HK1248149B - Preparation of acellular cartilage graft and uses thereof - Google Patents

Description

Preparation method and use of acellular cartilage graft

This application claims priority to U.S. Provisional Application No. 62/203,904 (filing date: August 1, 2015), the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of decellularized cartilage grafts, and particularly relates to a method for improving the preparation of decellularized cartilage grafts. The decellularized cartilage grafts are suitable as biological scaffolds for cell growth. Therefore, the decellularized cartilage grafts of the present invention can be used as xenografts for treating osteochondral diseases.

Background Art

Cartilage tissue damage often occurs in people who are very active and in the elderly, usually due to acute or repetitive injuries or aging. Current treatment options include rest, micro-joint microsurgery to remove the damaged area of cartilage, and other surgical treatments (such as microfracture, drilling, and abrasion). These treatments can only temporarily relieve symptoms, especially when the patient maintains the same level of activity as before the injury after treatment. For example, chronic knee cartilage damage may make the damage to the articular cartilage more severe, and eventually the entire knee joint must be replaced. In addition, osteochondral diseases or injuries still face other difficulties, and there are currently no effective medical procedures and methods to treat them. Furthermore, in addition to the cartilage damage caused by aging, injury and/or disease mentioned above, the demand for cartilage grafts required for plastic or cosmetic surgery is also increasing due to the need to repair or increase cartilage tissue, such as rhinoplasty, correction surgery for facial asymmetry, correction surgery around the eyelids and facial plastic surgery.

Engineered cartilage currently available on the market is formed by seeding hydrogels or natural or synthetic polymers. These methods do not possess the same mechanical properties as natural cartilage, as the primary component of natural cartilage is collagen, which possesses higher mechanical strength. To overcome this deficiency, crosslinking agents (e.g., glutaraldehyde) are often added during the preparation process to improve the mechanical strength of hydrogels or polymer scaffolds. However, most crosslinking agents are toxic to humans, limiting their use.

There is a need to provide an improved cartilage graft that can be used for corrective surgery or treatment of the above-mentioned injuries. The improved cartilage graft should not only have the mechanical strength and structure of natural cartilage, effectively restore the cartilage to its pre-injury state, but also not cause other complications (such as immunogenicity issues); in addition, the cartilage graft should be easy to manufacture and have low production costs.

Summary of the Invention

The present invention is made by the inventors to overcome the aforementioned problems of the prior art by manufacturing an improved cartilage graft, in particular a cartilage graft with better mechanical strength and a natural structure of natural cartilage.

One aspect of the present invention is a method for preparing a cell-free cartilage graft suitable for use in plastic surgery (e.g., rhinoplasty, facial correction surgery, etc.) or for treating osteochondral diseases. The method comprises the following steps:

(1) cutting cartilage of an animal to produce a cartilage matrix;

(2) treating the cartilage matrix of step (1) with an alkali;

(3) sterilizing the cartilage matrix treated with alkali in step (2); and

(4) Removing cells from the sterilized cartilage matrix in step (3) to prepare the acellular cartilage graft.

Furthermore, according to an embodiment of the present invention, in step (1), the cartilage is taken from a mammal, and the cartilage is nasal cartilage, costal cartilage, ear cartilage or knee joint cartilage.

According to an embodiment of the present invention, in step (2), the alkali treatment step comprises treating the cartilage matrix with a sodium hydroxide solution at room temperature for 3 to 24 hours.

According to an embodiment of the present invention, in step (3), the alkali-treated cartilage matrix is treated with a sterilizing agent, wherein the sterilizing agent is selected from the group consisting of: alcohols, oxidants, radiation, phenolic compounds, and quaternary ammonium compounds.

In certain embodiments, in step (3), the alkali-treated cartilage matrix is treated with an alcohol, and the alcohol is ethanol or isopropanol.

In another embodiment, in step (3), the alkali-treated cartilage matrix is treated with an oxidizing agent, and the oxidizing agent is hydrogen peroxide.

In another embodiment, in step (3), the alkali-treated cartilage matrix is treated with radiation, and the radiation is gamma rays.

In other embodiments, in step (3), the alkali-treated cartilage matrix is treated with a phenolic compound, wherein the phenolic compound is paraben, benzoic acid or salicylic acid.

In another embodiment, in step (3), the alkali-treated cartilage matrix is treated with a quaternary ammonium compound, wherein the quaternary ammonium compound is benzalkonium chloride, alkyldimethylbenzylammonium chloride, didecyldimethylammonium chloride or ammonium chloride.

According to an embodiment of the present invention, in step (4), the decellularization step includes treating the cartilage matrix sterilized in step (3) with a supercritical fluid (SCF) at a pressure of about 150-350 bar and a temperature of 30-50° C. for 20 minutes to 5 days.

The SCF is supercritical carbon dioxide (scCO ₂ ), supercritical nitrous oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkanes, supercritical alkenes, supercritical alcohols, or supercritical acetone. In one embodiment, the SCF is scCO ₂ . In other embodiments, the SCF is scN ₂ O.

According to a preferred embodiment, the decellularization step is performed at a temperature of 37° C. and a pressure of 350 bar for 100 minutes.

The prepared cartilage graft is primarily composed of collagen, retaining its natural structure and configuration. Therefore, after application (e.g., transplantation) to an individual, it can serve as a three-dimensional scaffold for cell growth. Furthermore, the cartilage graft is acellular, meaning it is free of any residual cellular material. Therefore, it is essentially non-immunogenic and does not induce any immune response in the host. Furthermore, the mechanical properties of the cartilage graft of the present invention are superior to those of natural cartilage, making it a preferred xenograft compared to natural cartilage and/or existing engineered cartilage.

Furthermore, a second aspect of the present invention provides a cartilage graft prepared using this method. The cartilage graft of the present invention can be used as a xenograft for plastic surgery or to treat cartilage defects caused by aging, injury, or disease. Examples of such plastic surgeries include, but are not limited to, rhinoplasty, correction of facial asymmetry, correction of eyelids, and facial plastic surgery.

The third aspect of the present invention is to provide a method for treating osteochondral diseases, comprising administering the decellularized cartilage graft of the present invention to a treatment site (eg, lesion) of a subject.

The osteochondral disease can be a congenital defect, a bone fracture, a meniscus injury or defect, a bone/spinal deformity, osteosarcoma, myeloma, bone dysplasia and scoliosis, osteoporosis, periodontal disease, cementum loss, osteomalacia, rickets, fibrous osteoma, renal bone dystrophy, spinal fusion, disc reconstruction or resection, Paget's disease, meniscus injury, rheumatoid arthritis, osteoarthritis, and traumatic or surgical cartilage injury.

After referring to the following embodiments, those skilled in the art will be able to easily understand the basic spirit and other objectives of the present invention, as well as the technical means and embodiments adopted by the present invention.

This summary is intended to provide a simplified summary of the present invention so that readers can have a basic understanding of the present invention. This summary is not a complete overview of the present invention and is not intended to identify important/critical elements of the present invention or to define the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

To make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

FIG1 is a photograph of a cartilage graft of the present invention stained with H&E and taken at a magnification of 20,000x according to one embodiment of the present invention, wherein (A) is a photograph taken before SCF treatment, and photographs taken after SCF treatment at different pressures of (B) 150 bar, (C) 200 bar, and (D) 350 bar, respectively;

FIG2 is a photograph of a cartilage graft of the present invention before (A) and after (B) SCF treatment, stained with H&E and DPAI according to one embodiment of the present invention;

FIG3 is a photograph of a cartilage graft of the present invention before (A) and after (B) SCF treatment, stained with Alcian blue, according to one embodiment of the present invention;

FIG4 is a photograph of a cartilage graft treated with SCF according to an embodiment of the present invention taken under a scanning electron microscope (SEM) at (A) 500k, (B) 1000k, and (C) 2,000k; and

FIG5 is a bar graph illustrating the compressive strength of the SCF-treated cartilage graft of the present invention and the natural cartilage graft according to Example 1.1, according to one embodiment of the present invention, wherein (A) is stress load and (B) is Young’s modulus.

DETAILED DESCRIPTION

To provide a more detailed and complete description of the present invention, the following provides illustrative descriptions of the embodiments and examples of the present invention; however, these descriptions are not intended to be the only ways to implement or use the embodiments of the present invention. The embodiments cover features of various embodiments, as well as the method steps and sequences for constructing and operating these embodiments. However, other embodiments may also be used to achieve the same or equivalent functionality and step sequences.

Unless otherwise indicated, words such as "a", "an" and "the" referring to the singular include the plural form.

The term "acellular" refers to a graft that is free of any residual cellular material, such as bioactive cells and/or fragments thereof. Furthermore, the term "acellular cartilage graft" refers to a cartilage graft that is derived from cartilage of an animal (e.g., a mammal) and that is substantially free of any cellular material. In one embodiment, the cartilage is derived from porcine nasal cartilage. In other embodiments, the cartilage is derived from porcine knee cartilage. In yet another embodiment, the cartilage is derived from porcine rib cartilage.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values of the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. As used herein, "about" generally refers to the actual value being within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard deviation of the mean, as determined by one skilled in the art. Except in the experimental examples, or unless expressly indicated otherwise, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the appended claims are approximate and may vary as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to apply normal rounding.

Broadly speaking, the present invention relates to the preparation of a decellularized cartilage graft suitable for use in cosmetic or plastic surgery, or for the treatment of osteochondral diseases. Specifically, the decellularized cartilage graft prepared by the present invention is transplanted into a cartilage lesion. Furthermore, the treatment of osteochondral diseases includes repairing or restoring damaged, injured, traumatized, or aged cartilage and/or repairing cartilage defects.

One aspect of the present invention relates to a method for preparing a decellularized cartilage graft that retains the mechanical strength, natural structure, and conformation of collagen. Thus, when the decellularized cartilage graft is transplanted into a host for tissue reconstruction or treatment of osteochondral diseases, it provides an optimal microenvironment for host tissue cells to grow. Furthermore, the cartilage graft produced by the present method is completely free of cellular material, making it essentially non-immunogenic and unlikely to induce an immune response in the host following transplantation.

Furthermore, the method of the present invention comprises at least the following steps:

(1) cutting cartilage of an animal to produce a plurality of cartilage matrices;

(2) treating the plurality of cartilage matrices produced in step (1) with an alkali;

(3) sterilizing the plurality of cartilage matrices treated with alkali in step (2); and

(4) Removing cells from any of the sterilized cartilage matrices in step (3) to obtain the acellular cartilage graft.

To prepare a suitable acellular cartilage graft, intact nasal cartilage, rib cartilage, ear cartilage, or knee cartilage can be removed from a non-human animal, preferably a commercial animal. Examples of commercial animal sources include, but are not limited to, pigs, cattle, cows, bulls, sheep, goats, donkeys, rabbits, ducks, geese, or chickens. Cartilage used as a cartilage graft is preferably harvested from a freshly slaughtered animal and immediately placed in a sterile isotonic solution or other preservative. The harvested cartilage is then cut into appropriate sizes and shapes to prepare cartilage matrices in the form of discs, strips, granules, or cones. In certain embodiments, each cartilage matrix is disc-shaped with a diameter of approximately 8-12 mm, preferably approximately 11 mm. In other embodiments, each cartilage matrix is strip-shaped with a length of approximately 6 cm, a width of approximately 10 mm, and a thickness of approximately 2-5 mm. Generally speaking, the size and shape of the cartilage graft are not critical to the present invention, as long as the size and shape of the cartilage graft conform to the shape of the treatment site.

The cut cartilage matrix is subjected to the treatments of steps (2) and (3) respectively, i.e., the alkali treatment in step (2) and the sterilization in step (3). Alkaline agents suitable for use in the method of the present invention include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, urea, sodium sulfide, and calcium thioacetate. In a preferred embodiment, the cartilage matrix is treated with a sodium hydroxide solution at room temperature for 3 hours. The sterilization step is to treat the cartilage matrix with a sterilizing agent to sterilize it, wherein the sterilizing agent can be an oxidizing agent, a phenolic compound, a quaternary ammonium compound, an antibiotic, and radiation. For example, sterilizing agents suitable for the method of the present invention include, but are not limited to, alcohols, isopropyl alcohol, sodium bicarbonate, hydrogen peroxide, acetic acid, sulfonamides, parabens, benzoic acid, salicylic acid, benzalkonium chloride, alkyldimethylbenzyl ammonium chloride, didecyldimethylammonium chloride, ammonium chloride, X-rays, gamma radiation, and ultraviolet light. In certain embodiments, the cartilage matrix is treated with a hydrogen peroxide solution. Furthermore, after each step is performed, the cartilage matrix is rinsed with a large amount of water to remove water-soluble substances therein before proceeding to the next step.

Next, the cells on the cartilage matrix that has been treated with alkali and sterilized in step (3) are removed. The purpose of decellularization is to remove the cellular material on the cartilage tissue while retaining the physical and biochemical properties of collagen so that it can be used as a tissue scaffold (e.g., a xenograft). In addition, in step (4), the cartilage matrix that has been sterilized in step (3) is treated with a supercritical fluid (SCF) at a pressure of about 150-500 bar, such as 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, , 300, 310, 320, 330, 340 and 350 bar; preferably about 150-350 bar, for example, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 and 350 bar. Furthermore, step (4) is carried out at a temperature between 30-50°C, for example, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50°C; preferably, the temperature is between 35-45°C, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45; and the treatment time is about 20 minutes to 5 days, for example, about 20, 30, 40, 50 and 60 minutes; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 hours; 2, 3, 4, and 5 days. In certain embodiments, the sterilized cartilage matrix in step (3) is treated with SCF for about 1 to 24 hours. In other embodiments, the sterilized cartilage matrix in step (3) is treated with SCF for about 2 to 5 days.

The SCF can be any of supercritical carbon dioxide (scCO ₂ ), supercritical nitrous oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkanes, supercritical alkenes, supercritical alcohols, or supercritical acetone. In one embodiment, the SCF is scCO ₂ . The supercritical conditions of scCO ₂ are a temperature of 37°C and a pressure of approximately 350 bar. Therefore, the removal of biomass is performed at or near the individual's body temperature (i.e., 37°C). In another preferred embodiment, the SCF is scN ₂ O.

In an optional embodiment, the SCF is applied to the sterilized cartilage matrix in step (3) together with a co-solvent. The co-solvent can be a C _1-4 alcohol, including, but not limited to, ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, and cyclobutanol. In certain preferred embodiments, the co-solvent is ethanol and is applied together with the SCF, wherein the volume ratio of the co-solvent to the SCF is 1:20 to 1:4, for example, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, and 1:4. In a preferred embodiment, the ethanol and SCF are applied in a volume ratio of 1:19. In other embodiments, the ethanol and SCF are applied in a volume ratio of 1:10. In another embodiment, the ethanol and SCF are applied in a volume ratio of 1:4. In a best embodiment, the ethanol and SCF are administered simultaneously, and the volume ratio of ethanol to SCF is about 1:10.

According to embodiments of the present invention, the cartilage graft treated with SCF in step (4) is substantially completely free of cellular material, eliminating the need for additional enzymatic digestion (e.g., digestion with proteases and/or glycosidases) to remove antigenic surface carbohydrates that could potentially serve as a source of xenograft rejection. According to embodiments of the present invention, the cartilage graft prepared by this method is completely free of nucleic acids and non-collagenous proteins.

Furthermore, the cartilage graft treated with SCF in step (4) is composed of collagen fibers and retains mechanical strength and collagen fiber integrity. Therefore, the cartilage graft prepared by the present invention is suitable as a biological scaffold for host cell growth thereon. According to the embodiments of the present invention, the cartilage graft of the present invention retains the natural structure and configuration of collagen and is capable of supporting the growth of fibroblasts.

It is important to note that the present invention differs from prior art methods in that it does not utilize commonly used steps such as hypertonic saline solution, dehydration, and/or freezing to prepare cartilage grafts. This not only simplifies the processing steps and facilitates the preparation process, but also maintains the integrity and/or mechanical strength of the collagen fibers in the cartilage grafts of the present invention. Furthermore, the present invention differs from prior art methods in that it does not utilize cross-linking agents (e.g., glutaraldehyde, formaldehyde, and the like) to cross-link the proteins in the cartilage grafts, thereby reducing or minimizing the toxicity and immunogenicity of the cartilage grafts.

According to optional embodiments of the present invention, the SCF-treated cartilage graft can be sterilized using any known method (e.g., treatment with a sterilizing agent as described above) and stored in a dry, sterile state until ready for use. In certain embodiments, the cartilage graft is sterilized by gamma irradiation at an intensity of 10-60 kGy, such as 10, 20, 30, 40, 50, and 60 kGy. In a preferred embodiment, the cartilage graft is sterilized by gamma irradiation at an intensity of 10-30 kGy, such as 10, 15, 20, 25, and 30 kGy.

The acellular cartilage grafts prepared by the present invention are suitable for use in cosmetic or plastic surgery, or for treating osteochondral diseases, i.e., the acellular cartilage grafts prepared by the method of the present invention are transplanted to a treatment site (e.g., a cartilage lesion). The treatment of osteochondral diseases includes repairing or restoring damaged, injured, traumatic, or aged cartilage and/or repairing cartilage defects.

Therefore, another aspect of the present invention is a method for treating an individual suffering from an osteochondral disease. The method comprises the steps of administering a cartilage graft of the present invention to a treatment site (e.g., a lesion) in a subject. For example, the osteochondral disease includes, but is not limited to, congenital defects, bone fractures, meniscal injuries or defects, bone/spinal deformities, osteosarcomas, myeloma, bone dysplasia and scoliosis, osteoporosis, periodontal disease, cementum loss, osteomalacia, rickets, fibrous osteoma, renal osteodystrophy, spinal fusion, disc reconstruction or resection, Paget's osteitis deformans, meniscal injuries, rheumatoid arthritis, osteoarthritis, or traumatic or surgical cartilage injuries. It is anticipated that upon implantation of the SCF-treated cartilage graft of the present invention at the lesion, adjacent chondrocytes will be activated and migrate to the pore structures of the cartilage graft. The cells will begin to grow until the damaged cartilage at the lesion is replaced, and the SCF-treated cartilage graft of the present invention will eventually degrade without leaving any toxic residues in the subject.

The following experimental examples illustrate certain embodiments of the present invention to facilitate practice by those skilled in the art. These experimental examples should not be construed as limiting the scope of the present invention. It is believed that after reading the description provided herein, a skilled artisan will be able to fully utilize and practice the present invention without undue interpretation. All publications cited herein are incorporated herein in their entirety as a part of this specification.

Example

Materials and methods

Cell culture

NIH-3T3 fibroblasts were cultured in high glucose DMEM (containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin) at 37°C and 5% CO2 in a humidified atmosphere.

Isolation of chondrocytes

Chondrocytes were isolated from porcine articular cartilage. The cartilage was rinsed with culture medium and then transferred to digestion medium (DMEM, 10% FBS, 1% P/S, 0.1 mg/ml streptomycin) containing type H collagen. The cells were cultured at 37°C for 24 hours. The cells were collected by centrifugation (300 × g, 5 minutes).

H&E staining

The sliced cartilage tissue was immersed in xylene for 10 minutes to remove the paraffin, and this step was repeated once. The deparaffinized tissue was dehydrated using a gradient of alcohol solutions, ranging from 100% to 95%, 85%, and 75%. During the dehydration step, the tissue sample was placed in each alcohol solution for only 2 minutes before being rinsed with a large amount of deionized water. After dehydration, the tissue was stained with hematoxylin for 3-5 minutes and then washed with deionized water and 75% alcohol. The hematoxylin-stained cartilage was then stained with 1% eosin Y for 30 seconds and then washed in deionized water, 95%, and 100% ethanol.

Alcian blue staining

Cartilage tissue sections were deparaffinized and dehydrated according to the procedures described in the "H&E Staining" section. Next, the samples were stained with Alcian blue for 30 minutes, rinsed in tap water for 2 minutes, and rinsed with deionized water. The Alcian blue-stained cartilage samples were counterstained with nuclear fast red solution for 5 minutes and then rinsed in natural water for 1 minute.

The cartilage samples were then sequentially passed through 95% alcohol and 100% alcohol (twice, 2 minutes each), and then fixed with resin mounting medium.

Growth of 3T3 cells on cartilage grafts

When 3T3 cells reached 80% confluent, they were harvested enzymatically (0.25% trypsin in 1 mM EDTA) and centrifuged at 500 x g for 5 minutes. The harvested cells were resuspended in culture medium at a final concentration of 2 x 10 ⁶ cells/ml. The SCF-treated cartilage grafts of the present invention were pre-moistened with culture medium and cultured overnight before being plated in low-attachment 24-well plates (Corning). Half of the cell suspension (200 μl) was transferred to one side of each SCF-treated cartilage graft and incubated in an incubator for 2 hours to ensure cell adhesion to the surface. Following the aforementioned procedure, the remaining cell suspension was transferred to the other side of the SCF-treated cartilage graft (i.e., the side not yet exposed to cells). Each cell-seeded cartilage graft was cultured in 2 ml of culture medium for 7 days and then fixed with 10% formalin for the following analyses.

Growth of primary chondrocytes on cartilage grafts (Recellularization)

The cartilage grafts were placed in a 24-well plate (200 mg/well), and freshly isolated chondrocytes were seeded onto the grafts. The final cell count in the culture medium in each well was 1.2 x ¹⁰⁷ cells. The culture medium was replaced three times a week. After two weeks of culture, the grafts were harvested and fixed with 10% formalin before analysis.

Compressive strength determination

Cartilage specimens were rehydrated in PBS for at least 16 hours. Depending on the specimen's shape, the height and/or diameter of the specimen were measured: if the specimen was strip-shaped, its length was measured; if the specimen was disc-shaped, its diameter was measured. Compressive strength was measured using a Lloyd machine (LRX Instrument, England). Each test began with a preload of 2 N and a loading rate of 0.18 mm/min. The test was terminated when the specimen's height and/or diameter decreased by 65% during loading.

Example 1 Preparation of cartilage grafts and analysis of their properties

1.1 Preparation of acellular cartilage grafts

After sterilizing the pig joint, remove the cartilage. Rinse the cartilage with water and cut it into the desired size and shape according to actual use.

First, the cartilage matrix was immersed in a sodium hydroxide solution (1N) at room temperature for 3 hours, and then in a hydrogen peroxide solution (5%) at 4° C. for 48 hours. After each solution treatment, the cartilage matrix was washed with plenty of water to remove any residual solution.

Next, the cartilage matrix was treated with _scCO₂ at 350 bar and 37°C for 100 minutes to completely remove residual cellular material and produce an acellular cartilage graft. The cartilage graft was then irradiated with gamma rays (30 kGy) and stored in a dry and sterile environment until use.

1.2 Characteristic Analysis of the Cartilage Graft of Example 1.1

Before SCF treatment, residual cellular material was observed within the cartilage cavities, i.e., chondrocytes were contained within the small spaces within the cartilage (Figure 1, (A)). However, after SCF treatment at different pressures, the remaining cellular material within the cavities was completely eliminated (Figure 1, (B), (C), and (D)). This result confirms that SCF treatment is an effective method for removing cellular material from cartilage. The SCF-treated cartilage was then stained with H&E, DAPI, and Alcian blue, revealing the absence of cell nuclei (Figure 2) or acidic polysaccharides (e.g., glycosaminoglycans in cartilage) (Figure 3). The results from Figures 1 to 3 confirm that the SCF-treated cartilage grafts of the present invention do not contain any residual cellular material (e.g., cell nuclei or carbohydrates).

Furthermore, the pores on the cartilage grafts treated with SCF were relatively intact ( FIG. 4 ), which supported the growth of 3T3 cells and primary chondrocytes on the cartilage grafts. Cell growth experiments revealed that primary chondrocytes not only migrated to the pores of the cartilage grafts of Example 1.1 but also embedded themselves in the cavities therein (data not shown).

The mechanical properties of the cartilage graft treated with SCF in Example 1.1 were superior to those of natural cartilage. In the compressive strength test, the cartilage graft treated with SCF in Example 1.1 could withstand at least twice the pressure as that of natural cartilage ( FIG. 5 ).

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those skilled in the art may make various changes and modifications thereto without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined in the appended claims.

Claims (9)

1. A method for preparing decellularized cartilage grafts, characterized in that it comprises: (1) Cutting the cartilage of an animal to produce multiple cartilage matrices; (2) Treat the plurality of cartilage matrices from step (1) with sodium hydroxide at room temperature for 3 to 24 hours; (3) Treating the plurality of cartilage matrices treated with sodium hydroxide in step (2) with a sterilizing agent, wherein the sterilizing agent is selected from the group consisting of: alcohols, oxidants, radiation, phenolic compounds, and quaternary ammonium compounds; and (4) Treat the multiple cartilage matrices sterilized in step (3) with supercritical carbon dioxide at a pressure of 150-350 bar and a temperature of 30-50°C for 100 minutes to remove cells from the multiple cartilage matrices and obtain the decellularized cartilage graft. The method does not employ hypertonic salt solutions, dehydration, or freezing treatment, and it does not use crosslinking agents. 2. The method as described in claim 1, wherein the cartilage is taken from a nasal cartilage, a rib cartilage, an ear cartilage, or a knee cartilage of a pig. 3. The method according to claim 2, wherein the shape of each of the decellularized cartilage grafts is disc-shaped, strip-shaped, cone-shaped, or granular. 4. The method according to claim 1, wherein the alcohol is ethanol or isopropanol. 5. The method according to claim 1, wherein the oxidant is hydrogen peroxide. 6. The method according to claim 1, wherein the phenolic compound is a paraben, benzoic acid, or salicylic acid. 7. The method according to claim 1, wherein the quaternary ammonium compound is benzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, dialcyl dimethyl ammonium chloride, and ammonium chloride. 8. The method as claimed in claim 1, wherein the temperature is 37°C and the pressure is 350 bar. 9. The method of claim 8, wherein each of the decellularized cartilage grafts can withstand a pressure at least twice that of natural cartilage.