TW202045105A - Acellular bone graft and artificial implant assembly comprising the same - Google Patents

Abstract

Disclosed herein is an acellular bone graft and an artificial implant assembly comprising the same for repairing a bone defect of a subject in need. The acellular bone graft has a three-dimensional (3-D) body characterized in having at least one coupling part formed on at least one surface of the 3-D body, wherein the coupling part has a structure that matches that of another coupling part of another acellular bone graft. The artificial implant assembly comprises at least two acellular bone grafts of the present aspect, wherein each acellular bone graft is connected to each other via joining the coupling parts respectively having matching structures that allow the coupling parts to be tightly joined.

Description

Decellularized bone graft and artificial graft component containing the decellularized bone graft

This disclosure is about the field of grafts. Specifically, it is about decellularized bone grafts used to repair bone defects in a much-needed individual.

Artificial grafts, especially bone grafts, are often used clinically to repair bone defects that are too serious and difficult to self-heal and therefore resort to surgical treatment, including fractures, dislocations, arthritis, congenital deformation, tumors, degenerative diseases, Tooth defect, etc. Currently commonly used artificial grafts include autograft, allograft, alloplastic graft, such as ceramic-based bone graft substitute and polymer substrate Polymer-based bone graft substitute (Polymer-based bone graft substitute) and xenograft (xenograft), the main purpose of these grafts is to assist the regeneration of bone cells and cartilage cells of the transplanted individual.

Xenogeneic bone grafts are bone pieces that are taken from non-humans or animals and are subjected to special treatments such as decellularization and removal of protein and fat. The remaining tissue is mainly composed of mineral components. Compared with other artificial grafts, xenogeneic bone grafts can be obtained from various sources, and it is more convenient for medical practitioners to obtain them. The xenogeneic bone graft can be used as a scaffold to allow the host bone cells to grow and gradually replace them with new bone tissue. This has a good effect on filling the bone defect space and maintaining the complete bone structure of the surgical incision position, thereby completing the bone heal. The advantages of xenografts not only shorten the operation time to avoid complications caused by the second operation, but also reduce the potential risk of infection of allografts.

However, because the decellularized xenograft is only a non-living tissue, it is often found clinically that when the area of the bone defect is larger, the healing efficiency of using a larger bone graft is lower. On the other hand, although segmented bone grafts can reconstruct delicate bone defects, they cannot provide sufficient strength and stability.

In view of this, there is an urgent need in the related art for an artificial graft component and a decellularized bone graft to repair the bone defect of the individual.

In order to provide readers with a basic understanding, the following provides a brief summary of the disclosure. This summary of the invention is not an extensive overview of the disclosure, and is not intended to identify the key/essential elements of the invention or outline the scope of the invention. Its sole purpose is to present some concepts of this disclosure in a simplified conceptual form as a prelude to the more detailed description presented later.

As implemented and broadly described herein, the present disclosure aims to provide a decellularized bone graft and an artificial graft component containing the decellularized bone graft to repair bone defects of individuals in desperate need.

Accordingly, one aspect of the present disclosure relates to a decellularized bone graft for repairing bone defects of individuals in desperate need. The decellularized bone graft has a three-dimensional body, which is characterized by having at least one coupling member formed from at least one surface of the three-dimensional body, wherein the coupling member has a structure that can be combined with another decellularized bone graft. The other structure of the coupling corresponds.

According to some embodiments of the present disclosure, the coupling member is a tenon formed convexly from the at least one surface, or a hole formed concavely from the at least one surface.

In certain embodiments of the present disclosure, the tenon may be a right-angle tenon, a dovetail tenon, a twin tenon, an oval tenon, or a round tenon.

According to specific embodiments of the present disclosure, the bone defect can be located in the frontal bone, temporal bone, cheekbone, jawbone, clavicle, scapula, sternum, ribs, spine, humerus, ulna, radius, wrist, hip, femur, patella, tibia, Any position of ankle bone, phalanx or a combination thereof.

Another aspect of the present disclosure relates to an artificial graft component, which includes at least two decellularized bone grafts of the foregoing aspects, wherein each decellularized bone graft is connected to each other by combining the coupling member, and the coupling member is respectively It has a corresponding structure that enables the coupling to be tightly combined.

According to some embodiments of the present disclosure, the coupling member is a tenon formed convexly from the at least one surface, or a hole formed concavely from the at least one surface.

In some preferred embodiments, the tenon may be a right-angle tenon, a dovetail tenon, a double finger tenon, an oval tenon, or a round tenon.

According to an optional embodiment of the present disclosure, the artificial graft component further includes a scaffold configured to cover at least a part of the at least two connected decellularized bone grafts.

In some preferred embodiments, the stent is a metal mesh, wherein the metal is selected from the group consisting of titanium, zirconium, cobalt-chromium alloy, gold-palladium alloy, and silver-palladium alloy.

According to some embodiments, the stent is a titanium mesh with a mesh of 200 to 300 mesh.

Through the above configuration, the produced bone graft and artificial graft component can provide a strong structure for individuals who need bone repair.

After referring to the following embodiments, those skilled in the art to which the present invention belongs can easily understand the basic spirit and other purposes of the present invention, as well as the technical means and implementation aspects of the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description for the implementation aspects and specific embodiments of the present invention; but this is not the only way to implement or use the specific embodiments of the present invention. The implementation manners cover the characteristics of a number of specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

I. Definition

For ease of description, here is a comprehensive description of specific terms described in this specification, embodiments, and the appended patent scope. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as understood and used by those with ordinary knowledge in the technical field of the present invention. In addition, without conflict with context, the singular nouns used in this specification cover the plural nouns; and the plural nouns also cover the singular nouns. Specifically, unless the context clearly indicates otherwise, the singular form "one" (a and an) used in the scope of the patent application herein and appended includes plural forms. In addition, in this specification and the scope of the patent application, expressions such as "at least one" and "one or more" have the same meaning, and both of them mean that they include one, two, and three. Or more.

Although the numerical ranges and parameters used to define the wider range of the present invention are approximate numerical values, the relevant numerical values in the specific embodiments are presented here as accurately as possible. However, any value inherently inevitably contains the standard deviation due to individual test methods. Here, "about" usually means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range. Or, the word "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of a person with ordinary knowledge in the technical field of the present invention. Except for the experimental examples, or unless otherwise clearly stated, all ranges, quantities, values and percentages used herein (for example, used to describe the amount of material, length of time, temperature, operating conditions, quantity ratio and other similar Those) have been modified by "about". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying patent scope are approximate values and can be changed as required. At least these numerical parameters should be understood as the indicated effective number of digits and the value obtained by applying the general carry method.

As used herein, "acellular" graft refers to a graft that does not contain any cellular material. The so-called cellular material is like biologically active cells and/or debris. Accordingly, "acellular bone graft" refers to a bone graft derived from bones of mammals (such as pigs, cows or sheep) and is substantially free of any cells and organic matter. In one embodiment, the bone is taken from the long bone of the hog. In other embodiments, the bone is taken from the femur of a hog. In another embodiment, the bone is taken from the hip bone of the hog.

II. Specific implementation

The present disclosure, in its broadest purpose, includes a decellularized bone graft and an artificial graft component containing the decellularized bone graft, by grafting at least one (preferably at least two) bone grafts of the present disclosure , Can be suitable for plastic surgery or can be used for the treatment of bone defects including fractures, dislocations, arthritis, congenital deformation, tumors, degenerative diseases and/or tooth defects.

Accordingly, the purpose of the present disclosure is to have a decellularized bone graft with a coupling member, and the coupling member has a corresponding structure that can connect one decellularized bone graft to another decellularized bone graft. In addition, the decellularized bone graft is prepared by a specific method, which essentially removes all cellular material and preserves the mineralized tissue, so the mechanical strength, natural structure and configuration of the bone are preserved.

The embodiments of the present invention will be described below with reference to FIG. 1A, FIG. 1B, and FIG. 2. FIG. 1A and FIG. 1B are schematic diagrams of the decellularized bone graft 1 drawn from the front and back perspectives, respectively, according to one embodiment of the present disclosure. Figure 2 is a schematic diagram showing the two decellularized bone grafts (1, 2) connected to each other by coupling the coupling members (15', 25) of the present disclosure.

With reference to Figures 1A and 1B, the decellularized bone graft 1 has a three-dimensional body 10 in its structure, which is characterized by having at least one coupling element (such as the two coupling elements 15 shown in Figures 1A and 1B) , 15'), which is formed on at least one surface (11, 13) of the three-dimensional body 10. In addition, the coupling members (15, 15') each have a structure that can correspond to the structure of another coupling member 25 of another decellularized bone graft 2, as shown in FIG. 2.

In order to prepare the decellularized bone graft of the present disclosure, it is preferable to obtain fresh bone from livestock that is a source of bone. Examples of suitable livestock include, but are not limited to, hogs, cows, sheep, goats, donkeys, rabbits, ducks, geese, and chickens. The separated bone as the source of bone graft should be taken from the sacrificed animal, immediately placed in a sterile isotonic solution or other preservation solution, and then subjected to various treatments, such as sequential alkali treatment, sterilization treatment and In the decellularized area, the desired decellularized bone graft is produced.

Alkaline reagents suitable for alkali treatment include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, urea, sodium sulfide, calcium thioacetate and the like. Preferably, the bone matrix is treated with a sodium hydroxide solution. The step of sterilization is to treat the bone with a sterilizing agent to make it sterile. Examples of suitable sterilizing agents include, but are not limited to, alcohols, isopropanol, sodium bicarbonate, hydrogen peroxide, acetic acid, ethanesulfonamide, parabens, benzoic acid, salicylic acid, benzyl chloride Alkylammonium, alkyldimethylbenzylammonium chloride, didecyldimethylammonium chloride and amine chloride. In some embodiments, the bone matrix is treated with hydrogen peroxide. In some optional embodiments, a large amount of water is used to rinse the bone matrix to remove water-soluble substances before performing each step.

As for the decellularization treatment, the purpose of its execution is to remove the cellular material on the bone tissue, and to retain the physical and biochemical properties of the mineralized structure, so that the product can be used as the bone graft expected in the present disclosure. Accordingly, a supercritical fluid (supercritical fluid, referred to as SCF hereinafter) is used to treat sterile bone under a pressure of about 150-350 bar (bar). Further, the decellularization treatment is performed at a temperature of 30-45°C for about 20 minutes to 5 days. In some instances, the sterilized bone can be SCF processed for 1 to 24 hours.

SCF can be any supercritical carbon dioxide (scCO ₂ ), supercritical nitric oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkanes, supercritical alkenes, supercritical alcohols or supercritical acetone. In one embodiment, the SCT is scCO _2; another embodiment, the SCT is scN ₂ O.

The bone graft produced after the decellularization treatment has substantially no need for any additional enzymes to remove cellular material, nucleic acid, protein and fat, so the surface carbohydrate portion of the antigen that may be a source of immune rejection of the xenograft is also completely removed .

It should be noted that bones collected from animals can be cut into small pieces before or after the aforementioned treatment. According to some specific embodiments of the present disclosure, the obtained bone can be cut into a suitable size by a conventional laser cutting machine, thereby making a decellularized bone graft 1 having a desired structure as described herein.

Still referring to Figures 1A, 1B, and 2, according to some embodiments of the present disclosure, the exemplary decellularized bone graft 1 has a three-dimensional body 10 having two surfaces (hereinafter referred to as Two coupling elements (hereinafter referred to as the first coupling element 15 and the second coupling element 15') of a surface 11 and a second surface 12). Furthermore, another decellularized bone graft 2 also has a three-dimensional body 20 characterized by having a coupling 25 formed on the surface 21. As described in Figure 2, the decellularized bone grafts (1, 2) can be connected to each other by the coupling of the coupling parts (15', 25), because of the coupling of the decellularized bone grafts (1, 2) The pieces (15', 25) respectively have corresponding structures that can be closely combined with each other. Specifically, the first coupling member 15 is a tenon formed convexly from the first surface 11, and the second coupling member 15' is a mortise formed concavely from the second surface 12, and the decellularized bone graft 2 The coupling member 25 is a tenon formed by protruding from the surface 21. According to this embodiment, the tenon 25 of the decellularized bone graft 2 and the eyelet 15' of the decellularized bone graft 1 are designed to correspond to each other, which makes any two decellularized bone grafts, whether in practice or in theory, The coupling of objects can be tightly combined.

It should be understood that although Figures 1A and 1B exemplarily show that the three-dimensional body 10 of the decellularized bone graft 1 is formed with three surfaces (11, 12, and 13), the decellularized bone graft ( The shape of 1, 2) is not limited to this, as long as it can meet the geometric definition of the three-dimensional structure determined by the three values on the coordinates. For example, the three-dimensional body of the decellularized bone graft can be a Platonic solid, such as tetrahedron, cube, octahedron, dodecahedron or icosahedron), a Kepler-Pan Kepler-Poinsot polyhedron, a sphere, a cylinder, or a combination thereof. Similarly, the decellularized bone graft of the present disclosure is not limited to a specific size, as long as it conforms to the structure and shape of the bone defect area. In some embodiments, each bone graft has a shape of a bone block with a length, width, and height of about 0.5-5 cm, 0.5-5 cm, and 0.5-2 cm, respectively, to be assembled into a complete bone shape (for example, Human long bones or mandibles). The shape of each decellularized bone graft can be the same or different from each other. In this embodiment, the acellular bone graft 1 and the acellular bone graft 2 are different.

As for the coupling parts (ie, the tenon (15, 25) and the mortise 15'), the present disclosure does not limit its size, as long as it meets the structural stability (for example, not easy to break easily) and corresponding requirements. Examples of tenons include, but are not limited to, a right-angle tenon, a dovetail tenon, a twin tenon, an oval tenon, or a round tenon. In a preferred embodiment, the tenon is an elliptical tenon, and the mortise has a corresponding shape that can fit the tenon. In other embodiments, the tenon may be a dovetail tenon.

In addition, the decellularized bone grafts (1, 2) produced by the aforementioned treatment preserve the natural structure and configuration of the combined minerals (refer to Figure 3), and are repaired by supporting the growth of osteoblasts and blood vessels in the bone defect site bone defect. According to the present disclosure, bone defects can be located in frontal bone, temporal bone, cheek bone, mandible, clavicle, scapula, sternum, and ribs. (costal bone), vertebral column, humeral bone, cubital bone, radial bone, carpal bone, hip bone, femur, patellar Any position of bone, shank bone, ankle bone, phalange, or a combination thereof.

The decellularized bone graft produced above can be used for plastic surgery or cosmetic surgery by transplanting at least one, or preferably at least two, decellularized bone grafts that can be assembled to a treatment site; it can also be used to include fractures , Dislocation, arthritis, congenital deformities, tumors, degenerative diseases and/or tooth defects and other bone defects. In this case, at least two decellularized bone grafts constitute an artificial graft component; preferably at least three bone grafts, such as at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, At least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, at least thirty, or at least fifty decellularized bone grafts constitute an artificial graft component. In fact, each decellularized bone graft used to assemble the artificial graft component of the present disclosure can be precisely designed so that the final artificial graft component is a complete bone structure, which can replace the individual's total bone injury. In an embodiment of the present disclosure, the artificial graft component is the complete shape of the human mandible.

In order to increase the stability of the assembled decellularized bone graft, according to some optional embodiments of the present disclosure, the artificial graft component further includes a scaffold, which is configured to cover at least a part of at least two connected decellularized bone grafts. In some embodiments, the stent may be a metal mesh, wherein the metal is selected from the group consisting of titanium, zirconium, cobalt-chromium alloy, gold-palladium alloy, and silver-palladium alloy. In some preferred embodiments, the stent is a titanium mesh. In other preferred embodiments, the mesh of the titanium mesh is 200 to 300 mesh; more preferably, it has 250 mesh.

Through the above configuration, the decellularized bone graft and the artificial graft component containing the decellularized bone graft provide a strong and strong structure to support the growth of osteoblasts and bone blood vessels without immune rejection. The decellularized bone graft of the present disclosure can be used as an ideal xenogeneic bone graft for human patients.

A number of embodiments are presented below to illustrate some aspects of the present invention, so as to facilitate those skilled in the art to which the present invention belongs to implement the present invention. These experimental examples should not be regarded as limiting the scope of the present invention. Without further explanation, it is believed that those with ordinary knowledge in the technical field can use the present invention to the fullest extent based on the description herein. All publications cited in this article are incorporated by reference in their entirety. Example

Materials and Methods

Collect bones

Fresh bones are separated and collected from pigs. The so-called bone sources include long bones, hip bones and femurs.

Example 1: Preparation of acellular bone graft and analysis of its characteristics

1.1 Preparation of decellularized bone graft 1

Obtain the pig's long bones, hip bones and femurs from a sacrificed pig. After washing the bones with water, they were immersed in sodium hydroxide solution (1N) at room temperature for 3 hours, and then placed in hydrogen peroxide solution (5%) at 4 °C for 48 hours. After processing each solution, wash the bones with plenty of water to remove the remaining solution.

Next, the bones were treated with scCO ₂ at 350 bar (bar), 37°C, and treated for 100 minutes to completely remove residual cellular material and produce acellular cartilage grafts; then gamma rays (30 kGy) The bone was irradiated to make it sterile to obtain acellular bone graft 1.

1.2 Preparation of decellularized bone graft 2

Obtain the pig's long bones, hip bones and femurs from a sacrificed pig. After washing the bones with water, they were immersed in sodium hydroxide solution (1N) at room temperature for 2 hours, and then placed in hydrogen peroxide solution (7%) at 4°C for 32 hours. After processing each solution, wash the bones with plenty of water to remove the remaining solution.

Next, the bones were treated with scCO ₂ at 250 bar (bar), 37°C, and treated for 180 minutes to completely remove residual cellular material and produce acellular cartilage grafts; then gamma rays (32 kGy) The bone was irradiated to make it sterile to obtain acellular bone graft 2.

1.3 Preparation of decellularized bone graft 3

Obtain the pig's long bones, hip bones and femurs from a sacrificed pig. After washing the bones with water, immerse them in sodium hydroxide solution (1N) at 20°C for 4 hours, and then place them in hydrogen peroxide solution (3%) at 4°C for 55 hours. After processing each solution, wash the bones with plenty of water to remove the remaining solution.

Next, the bones were treated with scN ₂ O at 150 bar (bar), 42°C, and treated for 200 minutes to completely remove residual cellular material and produce acellular cartilage grafts; then gamma rays ( 30 kGy) irradiate the bone to make it sterile to obtain decellularized bone graft 3.

According to the specific drawing of computer-aided design, the bone tissue produced from 1.1 to 1.3 is cut with a three-dimensional laser cutter. The length, width and height of each piece are between 0.5-5 cm, 0.5-5 cm and 0.5-2 cm, respectively. Small bone pieces of appropriate size between the small bone pieces, and each small bone piece tissue has at least one coupling element (ie, tenon and mortise) as shown in Figure 1A, Figure 1B and Figure 2. The obtained bone graft is stored in a dry and sterile state until subsequent use.

1.4 Characteristic analysis of the decellularized bone grafts of Examples 1.1-1.3

Under a scanning electron microscope, it is found that the porosity of the bone graft of the present invention remains relatively intact (Figure 3). This result also confirms that no residual cellular material (such as protein, fat, and living material) is present in the processed bone graft. Osteoblasts can also be observed in the transplanted individual. Go to Examples 1.1 to 1.3 The decellularized bone graft is embedded in the cavity structure and embedded in the chamber (data not shown).

Example 2: Assemble the decellularized bone grafts of Examples 1.1 to 1.3 to construct artificial graft components

Using computer-aided design software to accurately predict and describe the appearance of the human mandible, including its complete shape and each divided small piece, to cut the prepared bone tissue. According to this, the bones of Examples 1.1 to 1.3 were produced The graft is not only characterized by tenon and mortise, but the appearance also conforms to the appearance of a part of the mandible. The bone grafts of Examples 1.1 to 1.3 are connected to each other by tightly bonding the tenon and the eye (as shown in Figure 2), whereby a plurality of decellularized bone grafts 41 are placed on the titanium mesh 42 (the mesh is With the assistance of 250 mesh), the human mandible bone (artificial graft 4) was assembled.

It should be understood that the foregoing description of the implementation manners is only given in the form of examples, and various modifications can be made by those with ordinary knowledge in the technical field of the art. The above specification, examples and experimental results provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention, without departing from the principle and spirit of the present invention, Various changes and modifications can be made to it, so the protection scope of the present invention should be defined by the accompanying patent application.

1, 2, 41: Decellularized bone graft 10: Three-dimensional ontology 11, 12, 13, 21: surface 15, 15’, 25: coupling 4: Manually transplant components 42: Titanium mesh

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows:

Figures 1A and 1B are schematic diagrams of the decellularized bone graft 1 according to an embodiment of the present disclosure, wherein Figure 1A is a front view of the decellularized bone graft 1, and Figure 1B is a decellularized bone graft Rear view of bone graft 1;

Figure 2 is a schematic diagram showing two decellularized bone grafts (1, 2) connected to each other by coupling coupling members (15', 25) in the present disclosure;

Figure 3 is a scanning electron microscope (SEM) image of the decellularized bone graft of the present disclosure taken at magnifications of (A) 35k and (B) 100k respectively according to an embodiment of the present disclosure; and

FIG. 4 shows the artificial graft assembly 4 according to the embodiment of the present disclosure, which includes a plurality of decellularized bone grafts 41.

According to the usual working method, the various elements and features in the figure are not drawn to scale, and the drawing method is to best present the specific features and elements related to the present invention. In addition, between different drawings, the same or similar element symbols are used to refer to similar elements/components.

1, 2: Decellularized bone graft

12, 13, 21: surface

15, 15’, 25: coupling

Claims (10)

A decellularized bone graft for repairing a bone defect of a much-needed individual, wherein the decellularized bone graft has a three-dimensional body characterized by having at least one coupling member formed on at least one surface of the three-dimensional body , Wherein the coupling has a structure that can correspond to another structure of another coupling of another decellularized bone graft. The decellularized bone graft according to claim 1, wherein the coupling member is a tenon convexly formed from the at least one surface, or a hole is formed concavely from the at least one surface. The decellularized bone graft according to claim 2, wherein the tenon is a right-angle tenon, a dovetail tenon, a twin tenon, an oval tenon, or a round tenon. The decellularized bone graft according to claim 1, wherein the bone defect is located in frontal bone, temporal bone, zygomatic bone, jaw bone, clavicle, scapula, sternum, rib, spine, humerus, ulna, radius, carpal, hip, femur , Patella, tibia, ankle bone, phalanx or any combination thereof. A manual transplantation component, including: At least two decellularized bone grafts according to claim 1, wherein each of the decellularized bone grafts is connected to each other by combining the coupling member, and the coupling member respectively has a corresponding structure capable of tightly bonding the coupling member . The artificial transplant component according to claim 5, wherein the coupling member is a tenon formed convexly from the at least one surface, or a hole is formed concavely from the at least one surface. The artificial transplant component according to claim 6, wherein the tenon is a right-angle tenon, a dovetail tenon, a double-finger tenon, an oval tenon, or a round tenon. The artificial graft component according to claim 5, further comprising a scaffold configured to cover at least a part of at least two connected decellularized bone grafts. The artificial graft component according to claim 8, wherein the stent is a metal mesh, wherein the metal is selected from the group consisting of titanium, zirconium, cobalt-chromium alloy, gold-palladium alloy, and silver-palladium alloy. The artificial transplant component according to claim 9, wherein the stent is a titanium mesh with a mesh of 200 to 300 mesh.

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