Tissue filling materials

Abstract

To provide a tissue filler that allows a host cell to quickly get into the tissue filler and also allows the host cell to be engrafted in it, and has sufficient mechanical strength.SOLUTION: A tissue filler includes porous collagen sponge including a honeycomb structure inside it, and fibrillar collagen contained in the lumen of the honeycomb structure.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a tissue filler suitable for tissue reconstruction. [Background technology]

[0002] Artificial dermis is an artificial tissue filler that is used in regenerative medicine for wounds that cause extensive skin loss, such as severe burns. Artificial dermis is a completely artificial industrial product, which avoids the problem of donor shortages that accompanies transplant medicine and also controls the risk of rejection, making it an excellent transplant medicine. In regenerative medicine, where artificial extracellular matrix is ​​implanted in the body as a tissue filler, the extent and speed of host cell invasion and engraftment into the tissue filler are important. In artificial dermis, split-thickness epidermal grafting is possible only after sufficient host cells have engrafted, new blood vessels have invaded, and dermis-like connective tissue has formed.

[0003] Conventional artificial dermis includes products such as Pernac (Gunze), Terudermis (Olympus Terumo Biomaterials), and Integra (Integra Life Sciences). The collagen layer in these conventional artificial dermis is a cross-linked collagen sponge formed by freeze-drying, and has a honeycomb structure. In addition, a method for producing a tissue regeneration base material using a collagen sponge formed by freeze-drying and cross-linked has been reported (Patent Document 1).

[0004] The collagen layer with a honeycomb structure of the conventional artificial dermis and tissue regeneration scaffolds described above requires time for host cells to enter each lumen of the honeycomb structure because the walls of the lumen act as a barrier. As a result, the areas where host cells take longer to engraft become acellular tissue, which is one of the reasons why the artificial dermis falls off from the graft site, reducing the success rate of the procedure. Furthermore, it usually takes several weeks before split-thickness epidermal grafting is possible, and during that time, infections and fluid leakage must be prevented, which is also a factor in reducing the patient's quality of life.

[0005] Meanwhile, the present inventors have developed a tissue filler that reproduces a collagen fiber structure similar to that of living tissue by forming collagen fibers and vacuum drying it using 2-methyl-2-propanol (tert(t)-butylalcohol) (Non-Patent Document 1). This fibrous collagen has a dense fiber structure and has water absorption properties similar to absorbent cotton, allowing host cells to rapidly enter the tissue filler, and also has a scaffold structure suitable for cell engraftment, making it suitable for host cell engraftment. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2019-42280 A [Non-patent literature]

[0007] [Non-Patent Document 1] Advances in Wound Care, Vol.9, No.6 (2019) Summary of the Invention [Problem to be solved by the invention]

[0008] The fibrous collagen described in Non-Patent Document 1 is suitable for rapid infiltration and engraftment of host cells into the tissue filler, but has a dense fibrous structure, and cross-linking makes it difficult for cells to infiltrate. Therefore, there is room for improvement in terms of mechanical strength of the fibrous collagen. The present invention provides a method for the rapid ingression and engraftment of host cells into the interior of a tissue filler. The objective of the present invention is to provide a tissue filler having a sufficient mechanical strength. [Means for solving the problem]

[0009] As a result of intensive research into solving the above problems, the present inventors discovered that the above problems can be solved by accommodating the fibrous collagen described in Non-Patent Document 1 in the inner cavity of a honeycomb structure of a conventional porous collagen sponge containing an internal honeycomb structure, and thus completed the present invention. The present invention is as follows.

[0010] [1] A porous collagen sponge having an internal honeycomb structure, and Fibrous collagen contained within the lumen of the honeycomb structure. 23. A tissue filler comprising: [2] The tissue filler described in [1], having a plurality of through holes extending from one surface of the porous collagen sponge to the other surface. [3] The tissue filling material described in [1] or [2], wherein the fibrous collagen is arranged so as to span adjacent inner cavities of the honeycomb structure through the pores of the porous collagen sponge. [4] The tissue filling material described in [2] or [3], wherein the fibrous collagen is arranged so as to span two or more inner cavities of the honeycomb structure through the through holes. [5] The tissue filling material according to any one of [1] to [4], wherein the porous collagen sponge is thermally crosslinked and the fibrous collagen is not crosslinked. [6] The tissue filler according to any one of [1] to [5], wherein at least one surface of the porous collagen sponge is covered with a collagen thin film. [7] The tissue filler according to [6], which is an artificial dermis. [8] preparing a porous collagen sponge having an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; After the gel forming step, a step of replacing the solvent of the gel with 2-methyl-2-propanol; and removing the 2-methyl-2-propanol after the substitution step. A method for producing a tissue filler comprising: [9] preparing a porous collagen sponge having an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; applying a collagen solution to at least one surface of the porous collagen sponge after the gel forming step; After the coating step, a step of replacing the solvent of the gel body and the solvent of the coated collagen solution with 2-methyl-2-propanol; and removing the 2-methyl-2-propanol after the substitution step. A method for producing a tissue filler comprising:

[10] The manufacturing method described in [9], wherein the tissue filler is an artificial dermis. Effect of the Invention

[0011] According to the present invention, it is possible to provide a tissue filler that allows host cells to rapidly enter and survive inside the tissue filler, and has sufficient mechanical strength. The tissue filler has sufficient mechanical strength, and host cells can rapidly invade the tissue filler and are excellent in host cell engraftment, so that the tissue filler can be reduced from falling off due to engraftment failure. In addition, the time required for host cells to engraft sufficiently is short, and new blood vessels can rapidly invade and form connective tissue. Therefore, when transplanted as an artificial dermis, the time required for epidermal transplantation can be shortened, and the risk of bacterial infection and leakage of body fluids can be reduced. Furthermore, when the tissue filler is implanted, a structure similar to that of natural connective tissue is reproduced and can be maintained for a long period of time, making it less likely for scar contracture to occur, reducing pain and causing fewer cosmetic problems. [Brief description of the drawings]

[0012] [Figure 1] 1 is a photograph (a drawing substitute photograph) showing the results of Example 10 according to one embodiment of the present invention. [Diagram 2] 1 is a photograph (a drawing substitute photograph) showing the results of Example 10 according to one embodiment of the present invention. [Diagram 3] 1 is a photograph (a drawing substitute photograph) showing the results of Example 10 according to one embodiment of the present invention. [Figure 4] 1 is a photograph (a drawing substitute photograph) showing the results of Example 10 according to one embodiment of the present invention. [Diagram 5] 1 is a photograph (a drawing substitute photograph) showing the results of Example 10 according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] One aspect of the present invention is a tissue filler comprising a porous collagen sponge containing an internal honeycomb structure, and fibrous collagen contained within the lumen of the honeycomb structure.

[0014] The tissue filler according to this embodiment includes a porous collagen sponge having a honeycomb structure therein. The porous collagen sponge may be a conventional one, for example, the collagen sponge described in Patent Document 1. The honeycomb structure refers to a structure in which the porous collagen sponge contains a plurality of cavities (chambers) in a honeycomb shape within the outer wall, which is the surface of the porous collagen sponge. However, the terms "honeycomb structure" and "honeycomb shape" in this specification do not necessarily mean a structure in which regular hexagonal columns are arranged without gaps, and do not necessarily mean a structure whose cross section is a regular hexagon, but mean the "honeycomb structure" and "honeycomb shape" in conventional collagen sponges.

[0015] The honeycomb-structured outer wall of the porous collagen sponge provides mechanical strength to the tissue filler. After implantation in a living body, the outer wall swells after a certain period of time to form a persistent extracellular collagen matrix. This persistent collagen acts as a scaffold for cells for a long period of time, contributing to the maintenance of the reconstructed tissue.

[0016] The tissue filling material according to this embodiment contains fibrous collagen in the inner cavity of the honeycomb structure of the porous collagen sponge.

[0017] The fibrous collagen is preferably arranged so as to span adjacent inner cavities of the honeycomb structure through the pores of the porous collagen sponge. The pores of the porous collagen sponge include fissures caused by small holes and cracks that are naturally formed by low-temperature freezing, vacuum drying, etc. in the usual manufacturing method of the porous collagen sponge, and small holes and cracks opened in the outer wall of the lumen by artificial means. That is, the fibrous collagen may be arranged so as to span two or more lumens, including adjacent lumens, through fissures caused by small holes and cracks that are naturally formed by freezing, vacuum drying, etc. in the usual manufacturing method of the porous collagen sponge, or may be arranged so as to span two or more lumens, including adjacent lumens, through small holes and cracks opened in the outer wall of the lumen by artificial means. or a combination of these.

[0018] The fibrous collagen is arranged so as to span adjacent internal cavities of the honeycomb structure through the pores of the porous collagen sponge, so that bodily fluids can be rapidly permeated into the porous collagen sponge by capillary action. For example, bodily fluids can be made to permeate the wound site only with exudate, without a process of immersion in physiological saline or the like. In addition, since the fibrous collagen is suitable as a scaffold for host cells, host cells can rapidly enter the internal cavities and be engrafted. Furthermore, the fibrous collagen is decomposed and disappears inside the body together with the recruitment of host cells, but at the same time, the outer wall of the honeycomb structure swells to form persistent collagen fibers that are ideally mixed with the host cell group that has invaded. By using the tissue filler according to this embodiment, tissue with a structure close to that of normal tissue can be quickly reconstructed.

[0019] Regarding the diameter of the collagen fibers and the distance between the collagen fibers of the fibrous collagen accommodated in the lumen of the honeycomb structure, when the gaps between the collagen fibers are larger than the size of the host cells, the host cells can enter between the collagen fibers. On the other hand, even when the gaps between the collagen fibers are smaller than the size of the host cells, that is, when the collagen fibers are densely accommodated, the host cells can push aside the collagen fibers and enter.

[0020] Therefore, the diameter of the collagen fibers is preferably 60 nm or more, more preferably 80 nm or more, and even more preferably 95 nm or more, while it is preferably 140 nm or less, more preferably 120 nm or less, and even more preferably 105 nm or less.

[0021] The diameter of the collagen fibers can be determined, for example, as follows. A photograph of the fibrous collagen in the lumen is taken with an electron microscope, and the diameter of the collagen fiber is measured. The diameters of a plurality of collagen fibers are measured in the same manner, and the average value is calculated to be the "diameter of the collagen fiber." The number of samples is preferably 60 or more.

[0022] The distance between the collagen fibers is preferably 80 nm or more, more preferably 250 nm or more, and even more preferably 350 nm or more, while it is preferably 680 nm or less, more preferably 550 nm or less, and even more preferably 450 nm or less.

[0023] The distance between the collagen fibers can be determined, for example, as follows. A photograph of the fibrous collagen in the porous collagen sponge is taken with an electron microscope, and the intersecting collagen fibers are treated as a lattice, and the maximum width between the lattices on the same plane is measured. A number of maximum widths are measured in the same manner, and the average value calculated is the "distance between collagen fibers." The number of samples is preferably 60 or more.

[0024] The porous collagen sponge is preferably crosslinked. When crosslinked, the mechanical strength of the porous collagen sponge is improved. In addition, the crosslinked collagen contained in the porous collagen sponge is less likely to be decomposed than non-crosslinked collagen, and therefore functions as a scaffold for cells for a longer period of time.

[0025] Examples of crosslinking include chemical crosslinking (e.g., chemical crosslinking with glutaraldehyde), thermal crosslinking, etc. Among these, thermal crosslinking is preferred because it is easy for the outer wall of the honeycomb structure of the porous collagen sponge to swell, which makes it easy to prevent the accumulation of exudate, and there is little risk of the tissue filler falling off due to activation of the inflammatory response or accumulation of neutrophils.

[0026] On the other hand, the fibrous collagen may be cross-linked. However, as described above, the fibrous collagen is contained within the lumen, and cross-linking makes it difficult for cells to enter the tissue filler, so it is preferable that the fibrous collagen is not cross-linked.

[0027] The porous collagen sponge preferably has through-holes that extend from one surface to the other surface. Here, the porous collagen sponge can be used in a substantially spherical shape as in the examples described below, but in a normal manufacturing method, the porous collagen sponge is manufactured in a form that will eventually become a thick, sheet-like porous collagen sponge, and is therefore manufactured in a thick, sheet-like form. The term "penetrating from one surface to the other surface" may mean, for example, penetrating from one bottom surface to the other bottom surface in a thick, sheet-like porous collagen sponge, as shown in D of FIG.

[0028] The fibrous collagen is preferably arranged so as to span two or more inner cavities of the honeycomb structure through the through-holes. The fibrous collagen spanning two or more lumens of the honeycomb structure through the through-holes provides the following effects: In other words, tissue-forming substances such as body fluids, host cells, capillaries, nerves, and extracellular matrix can enter the lumen more efficiently and quickly than when they enter the lumen only through small holes and cracks in the porous collagen sponge. In addition, sufficient moisture such as body fluids can enter the porous collagen sponge, preventing it from shrinking during the process of exudate penetration after transplantation.

[0029] Therefore, the number of through-holes may be one or more, but it is preferable to have more than one since the above-mentioned effect can be obtained over a wide range of the porous collagen sponge. On the other hand, it is preferable to have a certain number or less in terms of the surface area of ​​the porous collagen sponge. For example, in the porous collagen sponge, 2 The number of pieces per unit may be, for example, 2 or more, 10 or more, 20 or more, 30 or more, 40 or more, or the like, while it may be, for example, 80 or less, 70 or less, 60 or less, or the like.

[0030] When there are multiple through holes, the spacing between them is preferably 1.0 mm or more, more preferably 1.5 mm or more, and even more preferably 2.0 mm or more, in order to obtain the above-mentioned effect over a wide area of ​​the porous collagen sponge and to minimize the effect on atrophy of the porous collagen sponge when water penetrates, making it easier to maintain a shape suitable for transplantation as a tissue filler, while on the other hand, it is preferably 5.0 mm or less, more preferably 3.0 mm or less, and even more preferably 2.5 mm or less.

[0031] The diameter of the through-hole is preferably 1.5 mm or less, more preferably 1.0 mm or less, and even more preferably 0.8 mm or less, in order to easily maintain a shape suitable for transplantation as a tissue filling material, since the impact on the atrophy of the porous collagen sponge when water penetrates is greater than the above-mentioned effect. On the other hand, in order to obtain the above-mentioned effect, the diameter is preferably 0.2 mm or more, more preferably 0.4 mm or more, and even more preferably 0.6 mm or more.

[0032] The tissue filler according to this embodiment is a material into which cells and blood vessels enter and take root, thereby causing tissue proliferation and regeneration, and includes so-called tissue fillers that are implanted into subcutaneous tissue or the body, and artificial dermis used for regenerating dermis.

[0033] In the tissue filler according to this embodiment, at least one surface of the porous collagen sponge is preferably covered with a collagen thin film. As described above, the porous collagen sponge can be used in a substantially spherical shape as in the examples described below. However, in the usual manufacturing method, the finished product is Since the porous collagen sponge is produced in a mold that will become a thick, sheet-like porous collagen sponge, the porous collagen sponge is produced in a thick, sheet-like form. The "at least one surface of the porous collagen sponge" may be at least one of the bottom surface and the other bottom surface of the thick, sheet-like porous collagen sponge, for example, as shown in Fig. 4D.

[0034] When at least one surface of the porous collagen sponge is covered with a collagen thin film, the tissue filler according to this embodiment can be used as an artificial dermis. Non-Patent Document 1 teaches that when the surface of a tissue filler is coated with a collagen thin film, re-epithelialization by epidermal cells can be promoted. When the tissue filler according to this embodiment is used as an artificial dermis, the coating can provide a similar effect. In this regard, for example, commercially available products such as Pernac (manufactured by Gunze) and Terudermis (manufactured by Olympus Terumo Biomaterials) use a silicon thin film instead of a collagen thin film. However, since epidermis cannot be regenerated on a silicon thin film, the tissue filler according to this embodiment is more advantageous than commercially available products when the surface of the tissue filler is coated with a collagen thin film.

[0035] The thickness of the collagen thin film is not particularly limited as long as it can promote re-epithelialization by epidermal cells, as described above, and does not have to be uniform, but is preferably 1 μm or more, more preferably 3 μm or more, and even more preferably 5 μm or more, while it is preferably 15 μm or less, more preferably 10 μm or less, and even more preferably 8 μm or less.

[0036] The volume of each cavity of the honeycomb structure may be the volume of each cavity of a porous collagen sponge manufactured by a conventional method for manufacturing a porous collagen sponge. For example, 3 More than 5 million μm, preferably 3 More preferably, 8 million μm 3 On the other hand, for example, 46 million μm 3 Less than 24 million μm, preferably 3 Less than or equal to 12 million μm 3 The following is the result.

[0037] The volume of each lumen of the porous collagen sponge can be determined, for example, as follows. A photograph of the porous collagen sponge is taken with an electron microscope, and each lumen is regarded as a rectangular parallelepiped and its maximum width is measured. In the same manner, multiple maximum widths are measured, and the average value calculated is taken as the average of one side. The average is then cubed to be the "volume of each lumen of the porous collagen sponge." The number of samples is preferably 60 or more.

[0038] The type of collagen contained in the porous collagen sponge, the type of fibrous collagen, and the type of collagen contained in the collagen thin film are not particularly limited as long as they can exert the effects of the present invention. Furthermore, these collagen types may all be the same type, all different types, or two of them may be the same type, and are not particularly limited as long as they can exert the effects of the present invention.

[0039] The collagen may be either fibrous collagen or non-fibrous collagen, but fibrous collagen is preferred. Examples of fibrous collagen include type I collagen, type II collagen, type III collagen, type V collagen, and type XI collagen. Among these, type I collagen, which has been used in clinical trials, is preferred. In addition, its properties are not particularly limited as long as it can exert the effects of the present invention, and it may be, for example, pepsin-soluble. Furthermore, atelocollagen, which is known to have low antigenicity (collagen in which the telopeptide region is cleaved and removed by protease treatment), is also preferred, and type I atelocollagen is more preferred. The collagen may be derived from bovine or porcine. It may be derived from jellyfish, and is not particularly limited as long as it can exert the effects of the present invention.

[0040] Another aspect of the present invention is providing a porous collagen sponge containing an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; After the gel forming step, a step of replacing the solvent of the gel with 2-methyl-2-propanol; and removing the 2-methyl-2-propanol after the substitution step. The method for producing a tissue filler includes the steps of: In this specification, this embodiment may be referred to as "production method 1."

[0041] The manufacturing method 1 according to this embodiment includes a step of preparing a porous collagen sponge having an internal honeycomb structure. In this step, the form of the porous collagen sponge is not particularly limited as long as the porous collagen sponge containing a honeycomb structure inside can be prepared. The collagen sponge may be a conventional one, for example, the collagen sponge described in Patent Document 1. Therefore, the collagen sponge can be produced by a conventional method, for example, the collagen sponge production method described in Patent Document 1.

[0042] This process may include a step of gelling collagen, and a step of freezing the gelled collagen at a low temperature and vacuum drying to obtain a porous collagen sponge containing a honeycomb structure inside. This may be done in a conventional manner. For example, collagen may be dissolved in low-concentration acetic acid or the like, dialyzed against high-concentration acetic acid or the like, and then gelled. Gelation may be performed, for example, by neutralizing the solution by adding phosphate-buffered saline (PBS) or the like to a final concentration of 1x, and heating to around 37°C. The gelled collagen may be frozen at a low temperature in the mold and then vacuum dried to obtain a porous collagen sponge. Specifically, this can be done as described in the Examples below.

[0043] The production method 1 according to this embodiment includes, after the preparation step, a step of crosslinking the porous collagen sponge. As for the preferred embodiment of the crosslinking, the above-mentioned contents are incorporated herein by reference.

[0044] When the crosslinking is thermal crosslinking, it can be carried out under the following conditions. By carrying out thermal crosslinking under the following conditions, a tissue filler is produced in which the outer wall of the honeycomb structure of the porous collagen sponge is likely to swell, exudate retention is likely to be prevented, and there is a low risk of the tissue filler falling off due to activation of an inflammatory response or accumulation of neutrophils. The temperature during crosslinking is preferably 80°C or higher, more preferably 90°C or higher, and even more preferably 95°C or higher, since irreversible deterioration occurs during crosslinking, while it is preferably 160°C or lower, more preferably 140°C or lower, even more preferably 120°C or lower, and even more preferably 110°C or lower. The time for the crosslinking treatment is preferably 3 hours or more, more preferably 4 hours or more, and even more preferably 5 hours or more, while it is, for example, 12 hours or less, preferably 10 hours or less, and more preferably 8 hours or less.

[0045] When the crosslinking is chemical crosslinking, the chemical crosslinking is carried out after the thermal crosslinking. Conventional crosslinking conditions can be used to chemically crosslink the porous collagen sponge. When the chemical crosslinking is, for example, crosslinking with glutaraldehyde, crosslinking can be performed under the following conditions: The concentration (v / v) of glutaraldehyde during crosslinking is, for example, 0.005% or more, preferably 0.5% or more, more preferably 2.0% or more, while it is, for example, 10% or less, preferably 5.0% or less, more preferably 3.0% or less. The temperature during crosslinking is, for example, 0° C. or higher, preferably 2° C. or higher, more preferably 3° C. or higher, and, for example, 10° C. or lower, preferably 8° C. or lower, more preferably 5° C. or lower. The time for the crosslinking treatment is, for example, 4 hours or more, preferably 8 hours or more, more preferably 10 hours or more, while it is, for example, 36 hours or less, preferably 24 hours or less, more preferably 16 hours or less. In addition, when the crosslinking is chemical crosslinking, the process may include a step of washing with water after crosslinking, a step of treating the functional group of the drug used for the chemical crosslinking, and a step of freezing at a low temperature and drying in a vacuum. For example, when the chemical crosslinking is crosslinking with glutaraldehyde, for example, as in the examples described later, washing with water after crosslinking, treating the aldehyde group with glycine, washing with water, and then freezing at a low temperature such as -30°C and drying in a vacuum may be included.

[0046] The production method 1 according to this embodiment includes, after the crosslinking step, a step of forming a plurality of through-holes penetrating from one surface to the other surface of the porous collagen sponge. As for the preferred embodiment for forming the through hole, the above-mentioned contents are incorporated herein by reference.

[0047] The through holes can be made with a tool for making holes, such as a hypodermic needle or a punch. The interval and diameter of the through holes can be controlled by adjusting the tool for making holes. For example, when a hypodermic needle is used, the diameter of the hole can be adjusted by the diameter of the needle, and holes can be made at any interval.

[0048] The manufacturing method 1 according to the present embodiment includes a step of immersing the porous collagen sponge in a collagen solution after the through-hole forming step, and a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body. The collagen impregnated into the porous collagen sponge by the immersion step and then gelled will ultimately become the fibrous collagen in the above-mentioned embodiment. The above-mentioned contents are incorporated herein by reference with regard to preferred embodiments of the collagen.

[0049] When the porous collagen sponge is immersed in a collagen solution, the concentration of the collagen solution is preferably 0.01 mg / mL or more, more preferably 0.1 mg / mL, and even more preferably 0.5 mg / mL or more, while it is preferably 10 mg / mL or less, more preferably 5 mg / mL, and even more preferably 2 mg / mL or less.

[0050] The gel is formed in a state where the porous collagen sponge is immersed in a collagen solution. The time required for gel formation may be any time sufficient for sufficient gelation to occur, and is, for example, 8 hours or more, preferably 10 hours or more, and more preferably 12 hours or more, while it is, for example, 24 hours or less, preferably 18 hours or less, and more preferably 16 hours or less. The temperature during gel formation is, for example, 30°C or higher, preferably 32°C or higher, and more preferably 35°C or higher, while it is, for example, 42°C or lower, preferably 40°C or lower, and more preferably 38°C or lower.

[0051] In addition, when the collagen is crosslinked, it becomes difficult for cells to invade the tissue filling material produced. Therefore, in this embodiment, the collagen contained in the porous collagen sponge is crosslinked. It may include a step, but preferably does not include such a step.

[0052] The manufacturing method 1 according to this embodiment includes, after the gel forming step, a step of substituting the solvent in the gel with 2-methyl-2-propanol. The replacement method includes washing the gel body with 2-methyl-2-propanol. Prior to this step, the gel body may be washed with alcohol, such as 100% ethanol. The washing method may be a conventional method.

[0053] The production method 1 according to this embodiment includes a step of removing the 2-methyl-2-propanol after the substitution step. The method for removing the 2-methyl-2-propanol includes, for example, a vacuum drying process. The vacuum drying process may be a low-temperature vacuum drying process. That is, the mixture may be frozen at a low temperature and then vacuum dried. For example, the mixture may be frozen at a low temperature such as -70°C and then vacuum dried.

[0054] Another aspect of the present invention is providing a porous collagen sponge containing an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; applying a collagen solution to at least one surface of the porous collagen sponge after the gel forming step; After the coating step, a step of replacing the solvent of the gel body and the solvent of the coated collagen solution with 2-methyl-2-propanol; and removing the 2-methyl-2-propanol after the substitution step. The method for producing a tissue filler includes the steps of: In this specification, this embodiment may be referred to as "production method 2."

[0055] The manufacturing method 2 according to the present embodiment differs from the manufacturing method 1 in the following respects: That is, manufacturing method 2 according to the present embodiment includes, after the gel-forming step of manufacturing method 1, a step of applying a collagen solution to at least one surface of the porous collagen sponge, and, after the application step, a step of substituting the solvent of the gel and the solvent of the applied collagen solution with 2-methyl-2-propanol.

[0056] The manufacturing method 2 according to this embodiment includes a step of applying a collagen solution to at least one of the surfaces of the porous collagen sponge after the gel formation step. By this step, at least one of the surfaces of the porous collagen sponge is covered with a collagen thin film. The above-mentioned contents are incorporated by reference for the preferred aspects of the collagen thin film.

[0057] The concentration of the collagen solution at the time of application is preferably 0.01 mg / mL or more, more preferably 0.1 mg / mL, and even more preferably 0.5 mg / mL or more, while it is preferably 10 mg / mL or less, more preferably 5 mg / mL, and even more preferably 2 mg / mL or less. The amount of coating should be set so as to achieve the thickness of the collagen thin film described above, and should be determined according to standard methods. Alternatively, various coating amounts may be applied in advance to clarify the relationship between the coating amount and the thickness, and then the coating amount may be set.

[0058] In addition, the method may include a step of washing the gel body with alcohol and a step of desalting the gel body before application. Examples of the alcohol include 85% ethanol. The washing method may be a conventional method. The desalting method may also be a conventional method.

[0059] The manufacturing method 2 according to this embodiment includes, after the coating step, a step of substituting the solvent of the gel body and the solvent of the coated collagen solution with 2-methyl-2-propanol. This step is the replacement step in Production Method 1, except that the solvent of the applied collagen solution is replaced with 2-methyl-2-propanol. The method of replacement is the same as the "step of replacing the solvent of the gel body with 2-methyl-2-propanol after the gel body formation step" in Production Method 1, and therefore the description thereof is hereby incorporated by reference.

[0060] The tissue filler produced by the production method 2 according to this embodiment can be used as an artificial dermis. That is, a preferred embodiment of this aspect is providing a porous collagen sponge containing an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; applying a collagen solution to at least one surface of the porous collagen sponge after the gel forming step; After the coating step, a step of replacing the solvent of the gel body and the solvent of the coated collagen solution with 2-methyl-2-propanol; and removing the 2-methyl-2-propanol after the substitution step. The present invention relates to a method for producing an artificial dermis, comprising the steps of:

[0061] In the manufacturing process 1 and manufacturing method 2, when the preparation process includes a process of gelling collagen, and a process of freezing the gelled collagen at a low temperature and vacuum drying to obtain a porous collagen sponge containing a honeycomb structure therein, the ratio of the collagen concentration in the collagen solution in the immersion process to the collagen concentration in the collagen solution containing the collagen in the gelling process is preferably 10 times or less, more preferably 5 times or less, and even more preferably 3 times or less, since the tissue filler produced can reproduce tissue closer to the dermis, while being, for example, 0.5 times or more, preferably 0.8 times or more.

[0062] The manufacturing method 1 and the manufacturing method 2 may not include the through-hole forming step. In that case, the tissue filler produced does not have a through-hole. That is, in the manufacturing method 1 and the manufacturing method 2, after the crosslinking step, the process may proceed to a step of immersing the porous collagen sponge in a collagen solution without including the through-hole forming step. EXAMPLES

[0063] The present invention will be described below with reference to specific examples, but the present invention is not limited to the following embodiments.

[0064] Example 1 The tissue filler was prepared as follows. As a collagen dissolving solution, pepsin-soluble dried type I collagen (Nippi Collagen, pepsin-solubilized bovine dermis collagen powder, model number PSC-1-100PW) derived from cows was added at 0. The mixture was dissolved in 5M acetic acid and dialyzed against 5 mM acetic acid. The collagen was then gelled using a mold (Nissin EM, Silicone Embedding Plate Type II, Model No. 4201). Gelation was carried out by adding 25-fold concentrated phosphate buffered saline (25xPBS) to a final concentration of 1x to neutralise the mixture, and heating to 37°C. The collagen concentration at this time was 1.0mg / ml. The collagen gel produced was then frozen in the mold at -30°C and vacuum dried to obtain a porous collagen sponge. The resulting porous collagen sponge was then subjected to thermal crosslinking by dry heat treatment at 100° C. for 6 hours, then treated with 2.5% glutaraldehyde at 4° C. for 12 hours, washed with water, treated with 50 mM glycine to remove aldehyde groups, washed with water again, frozen at −30° C., and vacuum dried to obtain a porous collagen sponge. Next, the porous collagen sponge was inserted into the 135 cm 2 Thirty-two (4 × 8) through-holes were drilled per base area. The diameter of each through-hole was 0.6 mm, and the spacing between the through-holes was 2.0 mm. Next, the porous collagen sponge was immersed in a 1.0 mg / ml neutralized type I atelocollagen solution, heated to 37°C, and gelled for 14 hours to form a gel. This gel was washed with 85% ethanol and desalted, and 10 μl of the 1.0 mg / ml neutralized type I atelocollagen solution was applied to one side of the gel surface to form a thin coating. Next, the gel was washed with 100% ethanol and then with 2-methyl-2-propanol (solvent substitution), frozen at -70°C, and vacuum-dried. The neutralized type I atelocollagen vacuum-dried in 2-methyl-2-propanol did not have a honeycomb structure, but was a fibrous collagen that maintained the shape of the collagen fibers.

[0065] Example 2 A tissue filler was produced in the same manner as in Example 1, except that 1.0% glutaraldehyde was used for chemical crosslinking.

[0066] Example 3 A tissue filler was produced in the same manner as in Example 1, except that 0.01% glutaraldehyde was used for chemical crosslinking.

[0067] Example 4 A tissue filler was produced in the same manner as in Example 1, except that the produced collagen gel was thermally crosslinked by dry heat treatment at 100°C for 6 hours to obtain a porous collagen sponge, instead of chemically crosslinking. The produced tissue filler is shown in Fig. 4D.

[0068] Example 5 A tissue filler was produced in the same manner as in Example 1, except that the collagen gel prepared was thermally crosslinked by dry heat treatment at 140°C for 6 hours to obtain a porous collagen sponge, instead of chemically crosslinking.

[0069] Example 6 A tissue filler was produced in the same manner as in Example 1, except that the collagen gel prepared was thermally crosslinked by dry heat treatment at 160°C for 6 hours to obtain a porous collagen sponge, instead of chemically crosslinking.

[0070] Example 7 When making a through hole in a porous collagen sponge, a 1.5 mm diameter punch hole was made at a distance of 135 cm. 2The tissue filler was produced in the same manner as in Example 4, except that two through-holes were opened per base area. The interval between the through-holes was 5 mm. The produced tissue filler is shown in FIG. 4D.

[0071] Example 8 Except for not forming through holes in the porous collagen sponge, a tissue filling material was produced in the same manner as in Example 4. The produced tissue filling material is shown in FIG.

[0072] Example 9 A tissue filler was produced in the same manner as in Example 4, except that the concentration of the pepsin-soluble type I collagen used to prepare the porous collagen sponge was 0.5 mg / ml.

[0073] Comparative Example 1 We used Terudermis (manufactured by Olympus Terumo Biomaterials), a commercially available artificial dermis. This product is a thermal crosslinking type artificial dermis.

[0074] Comparative Example 2 We used Pelnac (Gunze), a commercially available artificial dermis. This product is a chemically crosslinked artificial dermis.

[0075] Comparative Example 3 The tissue filler used was produced by the method described in Non-Patent Document 1. That is, it is a tissue filler of the present invention consisting only of fibrous collagen, and is not crosslinked.

[0076] Example 10 The tissue fillers produced were evaluated by the following method. A skin defect wound model mouse was prepared by drilling a hole with a diameter of 6 mm in the dorsal skin of the mouse, and each tissue filler made into an approximately spherical shape (diameter: about 6 mm) was placed on the wound to cover it and fixed with a bandage and suture. After 3 weeks, the wound healing on the tissue filler was observed histologically and skin regeneration was evaluated. Staining and observation methods were performed according to conventional methods.

[0077] The results are shown in Figure 1. For reference, images of normal skin tissue and scar tissue after wounding are also shown. In normal skin tissue, there was a moderate amount of collagen around the cells, and the collagen formed ripples in various directions. In scar tissue after injury, the number of cells was greater due to the infiltration of inflammatory cells, and no ripples were observed. When the tissue filler of Example 4 was used, the collagen fiber structure surrounded the cell population, forming ripples, and tissue with a structure similar to that of the dermis was formed. On the other hand, when Terudermis was used in Comparative Example 1, the cell penetration and the number of engrafted cells were insufficient. When Pernac was used in Comparative Example 2, the tissue was mixed with scar tissue. In both the cases of Terudermis in Comparative Example 1 and Pernac in Comparative Example 2, the honeycomb structure was filled with scar tissue, which was different from the structure of the dermis. When the tissue filler in Comparative Example 3 was used, most of the collagen was decomposed and the graft was replaced with scar tissue.

[0078] In order to verify that tissue reconstruction by the tissue filler of Example 4 was proceeding appropriately, vascular endothelial cells were stained by the following method. Vascular endothelial cells were stained based on CD31, a known marker. The staining method was performed according to a conventional method.

[0079] The results are shown in Figure 2. The cells that entered the porous collagen sponge established themselves and formed a cluster, using the collagen on the outer wall as a scaffold. At this point, the fibrous collagen had been decomposed or absorbed by the body. The honeycomb-structured outer wall of the porous collagen sponge swelled to form partitions of poorly degradable collagen, which formed ripples that prevented the cells from being oriented in one direction and prevented scar contracture. Staining with the marker CD31 revealed that capillaries were spread throughout the cell clusters.

[0080] The effect of different crosslinking methods on skin regeneration is shown in Figure 3. When the tissue filler of Example 4 was used, the outer wall of the honeycomb structure of the porous collagen sponge swelled sufficiently, and absorption of accumulated fluid and suppression of excessive immune response were observed. On the other hand, when the tissue fillers of Examples 1 to 3, 5, and 6 were used, accumulation of exudate and accumulation of a large number of neutrophils were observed in all cases, although all were usable as tissue fillers.

[0081] Furthermore, the effect of the form of the through-holes in the porous collagen sponge on skin regeneration is shown in Figure 4. The dashed line in the figure indicates the shape of the graft. In all cases where the tissue filler of Example 4 was used, the tissue filler of Example 7 was used, and the tissue filler of Example 8 was used, the infiltration of cells and the swelling of the outer wall of the honeycomb structure of the porous collagen sponge surrounding the cells were observed. When the tissue filler of Example 4 was used, the porous collagen sponge maintained its original shape, but when the tissue filler of Example 7 and the tissue filler of Example 8 were used, some atrophy of the porous collagen sponge was observed, although it was still usable as a tissue filler.

[0082] In addition, the effect on skin regeneration of the ratio of the collagen concentration in the collagen solution when the porous collagen sponge was immersed in the collagen solution to gel it, relative to the collagen concentration in the collagen solution used for gelling to create the porous collagen sponge, is shown in Figure 5. In both cases where the tissue filler of Example 4 and the tissue filler of Example 9 were used, the infiltration of cells and the swelling of the honeycomb structure of the porous collagen sponge surrounding the cells were observed. When the tissue filler of Example 4 was used, tissue with a density close to that of the dermis was formed. In addition, when the tissue filler of Example 9 was used, a looser tissue with more gaps was formed compared to when the tissue filler of Example 4 was used, so that the tissue filler is suitable for transplantation into soft tissues with low density.

Claims

1. A porous collagen sponge containing an internal honeycomb structure, and Fibrous collagen contained within the lumen of the honeycomb structure.

1. A tissue filler comprising:

2. The tissue filler according to claim 1 , having a plurality of through holes penetrating from one surface of the porous collagen sponge to the other surface.

3. 3. The tissue filler according to claim 1, wherein the fibrous collagen is arranged so as to span adjacent inner cavities of the honeycomb structure through pores of the porous collagen sponge.

4. The tissue filler according to claim 2 , wherein the fibrous collagen is arranged to span two or more internal cavities of the honeycomb structure through the through holes.

5. The tissue filler according to any one of claims 1 to 4, wherein the porous collagen sponge is thermally crosslinked and the fibrillar collagen is not crosslinked.

6. The tissue filler according to any one of claims 1 to 5, wherein at least one surface of the porous collagen sponge is covered with a collagen thin film.

7. The tissue filler of claim 6 which is an artificial dermis.

8. providing a porous collagen sponge containing an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; After the gel forming step, a step of replacing the solvent of the gel with 2-methyl-2-propanol; and removing said 2-methyl-2-propanol after said substitution step. Including, The method for producing a tissue filler, wherein the step of removing 2-methyl-2-propanol comprises a drying treatment.

9. providing a porous collagen sponge containing an internal honeycomb structure; after said preparing step, crosslinking said porous collagen sponge; forming a plurality of through holes penetrating from one surface to the other surface of the porous collagen sponge after the crosslinking step; After the through-hole forming step, a step of immersing the porous collagen sponge in a collagen solution; a step of gelling the collagen impregnated in the porous collagen sponge by the immersion step to form a gel body; applying a collagen solution to at least one surface of the porous collagen sponge after the gel forming step; After the coating step, a step of replacing the solvent of the gel body and the solvent of the coated collagen solution with 2-methyl-2-propanol; and removing said 2-methyl-2-propanol after said substitution step. Including, The method for producing a tissue filler, wherein the step of removing 2-methyl-2-propanol comprises a drying treatment.

10. The method of claim 9 , wherein the tissue filler is an artificial dermis.

Images