JPH08196618A - Cell invasive collagen formulation, artificial skin and their manufacture - Google Patents

Abstract

PURPOSE: To provide a bone-repairing collagen formulation which is strengthened in its collagenase resistance, does not induce foreign body reaction and has high biocompatibility with bone, by using a sponge-shaped collagen matrix in which many holes exist and specifying the diameter of the hole. CONSTITUTION: A cell invasive collagen formulation is composed of a sponge- shaped collagen matrix in which many holes are present. Here, diameter of the holes is 100 to 1000μm and the holes have an asymmetric structure. The collagen matrix is made out of a mixture of fibrous atherocollagen and denatured atherocollagen whose helix content is 0 to 80%. Furthermore, a bone- repairing material is formed out of a cell invasive collagen formulation. On the other hand, the cell invasive collagen formulation is manufactured by processing under reduced pressure and high temperature after a raw material collagen solution is foamed and freeze-dried. An injured part contact layer of an artificial skin is formed of the sponge-shaped collagen matrix.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell-penetrating collagen preparation, artificial skin, and a method for producing them.

[0002]

2. Description of the Related Art In the case where some abnormality or defect occurs in bone tissue, it is preferable to transplant a bone taken from another site of the patient, but it is often difficult to supply the bone. It has existed since ancient times. These artificial materials include metals such as titanium and stainless steel, and ceramics such as hydroxyapatite. However, metals cannot be directly bonded to living bones, and soft tissues are newly formed and intervene in the gaps with living bones, and thus lack long-term stability. Further, ceramics have a problem in terms of physical function of bone, because direct bonding with living bone can be obtained or the physical properties of the ceramic itself are fragile. The main components of bone are collagen as an organic substance and inorganic substances such as hydroxyapatite,
Several attempts have been made to utilize collagen preparations.

Collagen fleece is obtained by γ-irradiating freeze-dried atelocollagen derived from bovine skin.
It is commercially available under the name en fleece), and it is actually applied to bone reconstruction, compared to the case without this,
Ulrich Joos & Gertrued Ochs reported that premature bone formation at the bone defect site [Ulrich Joos & Gertrued Ochs, Biomaterials, 1 , 23
(1980)]. Further, bone collagen powder, purified atelopeptide reconstituted fine fibrous dermal collagen, purified atelopeptide reconstituted fine, characterized by being substantially non-crosslinked, comprising a purified atelopeptide preparation of collagen derived from bone from which inorganic components have been removed. The method of Donald Joe Wallace et al., Which uses a lyophilized gel of fibrous skin collagen and a collagen preparation comprising the above mixture, is disclosed in JP-A-3-2661.
No. 6 is disclosed.

When some abnormality occurs in living tissues such as skin, allogeneic transplantation from tissues of other parts of the body or individuals with less immunogenicity such as relatives is preferable.
Naturally, there are many cases where they are the subject of immune rejection reactions, so efforts are being made to find substances with low so-called tissue reactions that are insensitive to tissues and cells of the immune system. On the other hand, in order to protect wounds such as burns, trauma, and surgical wounds and promote healing, gauze, absorbent cotton, etc. have been conventionally used as artificial skin that is temporarily applied to the affected area. It has low prevention ability and absorbs exudate, so the wound surface sticks dry and bleeds when peeled off.
It was accompanied by pain and inhibition of healing. In addition, an ointment or the like may be used in combination, but in this case, on the contrary, storage of exudate and excessive wetting of the wound surface pose problems.

As alternatives to these, synthetic materials such as polyurethane film, silicone gauze, silicone rubber and synthetic fiber sheets such as nylon and Teflon having a velor surface, freeze-dried pig skin, chitin nonwoven fabric, Materials of biological origin such as collagen nonwoven fabric and polyleucine film are known. One example is research into the direction of making synthetic polymers typified by polyurethanes more permeable. However, artificial materials have good adhesion to the affected area,
There are problems with biocompatibility, anti-sticking effect, etc., while bio-derived materials show antigenicity, melting on the wound surface, infection, and deterioration due to exudate. In addition, a coating material consisting of collagen-treated nylon mesh and silicone film was developed,
Although it has adhesiveness to the wound surface and appropriate water permeation, it has a drawback that it strongly adheres to the wound surface and delays healing of the wound surface.
On the contrary, there is a concept that a substance is quickly assimilated with a tissue before an immune response is caused, thereby imparting an organ function. Choosing collagen, which is a biogenic material, as the material, infiltrate the fibroblasts at an early stage, build connective tissue, cover the target tissue, and avoid the immune reaction, the latter is more ideal. It is a close shape. However, in the case of collagen processed into a sponge, there is a mucopolysaccharide-containing collagen film, etc., but strong chemical cross-linking is performed to maintain the strength in vivo, but this makes the foreign substance high and cures. However, the operability was not good because of the delay in strength and weakness in strength.

[0006]

Re-fibrotic atelocollagen, for example, is a human bone tissue because the telopeptide portion, which should be called an immunoreactive determinant of collagen, has been cleaved / removed even in heterogeneous animals. Is considered to have a large affinity for. However, if cross-linking is not added between collagen molecules, not only is it easily decomposed and absorbed by collagenase in vivo, but gel freeze-dried
It is known that even if it is soaked in 37 ° C physiological saline, it will dissolve within several hours. Even if an attempt is made to apply such a collagen preparation to bone reconstruction, it is not stable in vivo, so that the initial purpose cannot be achieved.

On the other hand, irradiation with γ-rays or treatment with a chemical such as hexamethylene diisocyanate or glutaraldehyde can introduce crosslinks between collagen molecules and enhance physical properties and collagenase resistance. For example, if a freeze-dried collagen preparation is crosslinked with hexamethylene diisocyanate,
No change in morphology was observed even when immersed in a collagenase solution (100 unit / ml) at 37 ° C for 7 days. However, when such a strong cross-link is introduced, when implanted in a living body, only a foreign body reaction to collagen appears strongly, and phagocytosis by phagocytic cells is observed. In other words, it is a contradictory event that it is difficult to improve the biological performance such as the physical properties and the strength of collagenase resistance and the affinity for tissues and cells. Collagen preparations that are there for a certain period of time and that lead to bone reconstruction without foreign body reaction are present. It could not be obtained conventionally. Therefore, an object of the present invention is to provide a collagen preparation for cell invasive bone repair, which has enhanced collagenase resistance, does not induce foreign body reaction, and has high biocompatibility with bone.

As described above, in the artificial skin of the non-crosslinked collagen sponge, the rate of self-assembly due to the invasion of cells and blood vessels becomes a problem, but in the artificial skin of the chemically crosslinked collagen sponge, the cell invasion occurs. There is a problem in that the sponge structure with an appropriate pore size and its maintenance against pressure during use are used. For example, the biodegradable polyester is dispersed to maintain the sponge structure, or the temperature during freeze-drying is changed to control the sponge pore size to enhance cell invasion. There are problems such as cracks in itself. Therefore, if there are appropriate pores in the sponge for cell and blood vessel invasion, cells and blood vessels will invade and self-organization will be accelerated, resulting in a good mother bed for early transplantation of split-thickness skin grafts. It also becomes possible.

[0009]

Means for Solving the Problems The above object is achieved by a cell-penetrating collagen preparation having the following constitution, an artificial skin and a method for producing them. (1) It is composed of a sponge-like collagen matrix having a large number of pores, and the diameter of the pores is 100 to 100.
A cell-invading collagen preparation characterized by having a size of 0 μm. (2) The cell-invading collagen preparation according to (1) above, wherein the pores present in the collagen matrix have an asymmetric structure.

(3) The cell-penetrating collagen preparation according to (1) or (2) above, wherein the collagen matrix is a mixture of fibrotic atelocollagen and denatured atelocollagen having a helix content of 0 to 80%. (4) A bone repair material comprising the cell-penetrating collagen preparation according to any of (1) to (3) above. (5) Production of the cell-penetrating collagen preparation according to the above (1) to (3), which comprises foaming the raw material collagen solution, freeze-drying at −40 ° C. or lower, and then treating at low temperature and high temperature. Method. (6) A wound contact layer and a support layer laminated on the wound contact layer, wherein the wound contact layer comprises a sponge-like collagen matrix having a large number of pores, and the pores have a diameter of 100 to 1000 μm. An artificial skin characterized by being.

(7) The artificial skin described in (6) above, wherein the pores present in the collagen matrix have an asymmetric structure. (8) The artificial skin described in (6) to (7) above, wherein the collagen matrix is a mixture of fibrotic atelocollagen and denatured atelocollagen having a helix content of 0 to 80%. (9) The raw collagen solution is bubbled, poured into a container having an open upper surface and a flat bottom surface, and freeze-dried at -40 ° C or lower, and a large number of pores are present, and the upper surface side of the container is the upper surface and the lower surface side is the lower surface. A sponge-like collagen matrix is obtained, a thin film of a water-permeable substance is laminated on the upper surface of the collagen matrix, and the mixture is treated at a low pressure and a high temperature, and the artificial body according to the above (6) to (8). Method of manufacturing skin.

In the present invention, the collagen matrix has a sponge-like structure having a large number of pores, and the diameter of the pores is 100 to 1000 μm, more preferably 200 to 500 μm. The sponge-like structure preferably has an asymmetric structure. In the present invention, the asymmetric structure means that, within the scope of the present invention, one having a large diameter and one having a small diameter are formed as a group. Specifically, there are pores with a relatively large diameter on the surface side of the bone defect portion or the wound surface of the skin that comes into contact with biological tissue, and there are pores with a relatively small diameter on the opposite side. Indicates that.

In the present invention, the shape of the holes is not limited at all, and may be communication holes. The asymmetrical structure in the case of the communication hole means that the diameter of the surface side that is in contact with the living tissue is large and the diameter becomes tapered as it approaches the facing surface side.

The collagen used as a raw material in the present invention is not particularly limited, but a mixture of fibrotic atelocollagen and denatured atelocollagen having a helix content of 0 to 80% is particularly preferable. By mixing denatured collagen, it is possible to further enhance the initial cell invasion and prevent non-specific collagen negative trophic calcification.

In the present invention, the collagen matrix has a degree of crosslinking of 30 to 80%, more preferably 40.
It has a degree of crosslinking of ˜70%. When the degree of cross-linking is low, it is easily decomposed and absorbed by collagenase, and when a high-strength cross-link is introduced, when implanted in a living body, only a foreign body reaction to collagen appears strongly, and therefore 30 to 80%, more preferably 40 to A degree of crosslinking of 70% is good. The collagen matrix thus obtained is partially dissolved even when immersed in 37 ° C. saline, but the rest is not dissolved and maintains the sponge structure.

The crosslinking treatment in the present invention is not particularly limited as long as the degree of crosslinking can be adjusted within the above range. However, while the collagen matrix cross-linked with γ-rays or drugs uniformly induces foreign body reactions such as phagocyte infiltration, the heat-dehydration cross-linked collagen matrix shows undifferentiated mesenchymal cells and osteoblasts. Invasion of cells and capillaries is observed, but no foreign body reaction is observed and the biocompatibility is high. Therefore, thermal dehydration crosslinking is most preferable. By carrying out these cross-linking treatments, even if they are implanted in a bone defect part of an animal, a wound surface of skin, or other soft tissues or hard tissues, they can remain there for at least 4 weeks.

The degree of crosslinking in the present invention means that measured by the following method. Collagen matrix 0.5 mM
After stirring in HCl at 4 ° C for 15 to 16 hours, 3000 rpm
Centrifuge for 15 minutes to separate the supernatant and the residue. The supernatant at this time is designated as A. Next, add 8M urea to the residue,
Heat at 0 ° C. for 30 minutes and centrifuge at 3000 rpm for 15 minutes to separate the residue and the supernatant. The supernatant at this point is designated as B and the residue as C. Then, measure the protein concentrations in the liquids A and B, and multiply them by their respective volumes to obtain W _A , W _B
And The weight of _C is W _C.

The degree of crosslinking (%) = [(W _B + W _C ) /
(W _A + W _B + W _C )] × 100.

Next, a method for producing the collagen matrix of the present invention will be described. First, a collagen solution is prepared. This is divided into 2 minutes, one is left as it is, and the other is denatured collagen by denaturing this collagen solution by heating at 40 ° C. or higher. While maintaining the temperature at 36 ° C, homogenize with a Waring blender and beat. The solution left to stand is fibrillated at 37 ° C with a phosphate buffer solution, concentrated to a predetermined concentration, mixed with a foamed denatured collagen solution, poured into a container having a predetermined upper surface, and lyophilized. The collagen matrix of the present invention is prepared. At this time, in the collagen matrix, large-diameter pores are formed on the side (lower surface) in contact with the container, and small-diameter pores are formed on the open side (upper surface) of the container.

When the concentration of the denatured collagen is 1% or less in the production, the degenerated collagen that has been foamed disappears when mixed with the fibrillated collagen. Therefore, the concentration of the denatured collagen is preferably 1 to 2%. The frothing temperature is preferably 20 to 40 ° C, more preferably 30 to 3
It is 7 ° C.

In the present invention, a collagen matrix may be mixed with a physiologically active substance. The physiologically active substance is intended to promote healing of the wound surface, and is not particularly limited, and examples thereof include heparin, and other factors include platelet-derived growth factor (PDG).
F), fibroblast growth factor (FGF), endothelial cell growth factor (ECGF), transforming growth factor (TG)
F-β) and the like.

Next, the artificial skin of the present invention has a two-layer structure of a support layer and the above collagen matrix. The use of the artificial skin of the present invention on the wound surface may increase the outflow of exudate and float the membrane, possibly delaying healing. Therefore, the presence of pores in the matrix can improve the adhesion to the wound surface.

The support layer is not particularly limited, but its material includes silicone, polyurethane, polyolefin, styrene-butadiene block polymer, polytetrafluoroethylene and the like, and the thickness thereof is 50 to 5.
00 μm, preferably 100 to 200 μm, water vapor transmission rate 500 to 5000 g / m ² · 24 h, preferably 700
~ 2000 g / m ² · 24h.

In the artificial skin of the present invention, the support layer is reinforced with a fibrous reinforcing material made of the same material as or a material different from that of the support layer, whereby deformation of the collagen matrix due to external pressure or It is possible to prevent the peeling from the wound surface and prevent the decrease in cell invasion due to the deformation of the sponge structure. In addition, by imparting an appropriate rigidity during use, it is possible to prevent twisting and peeling during sticking. Furthermore, the presence of fibers in the membrane facilitates suturing when fixing the wound surface, and can reduce peeling from the wound surface. Furthermore, after a certain period of time after transplantation, the upper support layer is peeled off, and a self-divided split-thickness skin grafting is performed, whereby better survival and healing of the split-thickness skin graft can be obtained.

Next, a method for producing the artificial skin of the present invention will be described. (1) First, a collagen solution is prepared. This is divided into 2 minutes, one is left as it is, and the other is denatured collagen by denaturing this collagen solution by heating at 40 ° C. or higher. While maintaining the temperature at 36 ° C, homogenize with a Waring blender and beat. The solution left to stand is made into fiber at 37 ° C with a phosphate buffer solution, concentrated to a predetermined concentration, mixed with a foamed denatured collagen solution, poured into a container having a predetermined upper surface, and freeze-dried. The collagen matrix of the present invention is prepared.
At this time, in the collagen matrix, large-diameter pores are formed on the side (lower surface) in contact with the container, and small-diameter pores are formed on the open side (upper surface) of the container.

(2) Next, a solution of a water-permeable substance such as silicone is spread on a Teflon substrate to form a film, and in a state where the film still has an adhesive property, the above collagen matrix layer is formed above the substrate. The film is placed so that its surface adheres to the membrane, dried, and optionally heat-treated to cure the membrane, and the moisture permeation control membrane is adhered to the upper surface of the support layer. (3) Finally, the composite film is peeled off from the substrate and subjected to heat dehydration cross-linking under vacuum to obtain a desired artificial skin.

[0027]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

(Example 1) Preparation of collagen sponge Preparation of heat-denatured atelocollagen Atelocollagen (manufactured by Koken Co., Ltd.) was added at pH 3 at 4 ° C.
Dissolve it in the hydrochloric acid solution of, and adjust to a 0.2-0.3% solution. The solution was sterilized by filtration with a 0.2 μm filter and freeze-dried. The obtained atelocollagen powder was swollen with RO water and then denatured in a constant temperature bath at 60 ° C. for 30 minutes or more. Further, the heat-denatured atelocollagen was immersed in a constant temperature bath at 36 ° C. for 2 hours, then homogenized with a Waring blender and foamed to obtain a creamy heat-denatured atelocollagen solution.

Preparation of Fibrotic Atelocollagen Solution Atelocollagen was dissolved in a hydrochloric acid solution of pH 3 to 0.2
Adjust to ~ 0.3% solution. The solution was sterilized by filtration through a 0.2 μm filter and phosphate buffer (pH 7.4) was added to give a final phosphate concentration of 30 mM sodium phosphate, 100.
mM sodium chloride, collagen concentration 0.25%
Was adjusted so that This solution was left at 37 ° C. for 3 hours or longer to convert collagen into a fibrous state. The fibers were concentrated to about 2-4% by a centrifuge.

Preparation of Heparin / Fibrotic Atelocollagen Solution A 0.25% atelocollagen solution was prepared in the same manner as above. Heparin was added as a physiologically active substance so as to be 0.25% with respect to the amount of collagen. This solution was left at 37 ° C. for 3 hours or longer to convert collagen into a fibrous state. The fibers were concentrated to about 2-4% by a centrifuge.

Fibrotic Atelocollagen-Heat Denatured Atelocollagen The fibrotic atelocollagen solution prepared above and the creamy 1.5% heat denatured atelocollagen solution prepared above were mixed and thoroughly mixed. At this time, the creamy foam did not disappear even when mixed. This solution is poured into a plastic petri dish having a bottom on the bottom and an opening on the top, and the mixture is rapidly frozen at -40 ° C and then dried.
A collagen sponge was prepared by forming pores with a large diameter on the lower surface and pores with a small diameter on the upper surface.

Heparin / fibrotic atelocollagen-heat-denatured atelocollagen The heparin / heat-denatured atelocollagen solution prepared above and the creamy 1.5% heat-denatured atelocollagen solution prepared above were mixed and thoroughly mixed. At this time, the creamy foam did not disappear even when mixed. This solution is poured into a plastic petri dish having a bottom on the bottom and an opening on the top, and after rapid freezing at -40 ° C, drying is performed, and pores with large diameter are on the lower surface and pores with small diameter are collagen sponge. Was produced.

Example 2 Preparation of Cell-Invading Collagen Preparation
Preparation (1) The collagen matrix prepared in Example 1 was subjected to heat dehydration crosslinking at a temperature of not more than 0.05 Torr at 110 ° C. for 3 hours to obtain a cell-penetrating collagen preparation of the present invention.
At this time, the degree of crosslinking was 52%.

The obtained cell-penetrating collagen preparation was implanted on the cranial parietal bone of rat to evaluate bone remodeling. As the animal, about 700 g of Sprague-Dawlay (SD) male rat was used. Pentobarbital was intraperitoneally administered at 33 mg / kg BW and anesthetized, and then fixed in an abdomen. Craniotomy was performed and connective tissue was peeled off to expose the bone surface. A bone defect wound was prepared by removing the periosteum and polishing the skull. As the cell-penetrating collagen preparation, a comparative example was used in addition to the example. After implantation,
At the 4th week, autopsy was performed, and the skull was removed and fixed with formalin. After being cut to a thickness of about 2 to 3 mm, it was decalcified with a formic acid-formalin aqueous solution and thoroughly washed with water after completion of decalcification, and the implanted cell-penetrating collagen preparation and surrounding bone tissue were evaluated histopathologically. . Table 1 shows the results.

Example 3 Cell-Invading Collagen Preparation
Preparation (2) The collagen matrix prepared in Example 1 was thermally dehydrated and crosslinked at a temperature of not more than 0.05 Torr and 110 ° C. for 3 hours to obtain a cell-penetrating collagen preparation of the present invention.
The degree of crosslinking at this time was 65%. The obtained cell-penetrating collagen preparation was implanted by the cranioparietal bone embedding method as in Example 2, and the cell-penetrating collagen preparation and the surrounding bone tissue were evaluated histopathologically. Table 1 shows the results.

Comparative Example 1 Fibrotic atelocollagen was obtained by the same method as in Example 1-. Heat-denatured atelocollagen was obtained by the same method as in Example 1 except that homogenization was not performed with a Waring blender, and fibrillated atelocollagen was added to 15% of fibrillated atelocollagen to obtain a mixture of fibrillated atelocollagen-heat denatured atelocollagen. It was It is poured into a plastic petri dish having a bottom on the bottom and an opening on the top, and it is rapidly frozen at -40 ° C to obtain a collagen sponge, and then the collagen sponge is dried and thermally dehydrated and cross-linked at 110 ° C for 1 hour at 0.05 Torr or less. Then, a cell-penetrating collagen preparation was obtained. The degree of crosslinking at this time was 14%.

The obtained cell-penetrating collagen preparation was evaluated histopathologically for the cell-penetrating collagen preparation and peripheral bone tissue implanted by the cranioparietal bone embedding method as in Example 2. Table 1 shows the results.

Comparative Example 2 Fibrotic atelocollagen was obtained in the same manner as in Example 1 and heat-denatured atelocollagen was obtained in the same manner as in Example 1 to obtain 15% of the fibrotic atelocollagen. In addition to fibrotic atelocollagen, a mixture of fibrotic atelocollagen-heat denatured atelocollagen was obtained. It is poured into a plastic petri dish having a bottom on the bottom and an opening on the top, and it is rapidly frozen at -40 ° C to obtain a collagen sponge, and then the collagen sponge is dried and thermally dehydrated and crosslinked at 0.05 ° C or less and 110 ° C for 24 hours. Then, a cell-penetrating collagen preparation was obtained. At this time, the degree of crosslinking was 85%.

The obtained cell-penetrating collagen preparation was evaluated histopathologically for the cell-penetrating collagen preparation and peripheral bone tissue implanted by the cranioparietal bone embedding method as in Example 1. Table 1 shows the results.

[0040]

[Table 1]

In the cell-invading collagen preparations of Examples 1 and 2, remarkable bone reconstitution was observed, but in the cell-penetrating collagen preparation of Comparative Example 2, infiltration of inflammatory cells was observed, but the cells of Comparative Example 1 were In the invasive collagen preparation, the disappearance of collagen was clearly observed, and the bone remodeling was not observed.

Example 4 Preparation of Artificial Skin (1) Medical Grade Cylastic Type A silicone (manufactured by Dow Corning) is diluted with hexane to a concentration of 67% and dissolved. This solution is cast on a Teflon plate and 300 μm with a precision coating tool (applicator).
Then, the collagen sponge obtained in Example 1 was placed thereon and adhered. This composite film is 60 ℃, 3
Cure over time. Finally, below 0.05 Torr, 1
Heat treatment was performed at 10 ° C. or higher for 3 hours or longer to obtain the artificial skin of the present invention.

Next, the obtained artificial skin was applied to the rat back entire layer defect layer, and the fibroblast invasion was evaluated. Ha
An rtley-type guinea pig (8 weeks old) was shaved under Nembutal anesthesia to prepare a 2 cm square full-thickness layer with subcutaneous cutaneous muscle on the back, which was disinfected with isodine. After bleeding, artificial skin was applied. The edges of the silicone layer were fixed with knots at 16 places with sutures. Merolin (manufactured by S & N Co.) was placed on it and pressed and fixed with elastin tape. The cells were observed on the 7th, 10th and 14th days after transplantation and evaluated histopathologically. The results are shown in Table 2.

(Example 5) Preparation of artificial skin (2) The collagen sponge obtained in Example 1- and Comparative Example 1 was placed on the silicone film obtained in Example 4 and adhered thereto. The same treatment as above was performed to obtain the artificial skin of the present invention. The obtained artificial skin was histopathologically evaluated for the invasion of fibroblasts into the sample by the method of sticking to the rat dorsal full-thickness layer as in Example 4. The results are shown in Table 2.
Shown in

(Comparative Example 3) The collagen sponge obtained in Comparative Example 1 was placed on and adhered to the silicone film obtained in Example 4, and the same treatment as in Example 4 was performed below to give an artificial skin for comparison. Got The obtained artificial skin was histopathologically evaluated for the invasion of fibroblasts into the sample by the method of sticking to the rat dorsal full-thickness layer as in Example 4. The results are shown in Table 2.

Example 6 Preparation of Artificial Skin (3) The silicone / hexane solution obtained above was cast on a Teflon plate and stretched to 300 μm with an applicator, on which a polyester woven cloth (fiber thickness 100 μm , 70 g / cm ² ) of the basis weight, and the collagen sponge obtained in Example 1 was further placed thereon for adhesion. Then, the same treatment as in Example 4 was performed to obtain the artificial skin of the present invention. The obtained artificial skin was histopathologically evaluated for the invasion of fibroblasts into the sample by the method of sticking to the rat dorsal full-thickness layer as in Example 4. The results are shown in Table 2.

[0047]

[Table 2]

[0048]

As described above in detail, the cell-invading collagen preparation and the artificial skin can be obtained by the production method of the present invention. The cell-penetrating collagen preparation has an excellent cell-penetrating property because the collagen sponge constituting the cell-penetrating collagen has pores of 100 to 1000 μm. Further, by setting the degree of crosslinking of collagen to 30 to 80%, it has excellent physical strength. Therefore, the cell-penetrating collagen preparation can be used as a filling agent for a biological tissue defect such as a bone repair material, and when used as a bone repair material, undifferentiated mesenchymal cells, osteoblasts, and capillaries leading to bone remodeling. It can lead to invasion of blood vessels and achieve bone reconstruction. Further, by mixing denatured atelocollagen, the initial cell invasion property can be further enhanced, and by adding heparin, vascular invasion can be promoted, and as a result, bone remodeling can be enhanced.

The artificial skin of the present invention comprises a support layer and 100-
It consists of collagen sponge with pores of 1000 μm and has excellent cell invasion. Further, by setting the degree of crosslinking of collagen to 30 to 80%, it has excellent physical strength. Therefore, the artificial skin of the present invention is a wound,
When the tissue is damaged by burns, decubitus, etc., it is applied to the damaged surface to protect the wound surface, relieve pain, prevent bacterial infection, and early invasion of fibroblasts and capillaries. ,
Wound healing is promoted by building dermis-like tissue. Furthermore, after a certain period of time after the transplantation, the upper support layer is peeled off, and a self-divided split-thickness skin grafting is performed, whereby better survival and healing of the split-thickness skin graft can be obtained.

Claims (9)

[Claims] 1. A sponge-like collagen matrix having a large number of pores, wherein the diameter of the pores is 100 to 100.
Cell-penetrating collagen preparation characterized by having a size of 1000 μm. 2. The cell-penetrating collagen preparation according to claim 1, wherein the pores present in the collagen matrix have an asymmetric structure. 3. The collagen matrix is a mixture of fibrotic atelocollagen and denatured atelocollagen having a helix content of 0 to 80%.
The cell-penetrating collagen preparation according to any one of 1 to 3. 4. A bone repair material comprising the cell-penetrating collagen preparation according to claim 1. 5. The method for producing a cell-penetrating collagen preparation according to claim 1, wherein the raw material collagen solution is foamed and lyophilized, and then treated at low temperature and high temperature. 6. A wound contact layer and a supporting layer laminated on the wound contact layer, wherein the wound contact layer comprises a sponge-like collagen matrix having a large number of pores, and the pores have a diameter of 100. Artificial skin characterized by being ~ 1000 μm. 7. The artificial skin according to claim 6, wherein the pores present in the collagen matrix have an asymmetric structure. 8. The collagen matrix is a mixture of fibrotic atelocollagen and denatured atelocollagen having a helix content of 0 to 80%.
The artificial skin according to any one of 1 to 7. 9. A sponge-like composition in which the raw material collagen solution is bubbled, poured into a container having an open upper surface and a flat bottom surface and lyophilized, and a large number of holes are present, and the upper surface side of the container is the upper surface and the lower surface side is the lower surface. The method for producing artificial skin according to claim 6, wherein a collagen matrix is obtained, a thin film of a water-permeable substance is laminated on the upper surface of the collagen matrix, and the thin film is treated at a low pressure and a high temperature.