TW201545779A - Method for production of decellularized biological material and the decellularized biological material prepared - Google Patents

Abstract

The invention provides a method for decellularization of a biological material to obtain a decellularized biological material. Compared to untreated biological material, the content of DNA of the decellularized biological material of the invention decreases to a low level and there is no significant reduction of glycosaminoglycan and collagen. The invention also provides a decellularized biological material prepared from the method of the invention and a support comprising the decellularized biological material.

Description

Method for preparing decellularized biological material and decellularized biological material prepared therefrom

The present invention relates to decellularized biomaterials and methods for producing the decellularized biomaterials and their various applications. More specifically, the present invention relates to a decellularized extracellular matrix (ECM) prepared by decellularization with formic acid.

Articular cartilage allows the butt joint surface to move relatively under high load, and the cartilage defect is accompanied by persistent pain and functional limitations of the joint and is therefore considered a serious medical problem. Due to the low metabolic activity and weak proliferation rate of mature chondrocytes, and the lack of blood vessels or lymphatic vessels, the damaged articular cartilage exhibits limited self-regeneration ability. Cartilage transplantation has never been successful due to lack of tissue donated by autologous or allogeneic; in addition, surgical techniques (such as drilling subchondral bone, abrasion arthroplasty or osteochoplasty (mosaicplasty) )) only fibrocartilage and transient relief are produced, but when the repaired tissue fails, it develops into a progressive condition. In view of the serious damage to quality of life, the limits of today's treatment, and the increasing number of patients, there is a high degree of urgency to explore alternative treatment strategies for degenerative cartilage; therefore, this has been the main motivation for cartilage tissue engineering.

Cartilage is a unique extracellular matrix (ECM) that is produced or maintained by chondrocytes. The ECM includes collagen fibers, major type II collagen, and connexins (such as proteins). Proteoglycan, aggrecan, glycosamioglycan (GAG) and hyaluronic acid, which have structural functions to constitute the mechanical properties of cartilage, have feedback regulation on chondrocyte activity and also Become a feature of the chondrocyte phenotype. A variety of natural or synthetic scaffolds have been tested for cartilage engineering in animal models. Natural scaffolds interact with cells via cell surface receptors and regulate signal communication, but they may be mechanically poor and suffer from a variety of enzyme-led degradation. Synthetic polymers are more controllable and predictable by modifying mechanical and chemical properties. However, synthetic polymers do not benefit from direct cell-scaffold interaction unless specifically bound. Therefore, an alternative method is to use decellularized tissue.

The decellularized scaffold acts not only as a supporting substance but also as a supporting substance, such as cell adhesion, proliferation, secretion of ECM, and tissue regeneration by various existing bioactive molecules (eg, ECM, growth factors, and cytokines). The components of ECM are generally reserving between species and are fairly tolerant even to heterogeneous recipients. However, a heterologous or allogeneic cellular antigen is recognized by the host as an exosome, and thus may induce a severe inflammatory response or immune vector rejection. Removal of cells reduces the risk of immune response so that xenogeneic non-cellular ECM scaffolds can be used clinically, but vary widely depending on the type of tissue, the source of the species, and the method of decellularization.

WO 2008/146956 relates to a cell-derived ECM scaffold for the preparation of chondrocytes or stem cells, wherein a detergent, an organophosphorus compound or an interfacial agent is used to achieve decellularization. US 2012/0064043 A1 provides a method of producing a meniscus stent comprising simultaneously removing an unrelated substance with an oxidizing agent and a cleaning agent and increasing the pore size and porosity thereof to decellularize the meniscus tissue and in the biological reaction system Mechanical pressure is applied to the tissue. US 20140038290 A1 discloses an extracellular matrix comprising a modified polysaccharide, the modified polysaccharide consisting of repeating disaccharide units, at least 11% of which are oxidized to a carboxylic acid. US 20140023723 A1 discloses a method of preparing a composition comprising decellularized ECM, wherein 0.5% Triton X-100 The 20 mM ammonium hydroxide phosphate buffer (PBS) was treated at 37 ° C for 5 minutes to form a DM-coated tissue culture substrate, and the cells were isolated from the tissue culture substrate.

Elder et al. evaluated the effects of different decellularization treatments (sodium dodecyl sulfate (SDS), tributyl phosphate, Triton X-100, and hypotonic hypertonic solutions) on articular cartilage and found 2% SDS Treatment for 1 or 2 hours significantly reduced DNA content and maintained biochemical and biomechanical properties ( Elder BD, Eleswarapu SV, Athanasiou KA 2009. Extraction techniques for the decellularization of tissue engineered articular cartilage constructs. Biomaterials 30(22): 3749-3756 ) . In addition, SDS treatment for 6 or 8 hours resulted in complete removal of the nucleus, but the GAG content and compressive properties were significantly reduced at the same time. Thomas et al. ( College Engineering: Part A, Volume 14, Number 4, 2008, pp. 505-518 ) by exposing meniscus to multiple freeze-thaw cycles and cultivating in low tensile trishydroxymethylaminomethane A buffer solution, a 0.1% SDS low-tension buffer solution was added with a protease inhibitor, a nuclease, and a high-tension buffer, and then sterilized with peracetic acid to decellularize the meniscus. This result appears to retain its biomechanical properties (major structural protein) and does not exhibit a major heterologous epitope (galactose-α-1,3-galactose), but unfortunately, GAG has a 59.4% loss. Kheir et al. ( Journal of Biomedical Materials Research A, Nov 2011, Vol. 99A, Issue 2, pp. 283-294 ) applied a freeze-thaw cycle; followed by cycling in low- tension tris -hydroxymethylaminomethane buffer and 0.1 The low buffer of % (w/v) SDS was spiked with a protease inhibitor and nucleic acid plum (ribonuclease and deoxyribonuclease) and sterilized using 0.1% (v/v) peroxyacetic acid. This decellularization method has minimal effect on the collagen content of the cartilage. However, there was a significant decrease in the glycosaminoglycan content of decellularized tissues (98% loss).

Acid treatment is used to deplete cytoplasmic components of cells in a decellularization protocol and to remove nucleic acids ( Crapo PM, Gilbert TW, Badylak SF 2011. An overview of tissue and whole organ decellularization processes . Biomaterials 32(12): 3233-3243 ). However, some acids remove cells from biological materials while destroying the biological function of the biological material. For example, GAG and collagen are significantly reduced ( Xiaochao Dong et al., Mater Med (2009) 20: 2327-2336 ).

Even though several decellularization treatments have been uncovered, there is still a need to develop a decellularization method to obtain an ECM with satisfactory decellularization and strength that is functionally equivalent to untreated biological material. Degree of GAG and collagen.

The present invention provides a method for decellularizing biological material to obtain decellularized biological materials such as ECM, tissues and organs. The DNA content of the decellularized ECM of the present invention was reduced to a lower amount compared to the untreated biomaterial, and the GAG and collagen content were not significantly reduced.

In one aspect, the invention provides a method for producing a decellularized biomaterial comprising providing an untreated biomaterial having cells and efficiently removing cells and nuclear material from the biomaterial with a formic acid solution The biomaterial is treated at a concentration while maintaining a GAG of greater than about 85% compared to the untreated biomaterial. In one embodiment, the method further maintains greater than about 75% collagen compared to the untreated biomaterial. The decellularized biomaterial is preferably a decellularized ECM, tissue or organ.

In some embodiments, the biological material treated by the method of the present invention is skin, heart valve, pericardium, blood vessel, spinal cord, trachea, bladder, ligament, cartilage, meniscus, disc, bone, dura, small intestinal submucosa, spine Meninges, kidneys, liver, lungs or nerves. The tissues and organs treated by the method of the present invention are preferably articular cartilage, meniscus, disc tissue or bone tissue.

In some embodiments, the formic acid concentration in the solution is from about 10% (w/w) to about 100% (w/w), from about 15% (w/w) to about 100% (w/w), about 20% (w/w) to about 100% (w/w), about 30% (w/w) to about 100% (w/w), about 40% (w/w) to about 100% (w/ w), from about 50% (w/w) to about 100% (w/w), from about 60% (w/w) to about 100% (w/w), from about 70% (w/w) to about 100 %(w/w), about 80% (w/w) to about 100% (w/w), about 90% (w/w) to about 100% (w/w), about 60% (w/w) to about 99% (w/w), about 70% (w/ w) to about 99% (w/w), from about 80% (w/w) to about 99% (w/w) or from 90% (w/w) to about 99% (w/w). In one embodiment, the formic acid solution can further comprise other acids or cosolvents. In one embodiment, the biological material is treated with the formic acid solution for less than 15 hours, preferably less than 12 hours, more preferably from 2 to 12 hours. In some embodiments, the ratio of the biomaterial to formic acid is from about 1% (w/v) to about 5% (w/v), from about 丶% (w/v) to about 4% (w/v), From about 1% (w/v) to about 3% (w/v), from about 1% (w/v) to about 2% (w/v), from about 1.5% (w/v) to about 5% (w) /v), 1.5% (w/v) to about 4% (w/v), or 1.5% (w/v) to about 3% (w/v). The ratio of the biomaterial to formic acid is more preferably about 2%.

In one embodiment, the method of the invention removes cells and nuclear material from the biological material while maintaining the amount of GAG. Compared to untreated biomaterials, the DNA content was significantly reduced and the GAG content was not significantly reduced. In one embodiment, the DNA content is reduced to less than about 5% or less while maintaining greater than about 85% of the GAG compared to the untreated biomaterial. Preferably, the method of the invention further maintains greater than about 75% collagen.

The invention also provides a decellularized biomaterial wherein the DNA and GAG content are less than about 5% and greater than about 85%, respectively. In one embodiment, the decellularized biomaterial has greater than about 75% collagen. Preferably, the decellularized biomaterial is a decellularized ECM, tissue or organ. In some embodiments, the decellularized biomaterial of the present invention can be combined as part of one or more supports, such as pharmaceutical compositions, implants, tissue regeneration scaffolds, and medical devices.

The invention also provides a method for preparing an in vitro stent culture system and a method for treating an individual or treatment requiring implantation of a tissue or organ to prevent the risk of having an implanted tissue or organ, comprising using the invention Decellularize biomaterials (preferably decellularized ECM) or scaffolds.

The decellularized biomaterial (preferably decellularized ECM) or scaffold of the invention can be used to treat a defect, injury, injury, disease, aging or ischemic tissue or organ, including (but not limited to) cartilage, disc, articular cartilage, bone, dura mater, meniscus, head, neck, eyes, mouth, throat, esophagus, chest, bones, ligaments, tendons, lungs, colon, rectum, stomach, Prostate, pancreas, breast, ovary, fallopian tube, uterus, cervix, testicular or other reproductive organs, hair follicles, skin, diaphragm, thyroid, blood, muscle, bone marrow, heart, lymph nodes, blood vessels, large intestine, small intestine, kidney, liver , pancreas, brain, spinal cord and central nervous system.

Figure 1. Comparison of DNA, glycosaminoglycan, collagen, and type I and type II collagen of pig meniscus treated with formic acid for 2 hours compared to PBS-treated fresh meniscus. Values are presented as mean ± SD ( n = 5), * p < 0.05.

Figure 2. Giant view of fresh pig meniscus (left side) and decellularized meniscus (right side) treated with formic acid for 2 hours.

Figure 3. Fresh pig meniscus (A, C, E, and G) and meniscus treated with formic acid for 2 hours (B, D, F, and H) via H&E (A and B), Alcian blue Images of (C and D), Masson's trichrome (E and F) and immunohistochemistry (G and H) staining of type II collagen. Scale bar: 100 μm (AH) at 100x magnification and 50 μm (A1-H1) at 400x magnification.

Figure 4. SEM photograph of non-cellular ECM scaffolds on which human chondrocytes were inoculated for 0 days (A), 7 days (B), 14 days (C), 21 days (D), and 28 days (E). Scale bar: 200 μm (AE) at 300x magnification and 100 μm (A1-E1) at 500x magnification.

Figure 5. Fluorescent staining of viable/dead cells when human chondrocytes were cultured for 7 days (A), 14 days (B), 21 days (C) and 28 days (D) in a non-cellular ECM scaffold. Scale bar: 500 μm.

Figure 6. H&E staining images of non-cellular ECM scaffolds seeded on human chondrocytes for 7 days (A), 14 days (B), 21 days (C), and 28 days (D). Scale bar: 100 μm (AD) at 100x magnification and 50 μm (A1-D1) at 400x magnification.

Figure 7. Aers blue stained images of non-cellular ECM scaffolds seeded on human chondrocytes for 7 days (A), 14 days (B), 21 days (C), and 28 days (D). Scale bar: 100 μm (AD) at 100x magnification and 50 μm (A1-D1) at 400x magnification.

Figure 8. Masson trichrome staining images of non-cellular ECM scaffolds seeded on human chondrocytes for 7 days (A), 14 days (B), 21 days (C), and 28 days (D). Scale bar: 100 μm (AD) at 100x magnification and 50 μm (A1-D1) at 400x magnification.

Figure 9. Image of type II collagen immunohistochemical staining of non-cellular ECM scaffolds on human chondrocytes for 7 days (A), 14 days (B), 21 days (C) and 28 days (D). Scale bar: 100 μm (AD) at 100x magnification and 50 μm (A1-D1) at 400x magnification.

Figure 10. Contents of DNA (A), glycosaminoglycan (B), collagen (C) and type II collagen (D) in ECM non-cellular scaffolds from which human chondrocytes were cultured at different time points. Values are presented as mean ± SD (n = 5).

Figure 11. Aggrecan (A), type II collagen (B), type X collagen (C), and type I collagen (D) in decellularized pig meniscus inoculated with chondrocytes at different time points. ) The amount of gene expression. Values are presented as mean ± SD ( n = 3 ).

Figure 12. When bone marrow-derived human mesenchymal stem cells are cultured in monolayers (TCPS) or on non-cellular ECM scaffolds (S) and fed with growth medium (M) or chondrogenic medium (C) for 14 and 21 days. Gene expression levels of aggrecan (A), type II collagen (B), and type I collagen (C). Values are presented as mean ± SD (n = 3).

Figure 13. ECM non-cells stained with bone marrow-derived human mesenchymal stem cells for 21 days and immunohistochemical (C) stained with Ayers blue (A), Masson's trichrome staining (B) and type II collagen. The image of the stand. Scale bar: 100 μm at 100x.

Figure 14. When bone marrow-derived human mesenchymal stem cells are cultured in monolayer (TCPS) or on non-cellular ECM scaffolds (S) and fed with growth medium (M) or chondrogenic medium (C) for 14 and 21 days. Gene expression levels of aggrecan (A), type II collagen (B), and type I collagen (C). Values are presented as mean ± SD (n = 3).

Figure 15. Comparison of DNA and glycosaminoglycan content of pig meniscus treated with formic acid for different cycles compared to PBS treated fresh meniscus.

Figure 16. DNA content of pig meniscus treated with different concentrations of formic acid for 2 or 4 hours.

Figure 17. Comparison of the DNA content of pig skin after different decellularization treatments with fresh skin treated with PBS. Values are presented as mean ± SD (n = 3).

Figure 18. Contents of DNA (A), collagen (B) and glycosaminoglycan (C) in pig meniscus after different decellularization treatments at different time points with fresh PBS-treated meniscus Comparison. Values are presented as mean ± SD ( n = 3 ).

The present invention develops a method for decellularizing biological materials to obtain decellularized biological materials such as ECM, tissues and organs; the biological material is preferably cartilage, articular cartilage, meniscus, bone or skin. The decellularization method of the present invention has minimal adverse effects on the biomaterial (preferably ECM) used to prepare non-cellular scaffolds. The DNA content of the decellularized biomaterial of the present invention is reduced to a lower amount and the GAG and collagen content are not significantly reduced as compared to the untreated biomaterial. The decellularized biomaterial of the present invention not only provides temporary habitat, but also has different physiological functions and a native environment to promote cell proliferation and new tissue formation.

Various aspects of the invention are described in further detail in the following subsections. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise specified. Examples of suitable methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety.

As used herein, the singular forms "" For example, the term "a cell" includes a single cell as well as a plurality of cells, including mixtures thereof.

The term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items, and does not have a combination when interpreted as an alternative ("or").

As used herein, the term "about" when referring to a measurable value (such as a quantity or concentration and the like) means 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount. Variety.

As used herein, the term "GAG" means glycosaminoglycan.

As used herein, the terms "scaffold" and "matrix" are used interchangeably to refer to a natural or synthetic structure or a network having an open porosity that extends spatially and provides mechanical or other means for growth of living tissue in vivo or in vitro. support.

As used herein, the terms "extracellular matrix" and "ECM" refer to natural or artificial scaffolds for cell growth. Natural ECM (ECM found in multicellular organisms such as mammals and humans) is a complex mixture of structural and non-structural biomolecules including, but not limited to, collagen, elastin, laminin, glucosamine Glycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines and growth factors. In mammals, ECM typically contains about 90% collagen in a different form.

As used herein, the term "decellularization" refers to the partial removal of cells and related fragments from one of tissues or organs (eg, from an ECM).

As used herein, the term "individual" refers to an animal, a mammal or, in particular, a human patient.

As used herein, the term "treatment" means to ameliorate the condition of an individual by reducing, alleviating, reversing or preventing at least one adverse effect or symptom.

In one aspect, the invention provides a method for producing decellularized biological material a method comprising: providing an untreated biological material having cells and treating the biological material with a formic acid solution at a concentration effective to remove cells and nuclear material from the biological material while maintaining a higher than untreated biological material About 85% of GAG. In one embodiment, the method of the invention further maintains greater than about 75% collagen compared to untreated biomaterial.

In one embodiment, the biological material to be treated by the method of the present invention is tissue and organs. The tissue or organ is preferably a skin, heart valve, pericardium, blood vessel, spinal cord, trachea, bladder, ligament, cartilage, meniscus, disc, bone, dura mater, submucosa of the small intestine, spinal meninges, kidney, liver, lung or nerve. The tissues and organs treated by the method of the present invention are more preferably articular cartilage, meniscus, disc tissue or bone.

In one embodiment, the decellularized biomaterial of the invention is a decellularized ECM, a decellularized tissue, or a decellularized organ. The tissue or organ line is as mentioned above.

In one embodiment, the concentration of formic acid in the solution ranges from about 10% (w/w) to about 100% (w/w). The formic acid concentration is preferably from about 10% (w/w) to about 100% (w/w), from about 15% (w/w) to about 100% (w/w), about 20% (w/w) to About 100% (w/w), about 30% (w/w) to about 100% (w/w), about 40% (w/w) to about 100% (w/w), about 50% (w) /w) to about 100% (w/w), about 60% (w/w) to about 100% (w/w), about 70% (w/w) to about 100% (w/w), about 80% (w/w) to about 100% (w/w), about 90% (w/w) to about 100% (w/w), about 60% (w/w) to about 99% (w/ w), 70% (w/w) to about 99% (w/w), 80% (w/w) to about 99% (w/w) or 90% (w/w) to about 99% (w) /w).

In one embodiment, the formic acid solution may further comprise a cosolvent. The cosolvent preferably comprises any one of glycerin, ethanol, 1-propanol, polyethylene glycol 600, or a mixture thereof.

In one embodiment, the formic acid solution may further comprise an acid solution. The acid solution preferably comprises any one of acetic acid, peroxyacetic acid or citric acid or a mixture thereof.

In one embodiment, the ratio of biomaterial to formic acid is from about 1% (w/v) to about 5% (w/v), from about 1% (w/v) to about 4% (w/v), about 1% (w/v) to about 3% (w/v), about 1% (w/v) to about 2% (w/v), about 1.5% (w/v) to about 5% (w/v), 1.5% (w/v) to about 4% (w/v) or 1.5% (w/v) to about 3% (w/v). The ratio of biological tissue to formic acid is preferably from about 1% (w/v) to about 3% (w/v). The ratio of biological tissue to formic acid is preferably about 2% (w/v).

In one embodiment, the biomaterial is treated with a formic acid solution for less than 15 hours, preferably less than 12 hours. The treatment time is preferably less than any of about 1 hour to about 14 hours. The treatment time is more preferably from about 1 hour to about 14 hours, from about 1 hour to about 13 hours, from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about 1 hour to about 10 hours, from about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours , about 1 hour to about 2 hours, about 2 hours to about 15 hours, about 2 hours to about 14 hours, about 2 hours to about 13 hours, about 2 hours to about 12 hours, about 2 hours to about 11 hours, about 2 hours to about 10 hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours To about 4 hours or about 2 hours to about 3 hours.

In one embodiment, the method of the invention removes cells and nuclear material from the biomaterial while maintaining the amount of GAG. In one embodiment, the method of the invention further maintains collagen. Compared to untreated biomaterials, DNA content was significantly reduced and GAG and collagen levels were not significantly reduced. In one embodiment, the DNA content is reduced to less than about 5% or less compared to the untreated biomaterial. Preferably, the DNA content is reduced to less than about 4.5%, less than about 4.1%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%. Less than about 0.8% or less than about 0.5%. In one embodiment, the DNA content is reduced to from about 0.1% to about 5% compared to the untreated biomaterial; preferably to about 0.1% to about 5.0%, from about 0.1% to about 4.5%, to about 0.1. % to about 4.0%, from about 0.1% to about 3.0%, from about 0.4% to about 5.0%, from about 0.4% to about 4.5% or from about 0.4% to about 4.0%.

In one embodiment, after processing the biological material by the method of the present invention, GAG above about 85% can be maintained compared to treated biomaterials. Preferably, it is maintained above about 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, compared to the untreated biomaterial. 98% or 99% GAG; more preferably more than about 90%, 91%, 92%, 93%, 94% or 95% GAG. In one embodiment, from about 85% to about 99% of the GAG can be maintained. Preferably, it is maintained from about 85% to about 98%, from about 85% to about 97%, from about 85% to about 96%, from about 85% to about 95%, from about 85% to about 94%, from about 90% to about 99. %, about 90% about 98%, about 90% to about 97%, about 90% to about 96%, about 90% to about 95% or about 90% to about 94% of GAG.

In one embodiment, greater than about 75% of collagen can be maintained as compared to untreated biomaterial. Preferably, it can be maintained above about 76%, above about 77%, above about 78%, above about 79%, above about 80%, above about 82%, above about 84%, above about 86. %, above about 88%, above about 90%, above about 92%, above about 94%, above about 96%, above about 97%, above about 98% or above about 99% Collagen. In one embodiment, about 75% to 99%, about 77% to 99%, about 79% to 99%, about 80% to 99%, about 80% to 95%, or about 80% to 90% can be maintained. Collagen.

In one embodiment, the method of the present invention further comprises a pretreatment step prior to processing the biological material. In some embodiments, the pretreatment includes, but is not limited to, washing, disinfecting, separating, homogenizing, vibrating, sonicating, ultrasonic processing, perfusion, mechanical massage, pressurization, comminution, agitation, or agitation.

In one embodiment, the method of the invention further comprises the steps of physical decellularization, chemical decellularization or physical decellularization combined with chemical decellularization prior to processing the biological material. Physical decellularization preferably includes, but is not limited to, freeze-thaw, ultrasonic treatment, perfusion, or physical agitation. Chemical decellularization preferably includes, but is not limited to, an acidic solution, a hypotonic solution, an anionic, nonionic or cationic detergent, or an enzyme (deoxyribonuclease, ribonuclease or trypsin).

In one embodiment, the method of the present invention further comprises after processing the biological material Contains a washing step. Washing is a conventional wash of the decellularized matrix by perfusion or dialysis using physiological buffer, phosphate buffered saline or culture medium containing antibiotics. In one embodiment, the washing step is carried out for half a day to seven days or longer.

In another embodiment, after processing the biological material, the method of the present invention further comprises the step of forming a decellularized biomaterial as a scaffold by mixing the decellularized biomaterial with/or without a porogen, It is poured into a mold for forming the stent, and then the formed stent is freeze-dried.

In another aspect, the invention provides a decellularized biomaterial having a DNA and GAG content of less than about 5% or less and greater than about 85%, respectively, compared to an untreated biomaterial. In one embodiment, the decellularized biomaterial is maintained above about 75% compared to the untreated biomaterial. Preferred amounts of DNA, GAG and collagen are described herein. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. The tissue or organ line is as mentioned above.

In some aspects, the decellularized biomaterial of the invention (such as a decellularized ECM, tissue or organ) is associated with a support. In particular, herein includes methods for formulating decellularized biomaterials into one or more types of supports, such as pharmaceutical compositions, value-adding materials, tissue regeneration scaffolds, and medical devices. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. The tissue or organ line is as mentioned above.

The invention also includes methods for making and implanting decellularized biomaterials using the invention. The implant can be non-limiting.

In some aspects, the support is a biocompatible material such as a tissue regeneration scaffold. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. Decellularized tissues or organ lines are as mentioned above. One aspect of the invention provides for the incorporation of decellularized biomaterial into a biocompatible material for implantation into an individual. In one embodiment, the biocompatible material is in the form of a stent. The scaffold can be natural collagen, Decellularized, modified extracellular matrix, or synthetic polymer. In certain embodiments, the scaffold is used as a template for cell proliferation and ultimately tissue formation. In a particular embodiment, the scaffold allows for slow release of the decellularized extracellular matrix into the surrounding tissue. When the cells in the surrounding tissue begin to multiply, they fill the scaffold and grow into a three-dimensional tissue.

In some aspects, the support comprises a medical device. Decellularized biomaterials can be used to form medical devices or prosthetic devices in an implantable individual. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. Decellularized tissues or organ lines are as mentioned above. In one embodiment, the decellularized biomaterial can be incorporated into the substrate material needed to make a medical or prosthetic device. In another embodiment, the decellularized biomaterial can be used to coat or cover a medical or prosthetic device. Medical and prosthetic devices can be inserted or implanted into the patient's body.

In certain embodiments, the decellularized biomaterial can be used to treat a defect, injury, injury, disease, aging, or ischemic tissue or organ including, but not limited to, cartilage, disc, articular cartilage, bone, hard Membrane, meniscus, head, neck, eyes, mouth, throat, esophagus, chest, bones, ligaments, tendons, lungs, colon, rectum, stomach, prostate, pancreas, breast, ovary, fallopian tubes, uterus, cervix , test pills or other reproductive organs, hair follicles, skin, diaphragm, thyroid, blood, muscle, bone marrow, heart, lymph nodes, blood vessels, large intestine, small intestine, kidney, liver, pancreas, brain, spinal cord and central nervous system. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. The tissue or organ is preferably a skin, heart valve, pericardium, blood vessel, spinal cord, trachea, bladder, ligament, cartilage, meniscus, disc, bone, dura mater, submucosa of the small intestine, spinal meninges, kidney, liver, lung or nerve.

For example, for the treatment of defects, injuries, injuries, illness, aging or ischemic cartilage or skin, the decellularized biomaterial scaffold can be implanted into the injury site of the cartilage or by disease or age in a single operation. In the defect area caused.

For example, for the treatment of osteochondral defects, due to its anatomical arrangement, which is soft The subosseous bone is positioned directly beneath the injured cartilage and is damaged by cartilage damage and subchondral or subchondral bone damage, and thus is an extension of the methods described herein for treating cartilage lesions. A true bone defect, lesion, or fracture is a single lesion in the skeleton bone.

For example, articular cartilage is a unique tissue without a blood vessel, nerve or lymph supply. In addition to the formation of fibrous or fibrocartilageous tissue, the lack of blood vessels and lymphatic circulation can be a cause of why articular cartilage has such a weak healing ability. The unique mechanical function of articular cartilage never spontaneously recovers after significant damage, aging wear or disease, such as osteoarthritis (OA). The decellularized ECM scaffold of the present invention can be implanted into the defect articular cartilage for articular cartilage regeneration.

In one aspect, the invention provides a method for preparing an in vitro stent culture system, the method comprising (i) providing a decellularized biomaterial scaffold of the invention, (ii) a perfused cell population, including stem cells, progenitor cells Or a differentiated progenitor cell capable of differentiation, or a cell population capable of functional maturation for decellularization of a biomaterial scaffold, and (iii) re-cell differentiation and stem or progenitor cells in a perfusion decellularized biomaterial scaffold The perfusion decellularized biomaterial scaffold is contacted with the cell population for a period of time under conditions of differentiation and functional maturation or functional maturation of cells in the population. Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. Decellularized tissues or organ lines are as mentioned above. Preferably, the method further comprises providing a physiologically active substance capable of inducing differentiation of the cells into an organism. The physiologically active substance is selected from the group consisting of insulin-like growth factor (IGF), fibroblast growth factor (FGF), tissue growth factor (TGF), bone morphogenetic protein (BMP), nerve growth factor (NGF), Platelet-derived growth factor (PDGF) and tumor necrosis factor (TNF).

In another aspect, the invention provides a method for producing a tissue graft comprising the steps of: a) providing a decellularized biomaterial scaffold of the invention; b) allowing cells in the organism to infiltrate the decellularized organism In the material holder; c) cultivating the tissue in the organism for a time sufficient to differentiate the cells; and d) providing a physiologically active substance capable of inducing cell differentiation.

Decellularized biological material is preferably decellularized ECM, decellularized tissue or decellularized organ. Decellularized tissues or organ lines are as mentioned above.

The present invention uses formic acid to produce a protocol for decellularization of biological materials, preferably meniscus. The success of decellularization is determined by the lack of cells and the retention of ECM compared to untreated biomaterials. Initial in vivo biocompatibility studies of decellularized biomaterials seek to establish the possible clinical utility of cartilage regeneration. For example, after inoculation of human chondrocytes in non-cellular scaffolds, the content of DNA, GAG, collagen and type II collagen increased by 10.10 times, 7.11 times, 4.24 times and 5.11 times in the fourth week. An increase in the gene expression of type II collagen and aggrecan further confirmed the maintenance of the chondrocyte phenotype. In addition, when human bone marrow-derived mesenchymal stem cells cultured in a chondrogenic medium have signs of increased chondrogenesis markers, it is demonstrated that the scaffold effectively supports chondrogenesis. Finally, in vivo implantation was performed in rats to analyze biocompatibility. Non-cellular scaffolds not only provide temporary habitat, but also have different physiological functions and native environment to promote cell proliferation and new tissue formation.

The invention is further illustrated by, but not limited to, the following examples.

Instance

Example 1 Decellularization of meniscus with formic acid

The meniscus was collected from the adult pig knee joint, washed with phosphate buffered saline (PBS), and then freeze-dried (Eyela FD-5N, Tokyo, Japan). The lyophilized meniscus was finely disrupted and 0.2 g of meniscus was suspended and suspended in 10 mL of formic acid (>99%, Sigma-Aldrich, St. Louis, MO, USA) for 2 hours. The suspension is then homogenized and dialyzed. After removing excess acid and cell debris by dialysis, the meniscus slurry was placed in a cylindrical mold and lyophilized to make a stent. The stents were sterilized by ethylene oxide and subsequently degassed to analyze the features.

Characterization of non-cellular ECM scaffolds

Biochemical analysis

To measure the amount of DNA, GAG and collagen, the sample was first treated with papain solution at 60 ° C (in 150 mM sodium chloride, 55 mM sodium citrate. 2H ₂ O, 5 mM cysteamine hydrochloride and 5 mM sodium EDTA). Digestion of .6H/mL in .2H ₂ O for 16 hours. The double-stranded DNA content was measured by a PicoGreen Quantitative Kit (Molecular Probes, Eugene, OR, USA) and the number of cells was estimated using the previously reported value of 7.7 pg of DNA per chondrocyte. Fluorescence of the negative, DNA-free control group was subtracted from the fluorescence of the experimental group to obtain fluorescence of the individual scaffolds. The amount of GAG was determined by the Blyscan ^® GAG assay kit (Biocolor, Newtonabbey, UK) according to the manufacturer's instructions. The sample lysate with Blyscan ^® dye reagent are mixed and then collected by centrifugation GAG- dye complex. After removing the supernatant and draining the tube, the dissociation agent was added. The solution was then transferred to a 96 well plate and the absorbance relative to the background control was obtained by a microplate spectrophotometer at a wavelength of 656 nm. Chondroitin sulfate was used as a standard to calculate the concentration of GAG in the specimen.

The papain digest was further acidified and reacted with chloramine-T and p-dimethylaminobenzaldehyde solution to determine the amount of hydroxyproline. The hydroxyproline content was converted to collagen content using a mass ratio of 7.25. The optical density was detected at 560 nm by a microdisk reader. A standard curve was established using a range of concentrations of hydroxyproline. The amount of type I and type II collagen was studied by ELISA using Type I and Type II collagen detection kits (Chondrex Inc., Redmond, WA, USA) as described by the supplier. Briefly, samples were digested overnight by pepsin solution at 4 °C with gentle mixing for 48 hours and then mixed with pancreatic elastase solution overnight at 4 °C. The ELISA reaction optical density was read at 490 nm in a microdisk reader.

Histological and immunohistochemical staining

The meniscus was fixed overnight in 10% (v/v) neutral buffered formalin before and after decellularization, dehydrated via a series of ethanol and embedded in paraffin. The cross section of the 5-10 μm thick specimen was stained with hematoxylin and eosin (H&E) to observe the nucleus and stained with Ayers blue. The collagen was observed by observing GAG deposition or staining with Masson's trichrome staining. Immunization of type II collagen by anti-type II collagen antibody (bs-0709r, Bioss, MA, USA) diluted 1:100 in TBST solution (trishydroxymethylaminomethane buffered saline and Tween20) The signals were positioned and amplified using an UltraVision Quanto detection system (Thermo Scientific, MA, USA) for 10 minutes, followed by observation by DAB according to the manufacturer's instructions. The nuclei were contrast-stained with hematoxylin. Slides were scanned with ScanScop (Aperio, CA, USA).

In vitro study

In vitro cytotoxicity

The sterilized scaffold was incubated in serum-containing medium for 24 hours at 37 °C. 150 μL of scaffold extract was collected for in vitro cytotoxicity testing. NIH 3T3 mouse fibroblasts were seeded in 96-well plates at a seeding density of 1 × 10 ⁴ cells/well and incubated overnight at 37 °C. The medium was replaced with extract or fresh medium and incubated for an additional 24 hours. After the incubation period, the extract was replaced with fresh medium solution containing alamar blue (Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated for an additional 2 hours. The fluorescence intensity of the Alamar Blue/medium mixture was measured by a microdisk reader at an excitation wavelength of 560 nm and an emission wavelength of 620 nm. The fluorescence value is proportional to the number of viable cells and corresponds to cellular metabolic activity.

In vitro chondrocyte research

After formation of the confluent cell layer, human primary chondrocytes (ScienCell Research Laboratories, Carlsbad, CA, USA) were isolated using 0.025% trypsin plus EDTA-containing PBS and resuspended in supplemental medium (ScienCell Research Laboratories, Carlsbad, CA, In USA) and used for in vitro experiments. Prior to cell seeding, the scaffolds were each immersed in PBS and medium for 5 minutes, and excess liquid was removed by sterile tissue paper and then placed in a 96-well plate. A chondrocyte suspension (10 μL) (1 × 10 ⁵ cells) was directly dropped into the scaffold using a micropipette aid (pippetman). The cells/branches were placed in an incubator (37 ° C, 5% CO ₂ ) for 2 hours for cell adhesion, followed by the addition of 0.2 ml of fresh growth medium, and the medium was changed every three days. Cell-free scaffolds were also cultured in a similar manner and considered blank, and the values were subtracted from all assays to offset interference.

Live cell/dead cell staining

At week 1, week 2, week 3, and week 4, the cells/branches were washed with sterile PBS and analyzed by survival/death assay kits (Molecular Probes, Eugene, OR, USA) with 8 μM calcein- AM (calcein-AM) was incubated with 4 μM ethidium homodimer-1. Sections were immediately examined using an FITC/Texas red filter by an inverted fluorescent microscope attached to a confocal imaging system.

Scanning electron microscopy (SEM)

After a desired period of incubation, the attachment of chondrocytes was observed by SEM. The cells were fixed with 2.5% (w/v) glutaraldehyde/PBS, washed with PBS (pH 7.4), fixed with 1% (w/v) osmium tetroxide, and then used in a series of increasing concentrations. Dehydration of ethanol. The sample was then subjected to critical point drying and coated with gold before observation by SEM (Hitachi-2400, Tokyo, Japan).

Complete RNA extraction and real-time PCR

To quantify the gene expression of aggrecan, type I, type II, and type X collagen, according to the standard TRIzol (Invitrogen Life Technologies, Carlsbad, USA) protocol, at week 1, week 2, week 3, and Complete cellular RNA was extracted from chondrocytes grown on the scaffold for 4 weeks. The concentration and purity of the extracted RNA were evaluated by a NanoDrop ND-1000 spectrophotometer with UV absorption. Only samples exhibiting a ratio of 260 nm/280 nm of >1.8 were used in the following experiments. The isolated mRNA was transcribed into cDNA by the ReverTra Ace kit (Toyobo, Osaka, Japan).

The gene expression of aggrecan, type I, type II and type X collagen was quantified by real-time PCR on a LightCycler 480 system (Roche Applied Science, Indianapolis, USA). The primer was designed by the Human Probe Library's Roche Universal Probe Library Assay Design Center (www.roche-applied-science.com), and the human probe library No. 9 was used. And probe No. 80 (Roche Applied Science, Indianapolis, USA). Specific primers for aggrecan (NM_001135, positive: 5'-cagatggacacccccgg-3-3 and reverse: 5'-cattccactcgcccttctc-3'), type I collagen specific primer (NM_000088, positive: 5 '-gccaacctggtgctaaagg-3' and reverse: 5'-caggagcaccaacattacca-3'), type II collagen specific primer (NM_001844, positive: 5'-gtgaacctggtgtctctggtc-3' and reverse: 5'-tttccaggttttccagcttc-3 ') and type X collagen specific primer (NM_000493, positive: 5'-agcttcagaaagctgccaag-3' and reverse: 5'-gcagcatattctcagatggattct-3') amplify the target gene. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene (NM_002046, positive: 5'-acgggaagcttgtcatcaat-3' and reverse: 5'-catcgccccacttgatttt-3'), and all target genes were The performance is corrected to the GAPDH performance of the sample. For each reaction, a LightCycler Taqman Master kit (Roche Applied Science, Indianapolis, IN, USA) was used with 10 μL total volume of 400 nM primers, 0.2 μL probe and 900 ng cDNA. All amplifications were performed in LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) under the following conditions: preheating at 95 °C for 1 cycle for 15 minutes; amplification for 45 cycles: maintaining at 95 °C for 10 cycles Seconds, held at 58 ° C for 30 seconds, held at 72 ° C for 3 seconds; and finally cooled to 40 ° C. The relative amount of the target gene was quantified using a comparative C _T method based on the target cycle and the threshold cycle (C _T ) value of the housekeeping gene.

Chondrogenic bone marrow-derived human mesenchymal stem cells

Bone marrow-derived hMSC lines were purchased from the Texas A&M University Health Science Center (Temple, TX, USA). hMSC was cultured in a monolayer at 37 ° C, 5% CO ₂ with hMSC growth medium (Lonza, Walkersville, MD, USA) and the medium was changed every 3 days. After formation of the confluent cell layer, hMSCs were isolated using 0.05% trypsin plus EDTA (Lonza), resuspended in hMSC growth medium, and used for the following chondrogenesis induction experiments. The hMSC suspension (10 μL; 10 ⁵ cells) was directly dropped into a 96-well plate (tissue culture polystyrene [TCPS] group) or a scaffold in a 96-well plate using a dropper. The TCPS and the cell/support architecture were placed in an incubator for 2 hours for cell adhesion, followed by the addition of 0.2 mL of fresh medium. For chondrogenesis, add hMSC cartilage containing ITS+ supplements, dexamethasone, ascorbate, sodium pyruvate, proline, GA-1000, L-glutamic acid, and transforming growth factor (TGF)-β3 forming SingleQuots ^TM media (Lonza) for 14 and 21 days. The control group was maintained with hMSC growth medium (Lonza). All TCPS and cells/scaffolds were maintained at 37 ° C, 5% CO ₂ in a moisture containing incubator and the medium was changed every 3 days.

Cells were harvested at predetermined time points and subjected to histological, biochemical, and real-time PCR quantitative analysis.

In vivo immunobiocompatibility study

Fifteen male Sprague Dawley rats, aged 8 weeks to 10 weeks, were used. After anesthesia with isoflurane (Abbott, Queenborough, UK) and disinfection with iodophor and 70% ethanol, each rat was subcutaneously inserted into a small stent plug while three rats were treated as a sham treatment group. A 1 cm incision was made on the back of each rat using blunt dissection and four subcutaneous pockets were prepared. The stents were carefully placed in separate pockets and the wounds were closed with wound closure forceps and the animals were placed back into the enclosure facility where the animals were free to eat and drink. Weigh once a week and check the health of the rats. At week 1, week 2 and week 4, rats were sacrificed and specimens with constructs were collected and stained with H&E to assess whether an inflammatory response occurred. Because the ECM stent is not visible after implantation, a large enough area is removed to ensure that the stent remains in the specimen.

Figure 1 shows a significant reduction in the amount of DNA to 4.10% (p < 0.01) compared to fresh pig meniscus, indicating the efficiency of the decellularization process (formic acid treatment for 2 hours). The ECM content of the scaffold is also determined by individual measurements of GAG, total collagen, and type I and type II collagen. the amount. There was only a 7.16% loss of GAG content between the fresh meniscus and the non-cellular meniscus (p=0.087). The amount of collagen, type I and type II collagen in the non-cellular ECM scaffold decreased, but remained >80% (p=0.714, 0.092 and 0.087).

After decellularization of the meniscus for 2 hours by formic acid, a sponge-like porous scaffold was made; macroscopic photographs are shown in the right side of Figure 2, and freshly compacted pig meniscus is shown in the left side of Figure 2.

Decellularization of the scaffold and ECM maintenance (formic acid treatment for 2 hours) were further confirmed by histology. In fresh pig meniscus, H&E staining (Fig. 3(A) and Fig. 3(A1)) indicates that the chondrocytes are round and embedded in the cavity. In contrast, no cells or cell debris were present after decellularization, as shown in Figure 3 (B) and Figure 3 (B1). Aers blue staining gave positive results on fresh meniscus and decellularized meniscus, indicating residual GAG after decellularization (Figure 3 (C), Figure 3 (C1), Figure 3 (D), and Figure 3 (D1)). Stained by Masson's trichrome staining, collagen is present as a major tissue structure in fresh pig meniscus and decellularized scaffolds (Figure 3 (E), Figure 3 (E1), Figure 3 (F), and Figure 3 (F1). )). Figures 3(G), 3(G1), 3(H) and 3(H1) show the appearance of type II collagen after immunohistochemistry. We have demonstrated that formic acid treatment for 2 hours is capable of efficiently removing all cellular material while minimizing any adverse effects on the composition and amount of residual ECM.

In vitro cytotoxicity test

The absorbance obtained by Alamar Blue analysis is directly related to the metabolic activity of the cells and inversely proportional to the toxicity of the scaffold. After two days of culture, the fluorescence values of NIH 3T3 mouse fibroblasts inoculated in a monolayer (mean ± SD: 33134.42 ± 2573.10, n = 6) and NIH 3T3 mice inoculated in a non-cellular ECM scaffold There was no significant difference between the fluorescence values of fibroblasts (mean ± SD: 35195.14 ± 5417.12, n = 6) (p = 0.167), indicating that the non-cellular ECM scaffold was not cytotoxic.

Chondrocyte attachment and vitality

Human primary chondrocytes were seeded in non-cellular ECM scaffolds to assess cell proliferation and ECM production. The morphology and distribution of chondrocytes in the scaffolds were monitored by SEM throughout the experimental period. Figure 4 shows chondrocytes with circular or elliptical morphology attached to the well of the stent. Higher magnification shows the ECM-like components of the above captured cells and some cells have been found to migrate and attach to the interconnected pores. It was shown that the chondrocyte line was evenly distributed in the porous acellular scaffold and maintained its morphology even until the 28th day.

Regarding live cell/dead cell staining (Fig. 5), calcein AM is able to penetrate the cell membrane of living cells, which is cleaved by intracellular esterase and produces green fluorescence. Ethidium bromide homodimer-1 is capable of entering cells with damaged cell membranes and binding to fragmented nucleic acids, thereby producing red fluorescence in dead cells. On day 7, except for a few dead cells, most cells were stained with green fluorescence. Then look at the green living cells, and there are very few red dead cells. These results indicate that human chondrocytes exhibit high viability when cultured in a non-cellular ECM scaffold.

Histological and biochemical analysis of chondrocytes was performed to confirm the specific morphology of chondrocytes and ECM secretions that occurred throughout the study period, and cell/support architecture was assessed by histological staining. H&E staining was performed to observe the morphology, distribution, and viability of cells in the non-cellular ECM scaffold, and a number of uniformly distributed circular chondrocytes were shown in FIG. As shown in Figures 7-9, the significant deposition and accumulation of ECM components in the body is evident through the Aels Blue, Masson trichrome staining and immunohistochemical staining, indicating that a larger amount of GAG is synthesized separately. Collagen and type II collagen.

Measurement of DNA, GAG and collagen allows us to quantify chondrocyte proliferation and ECM synthesis. After inoculation of chondrocytes on days 14, 21, and 28, the DNA content showed an increase of 1.03, 1.89, and 2.62 (p < 0.01), respectively (Fig. 10(A)), and it indicates an increased number of cells. . On day 28, the total number of cells reached 10.10 × 10 ⁵ th, which corresponds to 10.10 times the month increased. Regarding GAG secretion, on days 7, 14, 21, and 28, the cells grown on the scaffold showed a significant increase of 1.05, 3.20, 5.85, and 7.11, respectively, compared to the blank scaffold ( p < 0.01) (Fig. 10 (B)). In Figure 10(C), the amount of collagen also showed similar increases of 1.06, 2.43, 3.39 and 4.24 at different time points. Regarding type II collagen, the content was also increased to 1.75 times, 3.82 times, 4.43 times, and 5.11 times (p < 0.01) over the entire study period as compared with the blank scaffold (Fig. 10(D)). In contrast, the amount of type I collagen (negative marker) was below the quantitative lower limit (LLOQ, <0.08 μg/mL). The increasing content of GAG, collagen and type II collagen is consistent with the amount of DNA; and these results correspond to the results of light microscopy histology and SEM observation.

Quantitative real-time PCR

To assess cartilage differentiation of chondrocytes seeded in non-cellular ECM scaffolds, changes in cartilage formation markers were determined by real-time PCR on days 7, 14, 21 and 28, respectively (Figure 11). Two positive markers of chondrogenesis, aggrecan and type II collagen were significantly increased (p < 0.01). In contrast, no significant changes in the negative markers of cartilage formation (type I and type X collagen) were observed (p = 0.155 and 0.245). The results of gene expression, together with histological staining and biochemical quantification, indicate that human chondrocytes maintain their normal phenotype and cartilage-like tissue is engineered when cultured in a non-cellular ECM scaffold.

Chondrogenic bone marrow-derived human mesenchymal stem cells

GAG, complete collagen, and type II collagen were used as markers for chondrogenic differentiation, and Figure 12 shows that when hMSCs were seeded on non-cellular ECM scaffolds or TCPS and cultured with growth medium or chondrogenic medium, day 14 and The relative ratio of the average amount of ECM at 21 days. Compared to hMSCs seeded on TCPS, hMSCs cultured on non-cellular ECM scaffolds and cultured with chondrogenic medium synthesize higher amounts of GAG, collagen and type II collagen. Histological images of aggrecan and type II collagen (Fig. 13) and increased gene expression (Fig. 14) confirmed consistent results for chondrogenic differentiation, while type I collagen (marker for dedifferentiation of chondrocytes) was observed. It is slightly down. In addition to human chondrocytes, non-cellular ECM scaffolds similarly provide signals to drive hMSCs toward chondrogenesis.

In vivo study

Non-cellular ECM scaffolds were implanted subcutaneously into rats for 7 days, 14 days, and 28 days to determine if the scaffold was biocompatible. No animals died during the experimental period. The immunogenic antigen in the non-cellular tissue is also removed as the cells are completely removed from the scaffold. Non-cellular ECM scaffolds are capable of allowing new tissue to form and are well tolerated by the host without adverse reactions, suggesting that allogeneic decellularized scaffolds can be an ideal source of cartilage engineering.

Example 2 to Example 7 Decellularization of meniscus with formic acid at different times

The scaffolds of Examples 2 and 7 were decellularized according to the method described in Example 1, wherein formic acid treatment was used as shown in Table 1 for different times (2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours). ). The minced meniscus was immersed in PBS for 12 hours and served as a control group. The suspension was homogenized at a predetermined time point, dialyzed, and then lyophilized to make a scaffold. The success of decellularization was determined by biochemical analysis comparing the amount of DNA and major ECM (GAG and collagen). Figure 15 shows that the longer the formic acid treatment time, the more effective the decellularization shown.

Examples 8 to 14 Decellularization of meniscus with formic acid at various concentrations

The scaffolds of Examples 8 and 14 were decellularized according to the method described in Example 1, using different concentrations of formic acid (12.5%, 25%, 37.5%, 50%, 62.5%, 75%, 87.5%) for 2 hours. Or 4 hours (see Table 1). Figure 16 shows that the higher the concentration of formic acid or the longer the treatment time, the more effective the decellularization shown.

Example 15 Making a decellularized skin scaffold

Skin was collected from adult pigs and washed by PBS. 0.2 g of skin was separately suspended in 10 mL of acetic acid (>99%), formic acid (>99%), peracetic acid (15%) and citric acid (60%) with stirring at room temperature for 24 hours. The skin was immersed in PBS for 24 hours as a control. After 24 hours, the skin was washed to remove excess acid and cell debris, and then lyophilized to make a scaffold. The stents were sterilized by ethylene oxide and subsequently degassed to determine DNA.

Figure 17 shows that the use of formic acid reduces the DNA content to less than 1%; however, the use of acetic acid and citric acid does not reduce the DNA content to a satisfactory level. Although the treatment with peracetic acid can significantly reduce the DNA, it corrodes the specimen.

Comparative Example 1 to Comparative Example 2

The stents of Comparative Example 1 to Comparative Example 2 were based on the method described in Example 1, wherein different concentrations of peroxyacetic acid and different treatment periods were employed as shown in Table 1. The success of decellularization was determined by biochemical analysis comparing the amount of DNA and major ECM (GAG and collagen).

Compare Example 3 to Comparative Example 6

The scaffolds of Comparative Examples 3 to 6 were decellularized according to the method described in Example 1, using acetic acid (>99%), malic acid (60%), succinic acid (5%) or citric acid (60). %) Treatment for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours or 12 hours. I tried to apply a higher concentration of acid solution to achieve optimal decellularization and shorten processing time. The purity of the commercially available acid product or its saturated solubility causes different concentrations of the acid used in the study. Compare the amount of DNA and major ECM (GAG and collagen) by biochemical analysis The success of decellularization.

Stent manufacturing analysis

To confirm cell removal, DNA quantification was performed. Figure 18 (A) shows the relative DNA content compared to control meniscus (12 hours of PBS treatment) after different acid treatments. Immersion in formic acid solution for 2 hours reduced the amount of DNA to 4.10% (p<0.01), while no significant effect was observed in the meniscus treated with acetic acid, 0.15% peracetic acid and 5% succinic acid for 2 hours. Each is p=0.095, 0.059 and 0.074).

Figure 18 (B) and Figure 18 (C) show the residual ratio of major ECM (collagen and GAG) after different treatments. There was no significant adverse effect on GAG or collagen content after treatment with formic acid for 2 hours (p=0.087 and 0.714). At 2 hours, acetic acid and citric acid caused extensive destruction of collagen (p=0.043 and 0.027, respectively), and acetic acid and 15% peroxyacetic acid greatly reduced the amount of GAG (p=0.038 and <0.01, respectively).

Claims (25)

A method of producing a decellularized biological material comprising providing an untreated biological material having cells and treating the biological material with a formic acid solution at a concentration effective to remove cells and nuclear material from the biological material, simultaneously with untreated The biomaterial maintains a GAG above about 85%. The method of claim 1, wherein the biological material treated by the method of the present invention is a tissue and an organ. The method of claim 1, wherein the biological material is skin, heart valve, pericardium, blood vessel, spinal cord, trachea, bladder, ligament, cartilage, meniscus, disc, bone, dura, small intestinal submucosa, spinal meninges, kidney, Liver, lung or nerve. The method of claim 1, wherein the concentration of formic acid in the solution is in the range of from about 50% (w/w) to about 100% (w/w). The method of claim 1, wherein the decellularized biomaterial is a decellularized ECM, a decellularized tissue, or a decellularized organ. As in the method of claim 1, it further maintains greater than 75% collagen. The method of claim 1, wherein the formic acid solution further comprises a cosolvent. The method of claim 1, wherein the formic acid solution further comprises an acid solution. The method of claim 1, wherein the ratio of the biomaterial to formic acid is from about 1% (w/v) to about 5% (w/v). The method of claim 1, wherein the biological material is treated with the formic acid solution for less than 15 hours. The method of claim 1, wherein the tissue and the organ are heterologous, homologous, allogeneic, autologous or xenogeneic tissues or organs. The method of claim 1, wherein the DNA content is reduced to less than about 5% or less compared to the untreated biological material. The method of claim 1 wherein greater than about 90% of the GAG is maintained. The method of claim 1, wherein more than about 80% of the collagen is maintained. The method of claim 1, further comprising a pretreatment step prior to processing the biological material. The method of claim 1, further comprising physical decellularization, chemical decellularization or a combination of the physical and the chemical decellularization prior to processing the biological material. The method of claim 1, after the processing of the biological material, further comprising a washing step. The method of claim 1, after the processing of the biological material, further comprising the step of forming a decellularized biomaterial as a scaffold by mixing the decellularized biomaterial with/without a porogen Pour into the mold used to shape the stent and then freeze dry the shaped stent. A decellularized biomaterial wherein the DNA and GAG content are less than about 5% or less and greater than about 85%, respectively. The decellularized biomaterial of claim 19 further maintains greater than 75% collagen. A decellularized biomaterial according to claim 19 which is a decellularized ECM, tissue or organ. The decellularized biomaterial of claim 19 is bound to a support. The decellularized biomaterial of claim 19, wherein the support is a pharmaceutical composition, an implant, a tissue regeneration scaffold, or a medical device. A method for preparing an in vitro stent culture system comprising (i) providing a decellularized biomaterial scaffold of the invention, (ii) a perfused cell population, including stem cells, progenitor cells, or partially differentiated progenitor cells capable of differentiating, Or a functionally mature cell population for decellularizing a biomaterial scaffold, and (iii) re-cell differentiation and stem cell or progenitor cell differentiation and function in a perfusion decellularized biomaterial scaffold The perfused decellularized biomaterial scaffold is contacted with the cell population for a period of time under conditions of maturity or maturity of the cells in the population. A method for producing a tissue graft comprising the steps of: a) providing a decellularized biomaterial scaffold of the present invention; b) infiltrating cells in the organism into a decellularized biomaterial scaffold; c) cultivating the organism Organizing a period of time sufficient to differentiate the cells; and d) providing a physiologically active substance capable of inducing cell differentiation.