Extracellular matrix powder derived from meniscus and graft for fibrocartilage tissue reconstruction

Abstract

The present invention relates to an extracellular matrix powder derived from the meniscus and a graft for fibrocartilage tissue reconstruction and, more specifically, to an extracellular matrix powder derived from the meniscus and a graft for fibrocartilage tissue reconstruction, wherein the extracellular matrix powder can enhance fibrocartilage differentiation ability and fibrocartilage regeneration effects by controlling a content of powder derived from a specific region of the meniscus.

Description

Extracellular matrix powder derived from the meniscus and graft material for fibrocartilage tissue reconstruction

[0001] The present invention claims the benefit of the filing date of Korean Patent Application No. 10-2024-0147481 filed with the Korean Intellectual Property Office on October 25, 2024, the entire contents of which are incorporated into the present invention. The present invention relates to an extracellular matrix powder derived from the meniscus and an implant material for fibrocartilage tissue reconstruction, wherein the extracellular matrix powder derived from the meniscus and the implant material for fibrocartilage tissue reconstruction can improve fibrocartilage differentiation ability and fibrocartilage regeneration effect by controlling the content of the powder derived from a specific location of the meniscus.

[0002] Recently, in the field of tissue engineering, a technology involving the decellularization of allogeneic or xenogeneic organs and tissues has been attracting attention. To date, various tissue-derived biomaterials, including small intestinal submucosal tissue, bladder, skin, amniotic membrane, bone, ligaments, and cartilage, have been commercialized or are currently under research.

[0003] Meanwhile, musculoskeletal tissues such as cartilage, ligaments, and menisci, although exhibiting a single form and structure as monolithic tissues, differ in their biochemical and physical properties depending on their anatomical location. In the case of the meniscus, a representative musculoskeletal tissue, its biochemical characteristics are broadly classified histologically into medial, intermediate, and lateral regions based on the structure and function of the extracellular matrix. Conventional tissue engineering techniques utilizing decellularization of organs and tissues employ a mixture of whole tissue extracellular matrix (ECM).

[0004] However, the effect varies depending on the part of the meniscus, and since the effect tends to differ depending on the content of different parts for the reconstruction of fibrocartilage tissue, research on this was necessary.

[0005] The technical problem to be achieved by the present invention is to provide an extracellular matrix powder derived from meniscus and a graft material for fibrocartilage tissue reconstruction that can enhance the fibrocartilage regeneration effect by dividing the meniscus into a first zone, a second zone, and a third zone, confirming the characteristic effects according to each of the zones, and improving the ability to induce fibrocartilage differentiation by adjusting the content of the powder derived from the first zone.

[0006] However, the problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0007] One embodiment of the present invention provides an extracellular matrix powder derived from a meniscus composed of a first zone, a second zone, and a third zone, wherein the first zone has a light transmittance of 60% or more and 100% or less based on a thickness of 2 mm, the second zone has a light transmittance of 48% or more and less than 60% based on a thickness of 2 mm, and the third zone has a light transmittance of 10% or more and less than 48% based on a thickness of 2 mm, and the powder derived from the first zone of the meniscus contains 20% by weight or more of the powder, and has improved fibrocartilage differentiation ability.

[0008] According to one embodiment of the present invention, the powder derived from the first zone of the meniscus may be included in an amount of 33.3% by weight or more and 80% by weight or less.

[0009] According to one embodiment of the present invention, the powder derived from the second zone of the meniscus may be included in an amount of 10% by weight or more and 33.3% by weight or less, and the powder derived from the third zone of the meniscus may be included in an amount of 10% by weight or more and 33.3% by weight or less.

[0010] According to one embodiment of the present invention, the weight ratio of powder derived from the first zone of the meniscus; powder derived from the second zone of the meniscus; and powder derived from the third zone of the meniscus may be 1 to 8:1:1.

[0011] According to one embodiment of the present invention, the moisture content of the first zone may be 77.0% (w / w) or more and 81.0% (w / w) or less, the moisture content of the second zone may be 63.0% (w / w) or more and 69.0% (w / w) or less, and the moisture content of the third zone may be 58.0% (w / w) or more and 67.0% (w / w) or less.

[0012] According to one embodiment of the present invention, the density of the first zone is 0.95 g / cm³ 3 Above 1.05 g / cm³ 3 It is less than or equal to, and the density of the above-mentioned second zone is 1.16 g / cm³ 3 Above 1.30 g / cm³ 3 It is less than or equal to, and the density of the above-mentioned third zone is 1.10 g / cm³ 3 Above 1.15 g / cm³ 3 It may be less than or equal to

[0013] According to one embodiment of the present invention, the denaturation temperature of the first zone may be 75 ℃ or higher and 85 ℃ or lower, the denaturation temperature of the second zone may be 100 ℃ or higher and 110 ℃ or lower, and the denaturation temperature of the third zone may be 93 ℃ or higher and 98 ℃ or lower.

[0014] According to one embodiment of the present invention, the first zone may comprise, for every 1 mg of the extracellular matrix powder, 750 μg or more and 850 μg of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 5 ng or more and 10 ng or less of TSP-1.

[0015] According to one embodiment of the present invention, the second zone may comprise, for every 1 mg of the extracellular matrix powder, 850 μg or more and 950 μg of collagen, 20 μg or more and 30 μg or less of sGAG, 17 μg or more and 23 μg or less of Elastin, 50 pg or more and 70 pg or less of bFGF, 80 pg or more and 130 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 15 ng or more and 25 ng or less of TSP-1.

[0016] According to one embodiment of the present invention, the third zone may comprise, for every 1 mg of the extracellular matrix powder, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF, and more than 0 ng and less than 5 ng of TSP-1.

[0017] One embodiment of the present invention provides a graft material for fibrocartilage tissue reconstruction comprising the extracellular matrix powder.

[0018] An extracellular matrix powder derived from meniscus according to one embodiment of the present invention can improve cell migration and adhesion ability to mesenchymal stem cells, improve the ability to induce fibrocartilage differentiation, and improve the fibrocartilage regeneration effect.

[0019] A graft material for fibrocartilage tissue reconstruction according to one embodiment of the present invention can enhance the fibrocartilage regeneration effect.

[0020] Figure 1 is a photograph of the meniscus. Specifically, Figure 1(A) is a photograph of the entire meniscus. Figure 1(B) is a photograph of the meniscus tissue taken on a light source plate. Figure 1(C) is a photograph of the meniscus tissue cut in the sagittal plane. Figure 1(D) is a planar photograph of the meniscus tissue cut in the transverse plane. Figure 1(E) is a lateral photograph of the meniscus tissue cut in the transverse plane.

[0021] Figure 2 is a photograph of the entire meniscus tissue taken on a light source plate. Specifically, Figure 2(A) is a photograph of the entire meniscus tissue. Figure 2(B) is a photograph showing zones 1 through 3 in the photograph of the entire meniscus tissue. Figure 2(C) is a graph showing the light transmittance of the entire meniscus tissue by zone.

[0022] Figure 3 is a photograph of a severed meniscus tissue taken on a light source plate. Specifically, Figure 3(A) is a photograph of a severed meniscus tissue. Figure 3(B) is a photograph showing zones 1 through 3 in the photograph of the severed meniscus tissue. Figure 3(C) is a graph showing the light transmittance of the severed meniscus tissue by zone.

[0023] Figure 4 is an enlarged photograph of the cross-section of the meniscus. Specifically, Figure 4(A) is an enlarged photograph of the sagittal plane of the tissue cut with respect to the sagittal plane of the meniscus. Figure 4(B) is an enlarged photograph of the cross-sectional plane of the tissue cut with respect to the sagittal plane of the meniscus.

[0024] Figure 5 is an enlarged photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane. Specifically, Figure 5(A) is a photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane stained with Safranin-O. Figure 5(B) is a photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane stained with Alcian Blue.

[0025] Figure 6 is a graph showing the biochemical component content of the entire meniscus and each of zones 1 through 3. Specifically, Figure 6(A) is a graph showing the collagen content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(B) is a graph showing the sGAG content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(C) is a graph showing the Elastin content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(D) is a graph showing the bFGF content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(E) is a graph showing the TGF-β content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(F) is a graph showing the VEGF content of the entire meniscus and each of zones 1 through 3 before and after decellularization. Figure 6(G) is a graph showing the TSP-1 content before and after decellularization of the entire meniscus and each of zones 1 to 3.

[0026] Figure 7 is a photograph showing the cell affinity of mesenchymal stem cells for each powder derived from the entire meniscus and zones 1 through 3. Specifically, Figure 7(A) is a photograph of surviving (green) or dead (red) mesenchymal stem cells 14 days after treatment with the powder derived from the entire meniscus and zones 1 through 3. Figure 7(B) is a graph showing the amount of mesenchymal stem cells proliferated over time by each powder derived from the entire meniscus and zones 1 through 3 as an absorbance.

[0027] FIG. 8 is a graph showing the cell migration and adhesion ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, FIG. 8(A) is a graph showing the cell migration ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 8(B) is a graph showing the cell adhesion ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0028] FIG. 9 is a graph showing the ability to induce fibrocartilage differentiation in mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, FIG. 9(A) is a graph showing the amount of COL2 expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 9(B) is a graph showing the amount of ACAN expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 9(C) is a graph showing the amount of SOX9 expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0029] FIG. 10 is a diagram showing the ability to induce in vivo fibrocartilage differentiation in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus, as the content of each powder is adjusted. Specifically, FIG. 10(A) is a bright field image taken of an artificial tissue created 4 weeks after transplantation in a mouse subcutaneous transplantation model, stained with safranin-O (SO) and type 2 collagen (COL2). FIG. 10(B) is a graph showing the content of sGAG expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 10(C) is a graph showing the content of COL2 expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0030] Figure 11 is a diagram confirming the fibrocartilage regeneration effect as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, Figure 11(A) is a bright field image taken after staining rabbit meniscus tissue with safranin-O (SO) and type 2 collagen (COL2) 8 weeks after transplantation into a rabbit meniscus tissue injury model. Figure 11(B) is a graph showing the content of COL2 expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Figure 11(C) is a graph showing the content of Scoring expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0031] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0032] In this specification, "A and / or B" means "A and B, or A or B".

[0033] In this specification, 'powder' and 'powder' refer to an aggregate of particles, and 'powder' is a superordinate concept to 'powder' and may refer to powder that includes powders.

[0034] In this specification, 'derived from A' may mean manufactured using A as a raw material.

[0035] In this specification, 'thickness' may refer to the distance through which light passes in the direction of light transmission.

[0036] In this specification, 'light transmittance', 'light transmittance', 'light transmittance', and 'light transmittance' may be used interchangeably to indicate the degree to which light penetrates the meniscus.

[0037] The present invention will be described in more detail below.

[0038] One embodiment of the present invention provides an extracellular matrix powder derived from a meniscus composed of a first zone, a second zone, and a third zone, wherein the first zone has a light transmittance of 60% or more and 100% or less based on a thickness of 1 mm or more and 2.5 mm or less, the second zone has a light transmittance of 48% or more and less than 60% based on a thickness of 1 mm or more and 2.5 mm or less, and the third zone has a light transmittance of 10% or more and less than 48% based on a thickness of 1 mm or more and 2.5 mm or less, and the powder derived from the first zone of the meniscus contains 20% by weight or more of the powder derived from the first zone of the meniscus, thereby providing an extracellular matrix powder with improved cartilage differentiation ability.

[0039] An extracellular matrix powder derived from meniscus according to one embodiment of the present invention can improve cell migration and cell adhesion capabilities to mesenchymal stem cells, improve the ability to induce fibrocartilage differentiation, and improve the fibrocartilage regeneration effect.

[0040] According to one embodiment of the present invention, the extracellular matrix powder is derived from a meniscus composed of a first zone, a second zone, and a third zone. As described above, by using the extracellular matrix powder derived from a meniscus composed of a first zone, a second zone, and a third zone, the differentiation of fibrocartilage can be increased, the formation of neovascularization can be inhibited, and the formation of fibrous tissue can be enhanced.

[0041] According to one embodiment of the present invention, the first zone has a light transmittance of 60% or more and 100% or less based on a thickness of 1 mm or more and 2.5 mm or less, the second zone has a light transmittance of 48% or more and less than 60% based on a thickness of 1 mm or more and 2.5 mm or less, and the third zone has a light transmittance of 10% or more and less than 48% based on a thickness of 1 mm or more and 2.5 mm or less. As described above, by controlling the light transmittance of the first, second, and third zones within the aforementioned ranges, biological and physical characteristics can be clearly distinguished in the tissues contained in the meniscus, and by controlling the effects of inducing fibrocartilage differentiation, inhibiting neovascularization, and forming fibrous tissue by including powder derived from the distinguished zones, customized treatment can be implemented by enhancing the necessary effects according to the symptoms.

[0042] In this specification, light transmittance may be defined by the following mathematical formula. Specifically, it may refer to the ratio of the intensity of the transmitted light to the intensity of the incident light by measuring the intensity of the light transmitted to the opposite side of the meniscus when the meniscus of the specific area is prepared with a thickness of 1 mm or more and 2.5 mm or less and then light is irradiated onto the meniscus. The thickness of the meniscus may be 1.1 mm or more and 2.4 mm or less, 1.2 mm or more and 2.3 mm or less, 1.3 mm or more and 2.3 mm or less, 1.4 mm or more and 2.2 mm or less, 1.5 mm or more and 2.1 mm or less, 1.6 mm or more and 2.0 mm or less, 1.7 mm or more and 2.0 mm or less, 1.8 mm or more and 2.0 mm or less, or 1.9 mm or more and 2.0 mm or less. Preferably, the thickness of the meniscus may be 2.0 mm.

[0043] [Mathematical Formula]

[0044] Light transmittance (%) = (Intensity of transmitted light / Intensity of incident light) X 100

[0045] According to one embodiment of the present invention, the extracellular matrix powder comprises 20% by weight or more of powder derived from the first zone of the meniscus. Specifically, the extracellular matrix powder may comprise 20% by weight or more and less than 100% by weight, 22% by weight or more and 95% by weight or less, 24% by weight or more and 90% by weight or less, 26% by weight or more and 85% by weight or less, 28% by weight or more and 80% by weight or less, 30% by weight or more and 75% by weight or less, or 32% by weight or more and 70% by weight or less of powder derived from the first zone of the meniscus within the above-described ranges, thereby improving the ability to induce fibrocartilage differentiation.

[0046] According to one embodiment of the present invention, the extracellular matrix powder may comprise a powder derived from a first zone of the meniscus, a powder derived from a second zone of the meniscus, and a powder derived from a third zone of the meniscus. By including all of the powder derived from the first zone of the meniscus, the powder derived from the second zone of the meniscus, and the powder derived from the third zone of the meniscus as described above, the ability to induce fibrocartilage differentiation can be improved.

[0047] According to one embodiment of the present invention, the content of the powder derived from the first zone of the meniscus in the extracellular matrix powder may be in excess compared to the powder derived from the second zone of the meniscus or the powder derived from the third zone of the meniscus, respectively. As described above, by including the powder derived from the first zone of the meniscus in excess compared to other powders, the ability to induce fibrocartilage differentiation can be improved.

[0048]

[0049] According to one embodiment of the present invention, the extracellular matrix powder has enhanced fibrocartilage differentiation ability. As described above, by including powder derived from the first zone of the meniscus in a specific amount, the ability to induce fibrocartilage differentiation can be enhanced.

[0050] According to one embodiment of the present invention, the powder derived from the first zone of the meniscus may be included in an amount of 33.3% by weight or more and 80% by weight or less. By controlling the content of the powder derived from the first zone of the meniscus within the above-described range, the ability to induce fibrocartilage differentiation can be improved.

[0051] According to one embodiment of the present invention, the powder derived from the second zone of the meniscus may be included in an amount of 10% by weight or more and 33.3% by weight or less. By controlling the content of the powder derived from the second zone of the meniscus within the above-described range, the formation of blood vessels within the tissue can be inhibited.

[0052] According to one embodiment of the present invention, the powder derived from the third zone of the meniscus may be included in an amount of 10% by weight or more and 33.3% by weight or less. By controlling the content of the powder derived from the third zone of the meniscus within the above-described range, the fibrous tissue-forming ability can be improved.

[0053] According to one embodiment of the present invention, the weight ratio of powder derived from the first zone of the meniscus; powder derived from the second zone of the meniscus; and powder derived from the third zone of the meniscus may be 1 to 8:1:1. Specifically, the weight ratio of powder derived from the first zone of the meniscus; powder derived from the second zone of the meniscus; and powder derived from the third zone of the meniscus may be 2 to 8:1:1, 3 to 8:1:1, 4 to 8:1:1, 5 to 8:1:1, 6 to 8:1:1, or 7 to 8:1:1. That is, powder derived from the first zone of the meniscus; powder derived from the second zone of the meniscus; The weight ratio of the powder derived from the third zone of the meniscus and the powder derived from the third zone of the meniscus may be 8:1:1, 5:1:1, 3:1:1, 2:1:1, or 1:1:1. By adjusting the weight ratio of the powder derived from the first zone of the meniscus, the powder derived from the second zone of the meniscus, and the powder derived from the third zone of the meniscus within the above-described range, the effect of inducing fibrocartilage differentiation can be enhanced more than the effect of inhibiting neovascularization and forming fibrous tissue, thereby improving the fibrocartilage regeneration effect.

[0054] According to one embodiment of the present invention, the moisture content of the first zone may be 77.0% (w / w) or higher and 81.0% (w / w) or lower. The moisture content may refer to the moisture content after the decellularization step described later, that is, the moisture content of the powder derived from the decellularized first zone. By controlling the moisture content of the first zone within the above-described range, the ability of the powder derived from the first zone to induce fibrocartilage differentiation can be improved.

[0055] According to one embodiment of the present invention, the moisture content of the second zone may be 63.0% (w / w) or higher and 69.0% (w / w) or lower. The moisture content may refer to the moisture content after the decellularization step described later, that is, the moisture content of the powder derived from the decellularized second zone. By controlling the moisture content of the second zone within the above-described range, the angiogenesis-inhibiting ability of the powder derived from the second zone can be improved.

[0056] According to one embodiment of the present invention, the moisture content of the third zone may be 58.0% (w / w) or higher and 67.0% (w / w) or lower. The moisture content may refer to the moisture content after the decellularization step described later, that is, the moisture content of the powder derived from the decellularized third zone. By controlling the moisture content of the third zone within the above-described range, the fibrous tissue-forming ability of the powder derived from the third zone can be improved.

[0057] According to one embodiment of the present invention, the density of the first zone is 0.95 g / cm³ 3 Above 1.05 g / cm³ 3 It may be less than or equal to the above. The above density may refer to the density after the decellularization step described later, that is, the density of the powder derived from the decellularized first zone. By controlling the density of the first zone within the above-described range, the ability of the powder derived from the first zone to induce fibrocartilage differentiation can be improved.

[0058] According to one embodiment of the present invention, the density of the second zone is 1.16 g / cm³ 3 Above 1.30 g / cm³ 3 It may be less than or equal to the above. The above density may refer to the density after the decellularization step described later, that is, the density of the powder derived from the decellularized second zone. By controlling the density of the second zone within the above-described range, the neovascularization inhibitory ability of the powder derived from the second zone can be improved.

[0059] According to one embodiment of the present invention, the density of the third zone is 1.10 g / cm³ 3 Above 1.15 g / cm³ 3 It may be less than or equal to the above. The above density may refer to the density after the decellularization step described later, that is, the density of the powder derived from the decellularized third zone. By controlling the density of the third zone within the above-described range, the fibrous tissue-forming ability of the powder derived from the third zone can be improved.

[0060] According to one embodiment of the present invention, the denaturation temperature (Td) of the first zone may be 75°C or higher and 85°C or lower. The denaturation temperature may refer to the denaturation temperature after the decellularization step described later, that is, the denaturation temperature of the powder derived from the decellularized first zone. By controlling the denaturation temperature of the first zone, the ability of the powder derived from the first zone to induce fibrocartilage differentiation can be improved.

[0061] According to one embodiment of the present invention, the denaturation temperature (Td) of the second zone may be 100°C or higher and 110°C or lower. The denaturation temperature may refer to the denaturation temperature after the decellularization step described later, that is, the denaturation temperature of the powder derived from the decellularized second zone. By controlling the denaturation temperature of the second zone within the above-described range, the neovascularization inhibitory ability of the powder derived from the second zone can be improved.

[0062] According to one embodiment of the present invention, the denaturation temperature (Td) of the third zone may be 93°C or higher and 98°C or lower. The denaturation temperature may refer to the denaturation temperature after the decellularization step described later, that is, the denaturation temperature of the powder derived from the decellularized third zone. By controlling the denaturation temperature of the third zone within the above-described range, the fibrous tissue-forming ability of the powder derived from the third zone can be improved.

[0063] According to one embodiment of the present invention, the first zone may comprise, per 1 mg of the extracellular matrix powder, 750 μg or more and 850 μg of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 5 ng or more and 10 ng or less of TSP-1. Specifically, the biochemical components of the first zone may refer to those identified through component analysis of the powder derived from the first zone. The first zone above may contain, per 1 mg, 750 μg or more and 850 μg or less of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 5 ng or more and 10 ng or less of TSP-1. That is, the powder derived from the first zone may comprise, per 1 mg of the powder derived from the first zone, 750 μg or more and 850 μg of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 5 ng or more and 10 ng or less of TSP-1. By controlling the biochemical components of the first zone as described above, the ability of the powder derived from the first zone to induce fibrocartilage differentiation can be improved.

[0064] According to one embodiment of the present invention, the second zone may comprise, for every 1 mg of the extracellular matrix powder, 850 μg or more and 950 μg of collagen, 20 μg or more and 30 μg or less of sGAG, 17 μg or more and 23 μg or less of Elastin, 50 pg or more and 70 pg or less of bFGF, 80 pg or more and 130 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 15 ng or more and 25 ng or less of TSP-1.

[0065] Specifically, the biochemical components of the second zone may refer to those identified through component analysis of the powder derived from the first zone. The second zone may comprise, per 1 mg, collagen of 850 μg or more and 950 μg or less, sGAG of 20 μg or more and 30 μg or less, Elastin of 17 μg or more and 23 μg or less, bFGF of 50 pg or more and 70 pg or less, TGF-β of 80 pg or more and 130 pg or less, VEGF of greater than 0 pg and 50 pg or less, and TSP-1 of 15 ng or more and 25 ng or less. That is, the powder derived from the second zone may comprise, per 1 mg of the powder derived from the second zone, 850 μg or more and 950 μg of collagen, 20 μg or more and 30 μg or less of sGAG, 17 μg or more and 23 μg or less of Elastin, 50 pg or more and 70 pg or less of bFGF, 80 pg or more and 130 pg or less of TGF-β, 0 pg or more and 50 pg or less of VEGF, and 15 ng or more and 25 ng or less of TSP-1. By controlling the biochemical components of the second zone as described above, the angiogenesis inhibitory ability of the powder derived from the second zone can be improved.

[0066] According to one embodiment of the present invention, the third zone may comprise, for every 1 mg of the extracellular matrix powder, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF, and more than 0 ng and less than 5 ng of TSP-1. The above third zone may contain, per 1 mg, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF, and more than 0 ng and less than 5 ng of TSP-1. That is, the powder derived from the third zone may comprise, per 1 mg of the powder derived from the third zone, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF, and more than 0 ng and less than 5 ng of TSP-1. As described above, by controlling the biochemical components of the second zone, the fibrous tissue-forming ability of the powder derived from the third zone can be improved.

[0067] One embodiment of the present invention provides a graft material for fibrocartilage tissue reconstruction comprising the extracellular matrix powder.

[0068] A graft material for fibrocartilage tissue reconstruction according to one embodiment of the present invention can enhance the fibrocartilage regeneration effect.

[0069] One embodiment of the present invention provides a method for producing extracellular matrix powder derived from meniscus, comprising: a) a step of preparing a meniscus; b) a step of separating the prepared meniscus into tissues of a first zone, a second zone, and a third zone, respectively; c) a step of freeze-drying and then grinding the cartilage tissues separated into the first zone, the second zone, and the third zone, respectively; d) a step of decellularizing the cartilage powders of the first zone, the second zone, and the third zone tissues formed by grinding, respectively; and e) a step of freeze-drying and then grinding the cartilage powders of the first zone, the second zone, and the third zone tissues formed by decellularization, respectively, to produce extracellular matrix powder.

[0070] A method for manufacturing extracellular matrix powder derived from meniscus according to one embodiment of the present invention can regenerate a tissue more similar to human tissue and improve the ability to induce fibrocartilage differentiation by recombining powders having different biochemical characteristics and producing them into a single composite tissue.

[0071] According to one embodiment of the present invention, in step a), a meniscus is prepared.

[0072] According to one embodiment of the present invention, it is preferable to prepare the prepared meniscus by purchasing pig knee cartilage from a facility that meets the standards by referring to EN 12442 “Animal tissues and their derivatives utilized in the manufacture of medical devices, part 1; Analysis and management of risk, part 2; controls on sourcing, collection and handling”.

[0073] According to one embodiment of the present invention, in step b), the prepared meniscus is separated into tissues of zone 1, zone 2, and zone 3, respectively.

[0074] According to one embodiment of the present invention, a method for separating the prepared meniscus into tissues of zones 1, 2, and 3, respectively, separates the meniscus using light transmittance. Specifically, the light transmittance changes depending on the anatomical location of the meniscus, the physical properties of the tissue, the degree of vascular infiltration, differences in microstructure, differences in biochemical component content, and the distribution and types of cells, and accordingly, biological and physical properties change. Consequently, the prepared meniscus can be separated into tissues of zones 1, 2, and 3, respectively.

[0075] According to one embodiment of the present invention, the first zone has a light transmittance of 60% or more and 100% or less when light is passed through it based on a thickness of 2 mm. Furthermore, the moisture content of the first zone may be 77.0% (w / w) or more and 81.0% (w / w) or less, and the density of the first zone is 0.95 g / cm³ 3 Above 1.05 g / cm³ 3 The above may be less than or equal to, and the denaturation temperature of the first zone may be 75 ℃ or higher and 85 ℃ or lower, and the first zone may contain, for every 1 mg of the extracellular matrix powder, 750 μg or more and 850 μg of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF and 5 ng or more and 10 ng or less of TSP-1.

[0076] According to one embodiment of the present invention, the second zone has a light transmittance of 48% or more and less than 60% based on a thickness of 2 mm. Furthermore, the moisture content of the second zone may be 63.0% (w / w) or more and 69.0% (w / w) or less, and the density of the second zone is 1.16 g / cm³. 3 Above 1.30 g / cm³ 3The above may be less than or equal to, and the denaturation temperature of the second zone may be 100 ℃ or higher and 110 ℃ or lower, and the second zone may contain 850 μg or more and 950 μg or less of collagen, 20 μg or more and 30 μg or less of sGAG, 17 μg or more and 23 μg or less of Elastin, 50 pg or more and 70 pg or less of bFGF, 80 pg or more and 130 pg or less of TGF-β, 0 pg or less and 50 pg or less of VEGF, and 15 ng or more and 25 ng or less of TSP-1 per 1 mg of the extracellular matrix powder.

[0077] According to one embodiment of the present invention, the third zone has a light transmittance of 10% or more and less than 48% based on a thickness of 2 mm. Furthermore, the moisture content of the third zone may be 58.0% (w / w) or more and 67.0% (w / w) or less, and the density of the third zone is 1.10 g / cm³. 3 Above 1.15 g / cm³ 3 The above may be less than or equal to, and the denaturation temperature of the third zone may be 93 ℃ or higher and 98 ℃ or lower, and the above third zone may contain, per 1 mg of the above extracellular matrix powder, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF and more than 0 ng and less than or equal to 5 ng of TSP-1.

[0078] According to one embodiment of the present invention, in step c), the cartilage tissues separated into the first zone, the second zone, and the third zone are each freeze-dried and then ground. Each meniscal tissue, cut and separated according to the tissues of the first, second, and third zones, is made into pieces of 10 to 20 mm in size, washed three times for 10 minutes each with physiological saline, and then freeze-dried for 3 days in an ultra-low temperature freezer at -80 ℃. The meniscal fragments of the first, second, and third zones that were dried therefrom are freeze-ground to a size of about 30 µm using a freeze-ground grinder (JAI, JFC-300, JAPAN), and then the meniscal powders of the first, second, and third zones obtained by grinding are stored again in an ultra-low temperature freezer at -80 ℃.

[0079] According to one embodiment of the present invention, in step d), the meniscus powders of the first, second, and third zone tissues formed by grinding are each decellularized. In order to remove cells and genetic material present in the meniscus powders of the first, second, and third zone tissues and to obtain only pure extracellular matrix (ECM) components, the decellularization process was performed as follows.

[0080] Specifically, each of the meniscus powders of the first, second, and third zone tissues prepared in step c) above is treated with 500 ml of hypotonic buffer per 10 g and stirred at 200 rpm for 4 hours in a 4 ℃ environment.

[0081] In order to precipitate and separate the meniscus powders from each storage solution after the above stirring process, a centrifuge (US-21SMT, Vision, Korea) is used to process them at 10,000 rpm for 30 minutes.

[0082] After removing the supernatant (storage solution) from the storage solution treated in this way, the meniscus powder is added to a 0.1% SDS (Sodium dodecyl sulfate, Bio-rad, USA) solution and stirred at 200 rpm for 2 hours in a 4 ℃ environment.

[0083] After the above SDS treatment is completed, the meniscus powder is washed five times repeatedly with triple distilled water, and it is preferable to replace the washing water under centrifugation conditions as described above.

[0084] Next, 200 ml of 500 U / ml DNase (Sigma, USA) was added and stirred at 200 rpm in a 37 ℃ incubator for 12 hours. After DNase treatment, the decellularization process was completed by washing 5 times with triple-distilled water as described above.

[0085] Next, in step e), the meniscus powders for each of the decellularized zone 1, zone 2, and zone 3 tissues are freeze-dried and then ground to produce meniscus-derived extracellular matrix powders for each of the zone 1, zone 2, and zone 3 tissues.

[0086] At this time, the meniscus powder decellularized by step d) is cooled in an ultra-low temperature freezer at -80°C and freeze-dried for 3 days, and then the dried meniscus powder is crushed in the manner described above to finally obtain cartilage powder of about 10 µm in size and stored in an ultra-low temperature freezer at -80°C until needed.

[0087] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention is not to be interpreted as being limited to the embodiments described below. The embodiments of this specification are provided to more completely explain the present invention to those with average knowledge in the art.

[0088]

[0089] <Example 1: Difference in light transmittance according to zone of meniscal tissue>

[0090] Figure 1 is a photograph of the meniscus. Specifically, Figure 1(A) is a photograph of the entire meniscus. Figure 1(B) is a photograph of the meniscus tissue taken on a light source plate. Figure 1(C) is a photograph of the meniscus tissue cut in the sagittal plane. Figure 1(D) is a planar photograph of the meniscus tissue cut in the transverse plane. Figure 1(E) is a lateral photograph of the meniscus tissue cut in the transverse plane.

[0091] Meniscus tissue was harvested from the knee joints of pigs using a surgical blade. Images of the collected meniscus tissue were captured using a digital camera on an LED light source plate (Mellow Buddy Happy Energy Light, China, 6500K, 2000 LUX) (Figs. 1(A) and 1(B)). In addition, to compensate for differences in light transmittance according to the anatomical thickness of the meniscus tissue, the meniscus was cut into equal thicknesses of 2 mm, and images were captured on the light source plate of the sagittal plane (Fig. 1(C)), transverse plane (Fig. 1(D)), and lateral plane (Fig. 1(E)).

[0092] Figure 2 is a photograph of the entire meniscus tissue taken on a light source plate. Specifically, Figure 2(A) is a photograph of the entire meniscus tissue. Figure 2(B) is a photograph showing zones 1 through 3 in the photograph of the entire meniscus tissue. Figure 2(C) is a graph showing the light transmittance of the entire meniscus tissue by zone.

[0093] The difference in light transmittance was quantitatively analyzed using Image J software for images of meniscal tissue captured on a light source plate (Fig. 2). To quantitatively analyze the intensity of light transmitted through the meniscus from the light source plate, the Integrated density value was measured. After capturing images of a light-blocking specimen (black square specimen) that does not transmit light, the image contrast was adjusted and corrected until the integrated density was 0 (minimum integrated density: negative control) and the integrated density of the light source plate in the area without the specimen was 255 (maximum integrated density: positive control). In the corrected image, the integrated density was measured in the vertical direction (sagittal plane) of the meniscal tissue to measure the intensity of light transmitted from the light source plate through the meniscal tissue. As a result of the light transmittance image analysis, the meniscus exhibited characteristics classified into three zones: Zone 1, where light transmits well to the lower part of the meniscus and appears white to off-white; Zone 2, where light transmits moderately to the middle part of the meniscus and appears yellow to orange; and Zone 3, where light does not transmit to the upper part of the meniscus and appears gray.

[0094] The measured integrated density showed light transmittances of 229–137 AU (Arbitrary Unit) in Zone 1, 121–68 AU in Zone 2, and 64–58 AU in Zone 3. When the integrated density was converted into light transmittance (%), it showed light transmittances of 89.8–53.73% in Zone 1, 47.45–26.67% in Zone 2, and 25.1–22.75% in Zone 3. Table 1 below shows the measured light transmittance of the entire meniscus. Additionally, for each tissue, the integrated density measurements for Zone 1, Zone 2, and Zone 3 were averaged by zone, and the average values ​​for Zone 1, Zone 2, and Zone 3 for five meniscus tissue samples were converted into light transmittance (%). Afterwards, the average light transmittance of each zone for the five samples was measured, and the light transmittance was 73.47±5.13% for Zone 1, 36.84±6.79% for Zone 2, and 26.32±2.09% for Zone 3, and it was confirmed that there were statistically different values ​​for each zone.

[0095] Location Area Integrated Density Light Transmittance (%) 1-00 2-255 100 3 Area 1 229 89.80 4 Area 1 210 82.35 5 Area 1 193 75.69 6 Area 1 183 71.76 7 Area 1 170 66.67 8 Area 1 154 60.39 9 Area 1 137 53.73 10 Area 2 121 47.45 11 Area 2 107 41.9 612 Zone 2 9637.6513 Zone 2 9035.2914 Zone 2 7930.9815 Zone 2 7228.2416 Zone 2 6826.6717 Zone 3 6425.1018 Zone 3 5822.7519 Zone 3 6224.3120 Zone 3 6123.9221 Zone 3 6224.3122 Zone 3 6324.71

[0096] Figure 3 is a photograph of a cut meniscus tissue taken on a light source plate. Specifically, Figure 3(A) is a photograph of a cut meniscus tissue. Figure 3(B) is a photograph showing zones 1 through 3 in the photograph of the cut meniscus tissue. Figure 3(C) is a graph showing the light transmittance of the cut meniscus tissue by zone. When specimens were cut to a uniform thickness of 2 mm to compensate for the difference in thickness between zones of the meniscus tissue and the integrated density and light transmittance were measured using the same method as above (Figure 3), zone 1 showed light transmittance (light transmittance) of 225–182 AU (88.24–71.37%), zone 2 showed 147–124 AU (57.65–48.63%), and zone 3 showed 122–94 AU (47.84–36.86%). Table 2 below shows the light transmittance of the transected meniscus. Similarly, an analysis of the average values ​​of light transmittance by zone for five specimens of the same thickness showed light transmittances of 75.89±6.5% in Zone 1, 52.95±4.38% in Zone 2, and 39.29±4.32% in Zone 3, and it was confirmed that there were statistically significant differences between each zone (Fig. 3(C)).

[0097] Therefore, it was confirmed that meniscal tissue can be separated into three zones based on differences in light transmittance.

[0098] Location Area Integrated Density Light Transmittance (%) 1-00 2-255 100 3 Zone 1 225 88.244 Zone 1 203 79.615 Zone 1 182 71.376 Zone 2 147 57.657 Zone 2 141 55.298 Zone 2 136 53.339 Zone 2 133 52.16 10 Zone 2 124 48.63 11 Zone 2 130 50.98 12 Zone 2 Zone 12850.2013 Zone 2 14054.9014 Zone 2 13552.9415 Zone 2 13552.9416 Zone 3 12147.4517 Zone 3 11645.4918 Zone 3 11043.1419 Zone 3 10641.5720 Zone 3 9436.8621 Zone 3 11043.1422 Zone 3 12247.84

[0099] <Example 2: Differences in Physicochemical Properties According to Zones of Meniscus Tissue> Example 2-1: Analysis of Microstructural Characteristics by Zone of Meniscus Tissue

[0100]

[0101] Scanning Electron Microscope (SEM) analysis was performed to observe the microstructure of the meniscus tissue by region. Figure 4 is a magnified image of the cross-section of the meniscus. Specifically, Figure 4(A) is a magnified image of the sagittal plane of the tissue cut along the sagittal plane of the meniscus. Figure 4(B) is a magnified image of the transverse plane of the tissue cut along the sagittal plane of the meniscus. The microstructure was observed using SEM by photographing and analyzing the sagittal plane (Figure 4(A)) and the transverse plane (Figure 4(B)) separately. As a result of observing the microstructure of the sagittal plane of the meniscus, Zone 1 had a relatively smooth surface compared to other zones, and high-magnification analysis confirmed that it exhibited a porous network form arranged randomly without a specific directionality (Figure 4(A)). Microstructural observation of Zone 2 revealed a greater number of pores compared to Zone 1, but a slightly denser microfiber arrangement in the cross-sectional direction compared to Zone 3. Microstructural observation of Zone 3 showed a significantly higher number of pores compared to Zones 1 and 2, reduced tissue density compared to Zone 2, and the most distinct microfiber arrangement in the sagittal and horizontal directions.

[0102] SEM analysis of the cross-sectional plane of meniscal tissue cut to a thickness of 2 mm revealed that, similar to the sagittal plane, Zone 1 exhibited a randomly arranged porous network structure, and among all zones, it had the smoothest surface and the thinnest fiber thickness (Fig. 4(B)). In Zone 2, the microfibers were arranged perpendicular to the cross-section and, consistent with the sagittal plane observation, showed a denser structure compared to Zone 3. Zone 3 had the largest number and size of pores among all zones, and, similar to Zone 2, the orientation of the microfiber structure perpendicular to the cross-section was confirmed.

[0103] Therefore, it was confirmed that there are differences in the microstructure and arrangement characteristics of the extracellular matrix depending on the anatomical zones of the meniscus tissue according to light transmittance.

[0104] Example 2-2: Analysis of Physical Characteristics by Zone of Meniscus Tissue

[0105] To analyze the physical properties of meniscal tissue by zone according to differences in light transmittance, water content, density, and denaturation temperature were analyzed before and after decellularization and summarized in Table 3 below. Tissues from each zone were sliced ​​to a thickness of 2 mm in the sagittal plane and prepared into cylindrical specimens using a 3 mm biopsy punch for physical property analysis. Water content was calculated according to the following formula after measuring the wet and dry weights of the meniscal tissue (Water content % = (Wet weight - Dry weight) / Wet weight x 100). The results of the water content measurement before decellularization showed that Zone 1 was 86.09%, Zone 2 was 69.07%, and Zone 3 was 64.26%, confirming that the water content was highest in the order of Zone 1, Zone 2, and Zone 3. Furthermore, the results of measuring the moisture content after decellularization showed that Zone 1 was 78.80%, Zone 2 was 65.91%, and Zone 3 was 62.63%, confirming that the moisture content was highest in the order of Zone 1, Zone 2, and Zone 3.

[0106] Density measurements were analyzed using a digital densimeter (DMA 5001, Anton Paar). The density results prior to decellularization showed that Zone 1 was 1.08 ± 0.04 g / cm³. 3 , Zone 2 is 1.24±0.04 g / cm³ 3 , Zone 3 is 1.17±0.004 g / cm³ 3 ...was indicated. Furthermore, the density measurement results after decellularization showed that Zone 1 was 1.01±0.03 g / cm³. 3 , Zone 2 is 1.21±0.05 g / cm³ 3 , Zone 3 is 1.14±0.005 g / cm³ 3 It represented.

[0107] The denaturation temperature was analyzed using a differential scanning calorimeter (Q 10; TA Instruments, Eschborn). The denaturation temperature measurements before decellularization showed Zone 1 at 78.54±2.54 ℃, Zone 2 at 102.29±1.28 ℃, and Zone 3 at 92.74±1.77 ℃. The denaturation temperature measurements after decellularization showed Zone 1 at 80.36±2.73 ℃, Zone 2 at 104.48±1.3 ℃, and Zone 3 at 95.03±1.02 ℃, confirming that thermal stability was highest in the order of Zone 2, Zone 3, and Zone 1.

[0108] Therefore, it was confirmed that there are differences in the physicochemical properties of the extracellular matrix depending on the anatomical zones of the meniscus tissue according to light transmittance.

[0109] Classification Total Zone 1 Zone 2 Zone 3 Before Decellularization After Decellularization Before Decellularization After Decellularization Before Decellularization After Decellularization Before Decellularization After Decellularization Water Content (%) 7 2.12±0.66 7 0.01±1.53 8 6.09±1.99 7 8.80±1.73 6 9.07±2.45 6 5.91±2.33 6 4.26±3.79 6 2.63±3.97 Density (g / cm³) 3)1.20±0.01 1.17±0.01 1.08±0.04 1.01±0.03 1.24±0.04 1.21±0.05 1.17±0.004 1.14±0.005 Transformation Temperature (Td, ℃) 90.99±0.96 94.39±1.29 78.54±2.64 80.36±2.73 102.29±1.28 104.48±1.39 2.74±1.77 95.03±1.02

[0110] Example 2-3: Analysis of histological characteristics of meniscal tissue by zone. Figure 5 is an enlarged photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane. Specifically, Figure 5(A) is a photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane stained with Safranin-O. Figure 5(B) is a photograph of the sagittal plane, transverse plane, and transverse lateral view of the tissue of the meniscus cut along the sagittal plane stained with Alcian Blue.

[0111] Safranin-O and Alcian Blue staining were performed to histologically analyze the degree of sulfated-glycosaminoglycan (sGAG) deposition in different zones of meniscal tissue according to differences in light transmittance (Fig. 5). As a result of the histological staining, it was confirmed that in Zone 1, red Safranin-O staining and blue Alcian Blue staining, which indicate sGAG deposition, were most predominantly expressed among all groups (Fig. 5(A) and Fig. 5(B)). In Zone 2, sGAG expression was found to be stronger compared to Zone 3, and Zone 3 showed the lowest sGAG expression among all groups.

[0112] Similar to the SEM analysis results, it was confirmed that the microfiber structures in zones 2 and 3 were arranged in a direction parallel to the sagittal plane; in particular, radial fibers composed of large collagen fibers were frequently observed in zone 3. On the other hand, radial fibers from zone 3 did not reach zone 2, and it exhibited a dense microstructure consisting of fibers with a smaller diameter than the large collagen fibers in zone 3. It was confirmed that zone 1 consisted of a finer fiber structure compared to zone 2 and was arranged in a random manner. Furthermore, as observed in the SEM analysis, zones 2 and 3 again showed an arrangement structure perpendicular to the plane of the cross-section.

[0113] Therefore, it was confirmed that there are differences in the histological characteristics of the extracellular matrix depending on the anatomical zones of the meniscus tissue according to light transmittance.

[0114] Examples 2-4: Analysis of Biochemical Characteristics by Zone of Meniscus Tissue

[0115] To analyze the biochemical characteristics of meniscal tissue by zone according to differences in light transmittance, the content of collagen, sGAG, elastin, and growth factors was analyzed (Fig. 6).

[0116] Figure 6 is a graph showing the biochemical component content of the entire meniscus and each of the first to third zones.

[0117] Collagen was measured using the Sirius Red assay (S1000, Biocolor), sGAG using the DMMB assay (B1000, Biocolor), and Elastin using the Elastin assay (Fastin, Biocolor).

[0118] Figure 6(A) is a graph showing the collagen content of the entire meniscus and zones 1 through 3 before and after decellularization. As a result of measuring collagen by zone after decellularization, it was confirmed that the collagen content was highest in zone 2 (935.02±21.35 μg / mg), followed by zone 1 (821.01±23.87 μg / mg) and zone 3 (786.05±26.06 μg / mg), and the collagen content of the entire meniscus tissue was 831.76±22.02 μg / mg (Figure 6(A)).

[0119] Figure 6(B) is a graph showing the sGAG content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. As a result of measuring sGAG after decellularization, it was confirmed that the sGAG content was highest in zone 1 (47.43±7.33 μg / mg), followed by zone 2 (25.7±2.85 μg / mg) and zone 3 (15.99±1.25 μg / mg), and the sGAG content of the entire meniscus tissue was 28.43±3.01 μg / mg (Figure 6(B)).

[0120] Figure 6(C) is a graph showing the elastin content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. After decellularization, the elastin content was found to be highest in zone 3 (24.44±1.8 μg / mg), followed by zone 2 (19.38±1.8 μg / mg) and zone 1 (8.57±0.93 μg / mg), and the total elastin content of the meniscus tissue was 17.72±2.08 μg / mg (Figure 6(C)).

[0121] To analyze the characteristics of growth factors in different zones of meniscal tissue according to differences in light transmittance, the content of Basic Fibroblast Growth Factor (bFGF, 100-18B50UG, Gibco), Transforming Growth Factor-beta (TGF-β, MBS8421038, MyBioSource), and Vascular Endothelial Growth Factor (VEGF, ES25RB, Invitrogen) was analyzed using Enzyme-Linked Immunosorbent Assay (ELISA). In addition, the content of Thrombospondin-1 (TSP-1, LS-F55808, LSbio) was analyzed as an anti-angiogenesis factor.

[0122] Figure 6(D) is a graph showing the bFGF content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. As a result of measuring bFGF after decellularization, it was confirmed that the bFGF content was highest in zone 3 (82.03±3.7 pg / mg), followed by zone 2 (59.59±3.66 pg / mg) and zone 1 (19.89±2.63 pg / mg), and the total bFGF content of the meniscus tissue was 66.82±9.6 pg / mg (Figure 6(D)).

[0123] Figure 6(E) is a graph showing the TGF-β content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. As a result of TGF-β measurement after decellularization, it was confirmed that the TGF-β content was highest in zone 1 (287.16±22.26 pg / mg), followed by zone 2 (120.1±13.36 pg / mg) and zone 3 (44.96±8.17 pg / mg), and the TGF-β content of the entire meniscus tissue was 95.89±6.06 pg / mg (Figure 6(E)).

[0124] Figure 6(F) is a graph showing the VEGF content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. As a result of measuring VEGF after decellularization, it was confirmed that the VEGF content was highest in zone 3 (1025.38±151.03 pg / mg), followed by zone 1 (11.33±4.39 pg / mg) and zone 2 (11.36±2.62 μg / mg), and the total VEGF content of the meniscus tissue was 609.04±84.12 pg / mg (Figure 6(F)).

[0125] Figure 6(G) is a graph showing the TSP-1 content before and after decellularization of the entire meniscus and zones 1 through 3, respectively. As a result of measuring TSP-1 after decellularization, it was confirmed that the TSP-1 content was highest in zone 2 (21.07±3.17 ng / mg), followed by zone 1 (7.68±1.72 ng / mg) and zone 3 (1.28±0.55 ng / mg), and the TSP-1 content of the entire meniscus tissue was 7.57±2.57 ng / mg (Figure 6(G)).

[0126] Therefore, it was confirmed that there are differences in biochemical content and composition depending on the anatomical zones of the meniscus tissue according to light transmittance.

[0127] <Example 3: Analysis of Cell Behavior and Differentiation Induction Characteristics of Meniscus Tissue-Derived Powder by Region on Mesenchymal Stem Cells>

[0128] Example 3-1: Preparation of decellularized powder derived from porcine meniscus tissue by zone

[0129] As described in Example 1 above, meniscal tissue was collected from a pig's knee joint using a surgical blade, and then divided into zones 1, 2, and 3 based on the light transmittance of the tissue on a light source plate and cut and separated using a surgical blade. The meniscal tissue from each zone was washed with distilled water and then dried using a freeze dryer (Bondiro, Ilshinlab, Daejeon, Korea). Subsequently, the freeze-dried tissue (lyophilized tissue) was obtained as a fine powder using a freezer mill (6870; SPEX, Metuchen, NJ, USA). Subsequently, the tissue powder was treated with a stock solution (10 mM Tris-HCl, pH 8.0) at room temperature for 12 hours, followed by decellularization with TBS buffer containing 0.1% sodium dodecyl sulfate (Tris-buffered saline containing 0.1% sodium dodecyl sulfate) for 2 hours at room temperature. The decellularized meniscus powder was centrifuged at 10,000 RCF for 10 minutes at 4°C and washed 7 times with distilled water to remove detergent. The collected cartilage tissue powder was treated with Dnase buffer (100 U / ml, Elpis Biotech, Daejeon, Korea) at 37°C for 12 hours to remove remaining genetic material. The final decellularized cartilage tissue powder was centrifuged at 10,000 RCF for 10 minutes at 4°C and washed 7 more times with distilled water. Decellularized meniscus powder was freeze-dried and prepared into a final powder form through a freezer mill, and then powder with a size of 100 μm or less was obtained using a molecular sieve.

[0130] Example 3-2: Analysis of Cell Behavior and Fibrochondrosis Induction Characteristics of Meniscus-Derived Powder by Region on Mesenchymal Stem Cells

[0131] To evaluate the differences in cell behavior regarding mesenchymal stem cells of meniscal tissue-derived powder by region, analyses regarding cell proliferation, migration, and adhesion were conducted. Human bone marrow-derived mesenchymal stem cells (MSCs) 4 x 10⁶ of meniscal tissue-derived powder from each region 6 It was added at a concentration of 1 mg / ml to a 6 cm diameter culture dish seeded with cells and cultured for 1, 7, and 14 days under conditions of 37 ℃ and 5% CO2.

[0132] Figure 7 is a photograph showing the cell affinity of mesenchymal stem cells for each powder derived from the entire meniscus and zones 1 through 3. Specifically, Figure 7(A) is a photograph of surviving (green) or dead (red) mesenchymal stem cells 14 days after treatment with the powder derived from the entire meniscus and zones 1 through 3. That is, a Live & Dead test was performed to confirm cell viability, where green fluorescence indicates surviving cells and red fluorescence indicates dead cells. Figure 7(B) is a graph showing the amount of mesenchymal stem cells proliferated over time by each powder derived from the entire meniscus and zones 1 through 3, expressed as absorbance.

[0133] MSC proliferation was analyzed using the WST assay, and it was confirmed that the group supplemented with meniscal tissue-derived powder significantly promoted cell proliferation up to day 14 of culture compared to the cell culture medium with no additions (Figs. 7(A) and 7(B)). Consequently, as most cells remained alive in Fig. 7(A), almost no red fluorescence signal was observed, confirming that the powders derived from zones 1 through 3 were cell-friendly. The difference in mesenchymal stem cell proliferation according to the meniscal tissue-derived powder by zone showed an increasing trend as one moved from zone 1 to zone 2 to zone 3.

[0134] FIG. 8 is a graph showing the cell migration and adhesion ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, FIG. 8(A) is a graph showing the cell migration ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 8(B) is a graph showing the cell adhesion ability of mesenchymal stem cells according to the control of the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. In FIG. 8, X:Y:Z represents the weight ratio of powder derived from zone 1: powder derived from zone 2: powder derived from zone 3. Furthermore, 'whole' means that the extracellular matrix powder is prepared by decellularizing the meniscus itself.

[0135] A Boyden Chamber assay was performed to evaluate whether the extracellular matrix powder derived from meniscus has biochemical chemotaxis and can promote the migration of stem cells (Fig. 8(A)). As a result, it was confirmed that as the content of the powder derived from zone 1 increased, it promoted cell migration to MSCs.

[0136] A cell adhesion test was conducted to evaluate the difference in cell adhesion affinity between MSCs and the extracellular matrix surface according to the weight ratio of powder derived from zone 1, powder derived from zone 2, and powder derived from zone 3 in the meniscus tissue extracellular matrix powder (Fig. 8(B)). For cell adhesion evaluation, extracellular matrix powders with adjusted content for each zone were suspended at a concentration of 1 mg / ml in an agarose gel and coated onto a culture dish. Subsequently, MSCs were inoculated, and after 2 hours, the culture dish was washed twice with PBS. The cells attached to the agarose were stained with Calcein AM, and the fluorescence intensity was measured.

[0137] As a result, cell adhesion significantly increased as the content of the powder derived from Zone 1 increased.

[0138] Therefore, it was confirmed that there are differences in the cellular behavior of stem cells depending on the anatomical zones of the meniscus tissue according to light transmittance.

[0139]

[0140] To determine the effect of adding meniscal-derived extracellular matrix powder according to zone content on mesenchymal stem cell differentiation, gene expression of type II collagen, Aggrecan, and SOX9 was analyzed via RT-PCR.

[0141] FIG. 9 is a graph showing the ability to induce fibrocartilage differentiation in mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, FIG. 9(A) is a graph showing the amount of COL2 expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 9(B) is a graph showing the amount of ACAN expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. FIG. 9(C) is a graph showing the amount of SOX9 expressed by mesenchymal stem cells by adjusting the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0142] As a result, it was confirmed that as the content of the powder derived from Zone 1 increased compared to the extracellular matrix powder derived from the entire meniscus, it significantly increased the gene expression of Type II collagen (COL2), Agrecan (ACAN), and SOX9, thereby having a specific effect on cartilage differentiation.

[0143] Therefore, it was confirmed that there are differences in the regulation of stem cell differentiation depending on the anatomical zones of meniscal tissue according to light transmittance.

[0144]

[0145] *<Example 4: Analysis of Fibrochondrogenic Differentiation Ability of Extracellular Matrix Powder According to Composition Ratio of Meniscus-Derived Powder by Zone in Mouse Subcutaneous Transplantation Model>

[0146] To determine how extracellular matrix powder according to the composition ratio of meniscus-derived powder by zone affects differentiation into fibrocartilage tissue, histological and biochemical tests were performed in a mouse subcutaneous transplantation model (Fig. 10).

[0147] FIG. 10 is a diagram showing the ability to induce in vivo fibrocartilage differentiation by controlling the content of each powder in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, FIG. 10(A) is a bright field image taken of an artificial tissue created 4 weeks after transplantation in a mouse subcutaneous transplantation model, stained with safranin-O (SO) and type 2 collagen (COL2).

[0148] For histological and biochemical tests, an extracellular matrix powder containing powders derived from menisci of zones 1, 2, and 3 in weight ratios of 1:1:1, 2:1:1, 3:1:1, 5:1:1, 8:1:1, 1:0:0, 1:2:1, 1:3:1, 1:5:1, 1:8:1, 0:1:0, 1:1:2, 1:1:3, 1:1:5, 1:1:8, and 0:0:1, and an extracellular matrix powder prepared from the whole meniscus (indicated as 'whole' in FIG. 10), were mixed with physiological saline at a concentration of 10 mg / ml, and then 1x10 6 200 μl of the artificial tissue was suspended with mesenchymal stem cells (MSCs) and implanted subcutaneously into mice. For histological examination, the artificial tissues created 4 weeks after implantation were stained with safranin-O (SO) and type 2 collagen (COL2) and bright field images were taken (Fig. 10(A)). As a result, it was confirmed that chondrogenesis was induced in the artificial tissues with a higher composition ratio in zone 1, as red Safranin-O staining, which indicates sGAG deposition, and type 2 collagen were most predominantly expressed among all groups.

[0149] To analyze the biochemical characteristics of the artificial tissue created subcutaneously, content analysis of sGAG and type 2 collagen was performed (Figs. 10(B) and 10(C)). Fig. 10(B) is a graph showing the content of sGAG expressed as the content of each powder is controlled in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Fig. 10(C) is a graph showing the content of COL2 expressed as the content of each powder is controlled in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0150] Collagen was measured using the Sirius Red assay (S1000, Biocolor) and sGAG using the DMMB assay (B1000, Biocolor).

[0151] As a result of collagen and sGAG measurements, it was confirmed that the content of type 2 collagen was higher as the content of powder derived from zone 1 increased (Figs. 10(B) and 10(C)).

[0152] <Example 5: Analysis of Fibrocartilage Regeneration Efficacy of Extracellular Matrix Powder According to Weight Ratio of Meniscus-Derived Powder by Zone in Rabbit Meniscus Injury Model>

[0153] To determine the effect of extracellular matrix powder on fibrocartilage tissue regeneration according to the weight ratio of meniscus-derived powder by zone, histological tests were analyzed after transplantation into a rabbit meniscus tissue injury model (Fig. 11).

[0154] Figure 11 is a diagram confirming the fibrocartilage regeneration effect as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Specifically, Figure 11(A) is a bright field image taken after staining rabbit meniscus tissue with safranin-O (SO) and type 2 collagen (COL2) 8 weeks after transplantation into a rabbit meniscus tissue injury model. Figure 11(B) is a graph showing the content of COL2 expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus. Figure 11(C) is a graph showing the content of Scoring expressed as the content of each powder is adjusted in an extracellular matrix powder containing powders derived from zones 1 to 3 of the meniscus.

[0155] For histological testing, extracellular matrix powder containing powders derived from zones 1, 2, and 3 menisci in weight ratios of 1:1:1, 2:1:1, 3:1:1, 5:1:1, 8:1:1, 1:0:0, 1:2:1, 1:3:1, 1:5:1, 1:8:1, 0:1:0, 1:1:2, 1:1:3, 1:1:5, 1:1:8, and 0:0:1, and extracellular matrix powder prepared from the whole meniscus (indicated as 'whole' in Fig. 11) were suspended in physiological saline at a concentration of 10 mg / ml, and 200 μl were implanted into rabbit meniscal injury sites with defects induced to a diameter of 1 mm. Eight weeks after transplantation, rabbit meniscal tissue was obtained and stained with safranin-O (SO) and type 2 collagen (COL2) to take bright field images. As a result, it was confirmed that as the content of powder derived from zone 1 increased, red Safranin-O staining, which indicates sGAG deposition in the meniscus, and type 2 collagen were most predominantly expressed among all groups, indicating regeneration into cartilage tissue (Fig. 11(A)).

[0156] In addition, images stained with type 2 collagen were semi-quantified using ImageJ software to compare the expression levels between groups (Fig. 11(B)). As a result, it was confirmed that the expression level of type 2 collagen increased as the content of powder derived from zone 1 increased.

[0157] Finally, the degree of meniscal regeneration between groups was compared by introducing the Zellner scoring method, which can evaluate the degree of meniscal regeneration based on histological images (Fig. 11(C)). As a result, it was confirmed that the expression of type 2 collagen increased as the content of powder derived from zone 1 increased.

[0158] Therefore, it was confirmed that there are differences in differentiation and regenerative efficacy when transplanted into damaged meniscal tissue depending on the anatomical zone of the meniscal tissue according to light transmittance.

[0159] Overall, it was confirmed that the higher the content of Zone 1, the more it promotes the induction of cartilage tissue differentiation.

Claims

1. An extracellular matrix powder derived from a meniscus composed of zone 1, zone 2 and zone 3, The above-mentioned first zone has a light transmittance of 60% or more and 100% or less based on a thickness of 1 mm or more and 2.5 mm or less, and The above-mentioned second zone has a light transmittance of 48% or more and less than 60% based on a thickness of 1 mm or more and 2.5 mm or less, and The above-mentioned third zone has a light transmittance of 10% or more and less than 48% based on a thickness of 1 mm or more and 2.5 mm or less, and An extracellular matrix powder with enhanced fibrocartilage differentiation ability, comprising 20 weight percent or more of powder derived from the first zone of the above-mentioned meniscus.

2. In Claim 1, An extracellular matrix powder comprising 33.3% by weight or more and 80% by weight or less of powder derived from the first zone of the meniscus.

3. In Claim 2, It contains 10% by weight or more and 33.3% by weight or less of powder derived from the second zone of the above meniscus, and An extracellular matrix powder comprising 10% by weight or more and 33.3% by weight or less of powder derived from the third zone of the meniscus.

4. In Claim 2, An extracellular matrix powder having a weight ratio of 1 to 8:1:1 of powder derived from the first zone of the meniscus; powder derived from the second zone of the meniscus; and powder derived from the third zone of the meniscus.

5. In Claim 1, The moisture content of the above-mentioned first zone is 77.0 %(w / w) or higher and 81.0 %(w / w) or lower, and The moisture content of the above-mentioned second zone is 63.0 %(w / w) or higher and 69.0 %(w / w) or lower, and An extracellular matrix powder having a moisture content of 58.0% (w / w) or more and 67.0% (w / w) or less in the third zone.

6. In Claim 1, The density of the first zone above is 0.95 g / cm³ 3 Above 1.05 g / cm³ 3 It is less than or equal to, The density of the above second zone is 1.16 g / cm³ 3 Above 1.30 g / cm³ 3 It is less than, The density of the above third zone is 1.10 g / cm³ 3 Above 1.15 g / cm³ 3 Extracellular matrix powder that is as follows.

7. In Claim 1, The transformation temperature of the above-mentioned first zone is 75 ℃ or higher and 85 ℃ or lower, and The transformation temperature of the above-mentioned second zone is 100 ℃ or higher and 110 ℃ or lower, and An extracellular matrix powder having a denaturation temperature of the third zone above of 93 ℃ or higher and 98 ℃ or lower.

8. In Claim 1, The above-mentioned first zone is an extracellular matrix powder comprising, per 1 mg of the above-mentioned extracellular matrix powder, 750 μg or more and 850 μg of collagen, 35 μg or more and 55 μg or less of sGAG, 6 μg or more and 10 μg or less of Elastin, 15 pg or more and 25 pg or less of bFGF, 250 pg or more and 310 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 5 ng or more and 10 ng or less of TSP-1.

9. In Claim 1, The above second section is an extracellular matrix powder comprising, per 1 mg of the above extracellular matrix powder, 850 μg or more and 950 μg of collagen, 20 μg or more and 30 μg or less of sGAG, 17 μg or more and 23 μg or less of Elastin, 50 pg or more and 70 pg or less of bFGF, 80 pg or more and 130 pg or less of TGF-β, more than 0 pg and 50 pg or less of VEGF, and 15 ng or more and 25 ng or less of TSP-1.

10. In Claim 1, The above third zone is an extracellular matrix powder comprising, per 1 mg of the above extracellular matrix powder, 750 μg or more and 830 μg of collagen, 10 μg or more and 20 μg or less of sGAG, 20 μg or more and 30 μg or less of Elastin, 65 pg or more and 90 pg or less of bFGF, 30 pg or more and 50 pg or less of TGF-β, 750 pg or more and 1250 pg or less of VEGF, and more than 0 ng and less than 5 ng of TSP-1.

11. A graft material for fibrocartilage tissue reconstruction comprising the extracellular matrix powder of Claim 1.

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