with the stump of the joint components (P2). The free bone can merge with the remaining joint components (sesamoid bone), and the merged bone does not merge with the partially missing joint components (P2). Therefore, the joint-like structure is retained for a long time and stably maintained. In the P3 removal model, ECM particles can regenerate the P3 bone, and the regenerated P3 bone forms joint structures with the sesamoid bone and P2 respectively.

[0009] In one aspect, the present invention relates to the following embodiments:

[0010] Embodiment 1. The main component of this inducer is ECM, and the characteristics of ECM are the products obtained from animal tissues and organs, including but not limited to: extracellular matrix taken from the small intestine, trachea, bladder, bone, or extracellular matrix from non-animal tissue and organ sources.

[0011] Embodiment 2. The main component of this inducer is ECM, and the characteristics of ECM are the products after being treated by physical or chemical methods in Embodiment 1. Such treatment methods include but not limited to: freeze-drying, pulverization, degradation, cross-linking, gelation, 3D printing, etc.

[0012] Embodiment 3. This inducer also contains excipients, and the excipients include but not limited to: pure water or neutral buffer solution; inorganic salts or inorganic salt solutions containing calcium, phosphorus, magnesium and other ions.

[0013] Embodiment 4. This inducer is an external medicine, which exerts its effect by being implanted into the damaged part of the tissue and is completely absorbed during the regeneration process.

[0014] Embodiment 5. The main component of this inducer is ECM, and ECM refers to any one of Embodiments 1 and 2.

[0015] In another aspect, the present invention relates to an inducer which contains extracellular matrix (ECM). In some embodiments, the inducer of the present invention contains extracellular matrix as the main component. In some embodiments, the inducer of the present invention contains extracellular matrix as the main active ingredient. In some embodiments, the inducer of the present invention contains extracellular matrix as the only active ingredient.

[0016] In some embodiments, the inducer of the present invention is used to induce joint reconstruction in a subject. In some embodiments, the inducer of the present invention is used to induce joint reconstruction in the case of joint component defects in a subject. In some embodiments, the inducer of the present invention is used to induce joint reconstruction in the case of complete absence of at least one joint component in a subject.

[0017] On the other hand, there is provided the use of the inducer of the present invention for inducing joint reconstruction in a subject.

[0018] On the other hand, there is provided the use of the inducer of the present invention in the preparation of a medicament for inducing joint reconstruction in a subject.

[0019] On the other hand, there is provided a method for achieving joint reconstruction in a subject, the method comprising contacting the damaged joint site of the subject with the inducer of the present invention. In some embodiments, the present invention relates to a method for achieving joint reconstruction in a subject, which comprises contacting the damaged joint site of the subject with the inducer of the present invention, shaping of the ECM, and delayed implantation.

[0020] In some embodiments, the joint reconstruction is joint reconstruction in the case of joint component defects, such as joint reconstruction in the case of partial or complete absence of joint components. Preferably, the joint reconstruction is joint reconstruction in the case of complete absence of at least one joint component (such as P3). In some embodiments, the joint component can be any joint component, such as P2 component, P3 component or sesamoid bone, etc.

[0021] In some embodiments, the joint component defect is a freshly generated joint component defect, such as immediately or shortly after joint resection (such as within 1, 2, 3, 4, 5, 6 days or within 1 week, etc.). In some embodiments, the joint component defect is an old joint component defect. In some embodiments, the joint component defect is a freshly generated joint component defect at the joint site of an old joint component defect, such as freshly generated by joint resection.

[0022] In some embodiments, the extracellular matrix is obtained from animal tissues and organs, including but not limited to extracellular matrix obtained from small intestine, trachea, bladder, bone. In some embodiments, the extracellular matrix is of non-animal tissue and organ origin.

[0023] In some embodiments, the extracellular matrix is obtained after being treated by physical or chemical methods, and the treatment methods include but are not limited to: shaping treatments such as freeze-drying, extrusion, crushing, degradation, crosslinking, gelation, 3D printing, etc.

[0024] In some embodiments, the inducer of the present invention further comprises excipients, which include but are not limited to inorganic salts such as phosphates, acetates, trehalose, hydroxyapatite micropowder, glucose, sorbitol, pure water, ethanol, inorganic salt solutions such as phosphate buffer, acetate buffer, citrate buffer. In some embodiments, the excipient is hydroxyapatite micropowder and / or optionally an inorganic salt or an inorganic salt solution may be added.

[0025] In some embodiments, the inducer of the present invention further comprises hydroxyapatite micropowder as an excipient. In some embodiments, the dry weight ratio of hydroxyapatite micropowder to ECM = 100:1 to 1:10000, such as 10:1 to 1:200, 5:1 to 1:100, 2:1 to 1:50, 1:10 to 1:50.

[0026] In some embodiments, the excipient is a diluent. The diluent may optionally add an inorganic salt or other bone-promoting components, including but not limited to: inorganic salts containing calcium, phosphorus, magnesium and other ions. The diluent includes but is not limited to pure water, phosphate buffer, acetate buffer, citrate buffer, etc., preferably pure water or a neutral buffer, more preferably a phosphate buffer containing 5% trehalose (preferably pH = 7.2).

[0027] In some embodiments, the inducer of the present invention further comprises other components, and the other components are, for example, selected from one or more of inorganic salts (such as inorganic salts containing calcium, phosphorus, magnesium and other ions), acetates, citrates, trehalose, hydroxyapatite micropowder, glucose, sorbitol or other bone-promoting components.

[0028] In some embodiments, the inducer of the present invention consists of an extracellular matrix and an excipient.

[0029] In some embodiments, the regeneration inducer of the present invention consists of an extracellular matrix and a diluent.

[0030] In some embodiments, the inducer of the present invention consists of an extracellular matrix.

[0031] In some embodiments, the inducer of the present invention is in the form of particles (such as microparticles), for example, formed by extruding a powder or a powder mixture of the particulate components. There is no particular limitation on the particle size of the particles as long as they can be suitably implanted into the damaged joint site of the subject. For example, the particles can have a particle size distribution in the range of 1 μm - 10 mm, such as 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or a range composed of any two of them.

[0032] There is no particular limitation on the method for preparing the particles. In some embodiments, the inducer particles are prepared by dry granulation or wet granulation. In dry granulation, the powders of all the particle components can be uniformly mixed and then directly extruded into particles. In wet granulation, the powders or powder mixtures of the particle components (such as one or more of ECM and / or the other components) can be wetted with a liquid (such as water, ethanol, solution or suspension), extruded into particles, and then dried. For wet granulation, the other components can be added to the powder mixture to be wetted, or can be added to (such as dissolved or suspended in) the liquid used for wetting, or a part of the other components can be added to the powder mixture to be wetted and the remaining part can be added to (such as dissolved or suspended in) the liquid used for wetting. In some embodiments, the liquid used for wetting is a buffer solution that optionally contains nutritional components or other osteogenic promoting components, such as a buffer solution with a pH of 6-8, including but not limited to phosphate buffer solution, acetate buffer solution or citrate buffer solution. In some embodiments, the buffer solution contains 5% trehalose.

[0033] In some embodiments, the inducer of the present invention is shaped into the shape of the bone to be regenerated, such as the shape of the P3 component or the shape of the missing part of the P2 component.

[0034] In some embodiments, the regeneration inducer of the present invention is an external medicine, which exerts its function by being implanted at the damaged part of the tissue and is partially or completely absorbed during the regeneration process.

[0035] In some embodiments, the inducer of the present invention is implanted into the damaged joint part of the subject immediately or shortly after the joint component defect of the subject (i.e., normal implantation). In some embodiments, the normal implantation of the present invention is carried out within 1 week after the joint component defect of the subject, such as within 1, 2, 3, 4, 5, 6 or 7 days after the joint component defect of the subject.

[0036] In some embodiments, the inducer of the present invention is implanted into the damaged joint part of the subject after a period of time (such as a period of more than 1 week) after the joint component defect of the subject (i.e., delayed implantation). In some embodiments, the delayed implantation of the present invention is carried out at any time point within the 2nd - 12th week, or the 2nd - 8th week, or the 2nd - 4th week after the joint component defect of the subject. In some embodiments, the delayed implantation of the present invention is carried out at any time point within the 2nd week, the 3rd week or the 4th week after the joint component defect of the subject. In some embodiments, the delayed implantation of the present invention is carried out after the wound at the joint defect site has healed.

[0037] In some embodiments, the inducer of the present invention can be implanted both normally and deferentially. That is, the inducer of the present invention is implanted into the joint defect site of a subject immediately or shortly after the occurrence of a joint component defect in the subject (e.g., within 1 week, such as within 1, 2, 3, 4, 5, 6, or 7 days) (normal implantation), and after a period of time (e.g., a period longer than 1 week, preferably at any time point within the 2nd - 12th week, or the 2nd - 8th week, or the 2nd - 4th week), the inducer of the present invention is implanted again (deferential implantation).

[0038] On the other hand, a kit including two containers is provided, wherein the first container contains the inducer of the present invention for implantation into the joint defect site of a subject immediately or shortly after the occurrence of a joint component defect in the subject (e.g., within 1 week, such as within 1, 2, 3, 4, 5, 6, or 7 days) (normal implantation), and the second container contains the inducer of the present invention for implantation into the joint defect site of a subject after a period of time after the occurrence of a joint component defect in the subject (e.g., a period longer than 1 week, preferably at any time point within the 2nd - 12th week, or the 2nd - 8th week, or the 2nd - 4th week), such as after the wound at the joint defect site has healed (deferential implantation).

[0039] In some embodiments, the subject is a mammal, bird, fish, or reptile. In some embodiments, the subject is a mammal. In some embodiments, the subject is selected from cats, dogs, sheep, goats, cows, horses, pigs, mice, rats, and guinea pigs. In some embodiments, the subject is a human being.

[0040] Effect of the inducer of the present invention: In a mouse P2 toe amputation model with soft tissue retained, the inducer can induce the generation of free bone at the stump of the amputated toe. The free bone can form a joint-like structure with P2, and the joint-like structure is stably maintained and exists for a long time. The free bone can also merge with the sesamoid bone into one bone, and the joint-like structure composed of the merged bone and P2 is stably maintained and exists for a long time.

[0041] Definition

[0042] It should be understood that the terms used herein are for the purpose of describing specific embodiments of the present invention only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0043] The term "in-situ regeneration" refers to a process in which, due to trauma, a part of an organism, tissue, or organ is partially missing or damaged, and on the basis of the remaining part, a structure that is the same or similar in form and function to the missing or damaged part grows, and this repair process is called in-situ regeneration.

[0044] The term "joint component" refers to a single bone in a joint as a joint component, including the articular cartilage on the surface of the bone. For example, the distal joint of the phalanges / toes of a human or a mouse consists of three joint components, namely P2, P3, and the sesamoid bone.

[0045] The term "joint component defect" or "joint mutilation" includes one or both of "partial loss of joint components" and "complete loss of joint components", both of which refer to structural defects of the joint, which are different from structurally intact injuries such as microdrilling. In the P2 toe amputation trauma model, P3 was completely removed, so P3 is a complete loss of joint components; the distal part of P2 was removed but the proximal part was retained, so P2 is a partial loss of joint components.

[0046] The term "chronic joint component defect" refers to a joint component defect that has occurred for more than 4 weeks, such as more than 1 month, 4 months, 8 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, 30 years, 40 years, or longer.

[0047] The term "freshly generated joint component defect" refers to a joint component defect that has occurred within 4 weeks, such as within 1, 2, 3, 4, 5, 6 days or within 1, 2, 3, or 4 weeks. In this article, "freshly generated joint component defect" also includes a freshly generated joint component defect at the joint site of a chronic joint component defect, such as freshly generated by arthrodesis.

[0048] The terms "joint structure" and "joint-like structure" are used interchangeably and refer to a structure composed of two adjacent bone ends and a bone gap, with cartilage layers covering both bone ends.

[0049] The term "free bone" refers to a regenerated bone that is not connected to the original bone and is wrapped by connective tissue.

[0050] The term "regenerated P3 bone" refers to the free bone induced by ECM in the P3 removal model. There is usually only one free bone regenerated in the P3 removal model. Without merging with the sesamoid bone, its position, shape, and other characteristics are close to those of the P3 bone, so it is called the regenerated P3 bone.

[0051] The terms "joint reconstruction", "joint structure reconstruction", and "joint-like structure reconstruction" are used interchangeably and refer to re-establishing a joint-like structure at the joint defect site of a subject with a complete or partial loss of joint components.

[0052] The terms "extracellular matrix" and "ECM" and similar terms are used interchangeably herein and, in a narrow sense, refer to a collagen-rich substance found between cells in animal tissues and serving as a structural element in the tissue. It typically comprises a complex mixture of polysaccharides and proteins secreted by cells. The extracellular matrix can be isolated and processed in various ways. The extracellular matrix (ECM) can be obtained by isolation from animal tissue organs, including mammals such as domesticated or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs, primates; birds; laboratory animals such as mice, rats, and guinea pigs; and the animal tissue organs include but are not limited to: small intestine, trachea, bladder, bone, submucosa of small intestine, submucosa of stomach, submucosa of bladder, tissue mucosa, dura mater, hepatic basement membrane, pericardium, or other tissues. After isolation and processing, it is generally referred to as extracellular matrix or ECM. In a broad sense, the extracellular matrix also includes extracellular matrix derived from non-animal tissue organs, such as artificial extracellular matrix, extracellular matrix derived from cells cultured in vitro, etc. Preferably, the extracellular matrix can be prepared according to the method described in Agrawal V, Johnson SA, Reing J. Epimorphic regeneration approach to tissue replacement in adult mammals. [J]. Proc Natl Acad Sci, 2010, USA 107: 3351 - 3355.

[0053] The terms "inducer" and "regeneration inducer" are used interchangeably and refer to a composition that induces the regeneration of a subject's tissue and / or organ, particularly a composition that induces joint reconstruction.

[0054] The term "excipient" refers to a substance added during the preparation or formulation of a pharmaceutical preparation other than the active ingredient such as ECM. The excipient can be liquid, solid, or gas, and can be a single substance or a mixture of multiple substances. Excipients include fillers, stabilizers, antibiotics, antioxidants, preservatives, diluents (including diluents), etc. The excipients of the present invention are excipients known to those skilled in the art, including but not limited to hydroxyapatite micropowder, phosphates, acetates, trehalose, glucose, sorbitol, pure water, ethanol, phosphate buffer, acetate buffer, citrate buffer, etc. For more information on excipients, reference can be made to reference books in the art, such as Luo Mingsheng, Gao Tianhui, eds., Encyclopedia of Pharmaceutical Excipients, 2nd Edition, Sichuan Science and Technology Press.

[0055] The term "diluent" refers to a liquid used to dilute or moisten the ECM, such as an organic or inorganic solvent like water, ethanol, or a mixture thereof. The diluent may optionally contain inorganic salts or other osteogenic components, including but not limited to: inorganic salts containing calcium, phosphorus, magnesium ions, etc. The diluents of the present invention include but are not limited to pure water, phosphate buffer solution, acetate buffer solution, citrate buffer solution, etc., preferably pure water or a neutral buffer solution, such as a phosphate buffer solution (pH = 7.2) containing 5% trehalose.

[0056] The terms "subject" and "patient" are used interchangeably and include mammals, such as domesticated animals or farm animals, such as cats, dogs, sheep, goats, cows, horses, and pigs, humans and primates; birds; laboratory animals, such as mice, rats, and guinea pigs: fish; reptiles, preferably humans.

[0057] Application

[0058] The inducer of the present invention can be formulated into any suitable dosage form and administered by any suitable route. The inducer of the present invention can be formulated into solutions, suspensions, emulsions, freeze-dried preparations, etc. for injection, formulated into granules, gels, ointments, creams, suppositories, patches, etc. for local administration, and formulated into aerosols, sprays, powders, etc. for local administration. The preferred administration method is generally direct local administration at the tissue defect site. More preferably, the inducer of the present invention acts by being implanted into the damaged area of the tissue and is completely absorbed during the regeneration process. Description of the Drawings

[0059] Figure 1 Shows a normal mouse toe, where the position of P2 truncation is indicated by the black arrow, the sesamoid bone is pointed to by the white arrow, and the part removed after toe amputation is within the dotted line.

[0060] Figure 2 Shows a normal mouse toe (A) and a comparison on the 20th day after normal implantation of ECM in the P2 amputation model (B) and the control group (C) (Example 1). Normal implantation is to implant ECM particles at the same time as toe amputation. The arrow in A and C points to the sesamoid bone, and the arrow in B points to the free bone.

[0061] Figure 3 Shows a photograph on the 21st day after delayed implantation of ECM in the P2 amputation model (Example 2). Delayed implantation is to implant ECM particles 2 weeks after toe amputation. The white arrows point to the sesamoid bone and the free bone respectively, and the soft tissue (connective tissue) in the bone gap between P2 and the sesamoid bone is within the dotted box.

[0062] Figure 4Shows a photo taken on the 33rd day after delayed implantation of ECM in the P2 resection model (Example 2). The black arrows point to the sesamoid bone and the free bone respectively, and the soft tissue (connective tissue) in the bone gap is within the dashed line.

[0063] Figure 5 Is a photo taken on the 33rd day after delayed implantation of ECM in the P2 resection model ( Figure 4 Magnification). The chondrocytes are indicated by the black arrows. Chondrocytes are mainly distributed in cartilage and also in the soft tissue adjacent to the cartilage. The edge of the cartilage is indicated by the white arrow.

[0064] Figure 6 Is a photo taken on the 65th day after delayed implantation of ECM in the P2 resection model (Example 2). The white arrows point to the sesamoid bone and the free bone respectively, and the edge of the regenerated cartilage is indicated by the black arrow. Since the thickness of the regenerated cartilage layer is uneven, the edge line of the regenerated cartilage is an irregular curve.

[0065] Figure 7 Is a photo taken on the 98th day after delayed implantation of ECM in the P2 resection model (Example 2). The white arrows point to the sesamoid bone and the free bone respectively, and the chondrocytes are indicated by the black arrows.

[0066] Figure 8 Shows photos of the control group (A), the ECM normal implantation group (B), and the ECM normal implantation + delayed implantation group (C) in the P3 removal model (Examples 3 and 4), where s is the sesamoid bone, and the regenerated P3 bone is within the white dashed box. Scale bar = 200 μm. Detailed implementation mode

[0067] The following examples are used to elaborate the present invention in more detail, but they should not be construed as limiting the scope defined by the claims of the present invention.

[0068] Example 1: Normal implantation of ECM in the P2 resection model

[0069] Preparation of ECM. Take fresh porcine bladder and prepare ECM freeze-dried powder according to the literature method [Agrawal V, Johnson SA, Reing J. Epimorphic regeneration approach to tissue replacement in adult mammals. [J]. Proc Natl Acad Sci, 2010, USA 107: 3351 - 3355]. Extrude the ECM freeze-dried powder directly into particles, or wet it with pure water and then extrude it into particles and dry it in the air (about 0.125 μL of pure water per particle). The dry weight of the ECM particles is about 0.25 mg per particle, and the particle size is about 0.5 mm.

[0070] P2 resection model and use of the inducer: Adult mice were anesthetized, and P2 resection with soft tissue preservation was performed on the second / fourth toe of the hind foot: that is, the mouse toe was transected on the P2 phalanx, and the excised bone tissue was removed (i.e., the distal part of P2 and P3 were removed), and the soft tissue was preserved as much as possible. Immediately after P2 resection in the ECM group, ECM particles (1 particle / toe) were implanted, and there was no implantation in the control group after P2 resection, and no further treatment was given thereafter.

[0071] Twenty days after implantation, free bone formation was observed in the ECM group, while no free bone formation was observed in the control group throughout the experiment ( Figure 2 ).

[0072] Example 2: Delayed implantation of ECM in the P2 resection model

[0073] Adult mice were subjected to P2 resection (see Example 1), and no further treatment was given thereafter. Two weeks after P2 resection, the mice were anesthetized again, and the skin at the end of the toe was incised without damaging the bone tissue. ECM particles were implanted through the skin incision in the ECM group, and no implantation was performed after skin incision in the control group. The ECM particles were directly extruded from ECM freeze-dried powder.

[0074] Twenty-one days after the implantation operation, free bone formation was observed in the ECM group, while no free bone formation was observed in the control group throughout the experiment. In the ECM implantation group, a bone gap was observed between the free bone, sesamoid bone and P2, and there was soft tissue in the bone gap ( Figure 3 , 21 days after implantation). As time passed, the soft tissue in the bone gap decreased, and finally the bone gap became transparent ( Figure 4 , 33 days after implantation), and new cartilage was generated at the edge of the sesamoid bone. The newly generated cartilage could differentiate into bone through endochondral ossification, increasing the volume of the sesamoid bone ( Figure 5 , 33 days after implantation). On the 65th day after implantation, it was observed that the sesamoid bone increased and merged with the free bone to form a single bone, but the sesamoid bone did not merge with the P2 bone ( Figure 6 ). On the 98th day after implantation, it was observed that the bone gap between the merged bone and P2 still existed, and the joint-like structure remained unchanged ( Figure 7 ). The bone surfaces on both sides of the bone gap were covered with cartilage, and the cartilage edge line was an irregular curve. The surface of the regenerated cartilage had non-transparent soft tissue, and chondrocytes were also distributed in these soft tissues ("eyeball-shaped" cells are chondrocytes).

[0075] Example 3: Normal implantation of ECM in the P3 removal model

[0076] P3 removal model: Adult mice were anesthetized, and the entire P3 bone was removed without damaging the P2 bone, and the skin and other soft tissues were preserved as much as possible.

[0077] The ECM particles prepared as in Example 2 were immediately implanted after P3 removal in the ECM group, and no implantation was performed after P3 removal in the control group. On the 35th day after the implantation operation, the transparent specimens of the mouse toes showed that there was no new P3 bone formation after P3 removal in the control group ( Figure 8 .A), and there was new P3 bone formation in the ECM group ( Figure 8 .B). There was a bone gap between the regenerated P3 bone and the P2 bone. A joint-like structure was formed between the P2 and the regenerated P3 bone. The P2 / P3 joint structure was reconstructed, and the diameter of the distal bone of the P2 increased.

[0078] Example 4: Normal implantation and delayed implantation of ECM in the P3 removal model

[0079] Adult mice were normally implanted with ECM particles in the P3 removal model (see Example 3). Two weeks after P3 removal, the mice were anesthetized again. Without damaging the bone tissue, the skin at the end of the mouse toe was incised, and ECM particles prepared as in Example 2 were implanted again through the skin incision. No implantation was performed after the skin incision in the control group. On the 35th day after the second implantation (i.e., delayed implantation), the transparent specimens of the mouse toes showed that there was no new P3 bone formation after P3 removal in the control group, and there was new P3 bone formation in the ECM group. Compared with Example 3, the bone mass of the regenerated P3 bone in Example 4 was more, and the shape of the regenerated P3 bone was closer to that of the normal P3 ( Figure 8 .C). In addition, the diameter of the distal bone of the P2 increased.

Claims

1. Use of an inducer comprising an extracellular matrix (ECM), preferably comprising the extracellular matrix as the main active ingredient, for example comprising the extracellular matrix as the only active ingredient, in the preparation of a medicament for joint reconstruction in a subject, preferably the medicament is used for joint reconstruction in the case of joint component defect, more preferably the medicament is used for joint reconstruction in the case of complete loss of at least one joint component.

2. The use according to claim 1, wherein the extracellular matrix is ​​obtained from animal tissues and organs, including but not limited to extracellular matrix obtained from small intestine, trachea, bladder, bone, or wherein the extracellular matrix is ​​of non-animal tissue and organ origin.

3. The use according to claim 1 or 2, wherein the extracellular matrix is ​​obtained by physical or chemical treatment, and the treatment method includes but is not limited to: freeze-drying, extrusion, crushing, degradation, cross-linking, gelation, and 3D printing.

4. The use according to any one of the preceding claims, wherein the inducing agent further comprises other components, the other components being selected from one or more of inorganic salts (e.g., inorganic salts containing calcium, phosphorus, magnesium ions), acetate, citrate, trehalose, hydroxyapatite powder, glucose, sorbitol or other osteogenesis-promoting components.

5. The use according to any one of the preceding claims, wherein the inducing agent is in the form of particles, for example formed by dry granulation or wet granulation; or the inducing agent is shaped into the shape of the bone to be regenerated.

6. The use according to claim 5, wherein for wet granulation, the further components are added to the powder mixture to be wetted, or added (e.g. dissolved or suspended) in the liquid for wetting, or a part of the further components is added to the powder mixture to be wetted and the rest is added (e.g. dissolved or suspended) in the liquid for wetting.

7. The use according to any one of the preceding claims, wherein the drug is an external drug, preferably exerts its effect by being implanted into the damaged part of the tissue and is partially or completely absorbed during the regeneration process.

8. The use according to any one of claims 1-7, wherein the inducing agent is implanted into the joint damaged part of the subject within 1 week after the joint component of the subject is defected; or wherein the inducing agent is implanted into the joint damaged part of the subject at any time point within 2-12 weeks, or 2-8 weeks, or 2-4 weeks after the joint component of the subject is defected, for example, after the wound at the joint defect site is healed; or wherein the inducing agent is implanted into the joint damaged part of the subject within 1 week after the joint component of the subject is defected, and is implanted into the joint damaged part of the subject at any time point within 2-12 weeks, or 2-8 weeks, or 2-4 weeks after the joint component of the subject is defected, for example, after the wound at the joint defect site is healed.

9. An inducing agent for use in joint reconstruction in a subject, wherein the inducing agent is as defined in any one of claims 1 to 7.

10. A kit comprising two containers, wherein the first container contains the inducing agent defined in any one of claims 1-7 for implantation into a joint defect site of a subject within 1 week after a joint component defect of the subject, and the second container contains the inducing agent defined in any one of claims 1-7 for implantation into a joint defect site of a subject at any time point within 2-12 weeks, or 2-8 weeks, or 2-4 weeks after a joint component defect of the subject, for example, after the wound of the joint defect site has healed.

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