JP2007130118A - Grafting material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To lift up the skin permanently, to reconstruct or restore tissue and to three-dimensionally construct regeneration tissue for grafting. <P>SOLUTION: The grafting material for the treatment of lifting up the skin includes at least one kind of cells selected from a group composed of osteoblasts and chondrocytes and a gelling material or a gelling precursor material which can be gelified inside a living body. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an implant material.

  The transplant material of the present invention can retain the function of cells for a long time even after transplantation.

  Regenerative medicine in which various cells are cultured and proliferated and then transplanted into a living body is being performed. Many methods perform cell culture growth on a two-dimensional plane such as the surface of a flask, collect the cells and transplant the cells themselves, or collect the collected cells with various inorganic materials such as ceramics of various porous bodies. After seeding in a bioabsorbable polymer such as polylactic acid to construct a three-dimensional cultured tissue, it is transplanted in vivo.

  By these methods, it is possible to produce a three-dimensional cultured tissue using various biomaterials, but inorganic materials such as ceramics are hard and are excellent in repairing bone tissues and the like that are load sites. It is difficult to use for tissue / organ repair. In addition, bioabsorbable polymers such as polylactic acid can control hardness, in vivo degradation rate, etc. by modifying the synthesis process, but the cell adhesion site is scarce and effective cell seeding is difficult. is there.

  In addition, there are cases where a body part is lost due to a surgical operation such as a mastectomy for breast cancer treatment, an accident, or the like, and there is a case where the body part is desired to be repaired, particularly raised for cosmetic purposes.

  It is an object of the present invention to perform permanent skin ridges and to reconstruct or repair tissue.

  Another object of the present invention is to three-dimensionally construct a regenerative tissue for transplantation.

The present invention relates to the following transplant materials.
1. A transplant material for treatment for raising skin, comprising at least one cell selected from the group consisting of osteoblasts and chondrocytes and a gel material or a gel precursor material that can be gelled in vivo.
2. Item 2. The transplant material according to Item 1, wherein the gel material or gel precursor material is a natural or synthetic or semi-synthetic protein, peptide, polysaccharide or hydrogel.
3. Item 3. The method according to Item 1 or 2, wherein the gel material is at least one selected from the group consisting of various synthetic peptides.
4). Item 4. The method according to any one of Items 1 to 3, wherein the volume of cells in the volume of the gel material or gel precursor material is 5-95%.
5. Item 5. The method according to any one of Items 1 to 4, wherein the gel has a gel viscosity of 1 to 1 × 10 ⁶ mPa · s (cP).
6). Item 6. The transplant material according to any one of Items 1 to 5, wherein the skin bulge is performed to reconstruct a tissue lost due to a surgical operation, a disease, or an accident.
7). Item 6. The transplant material according to any one of Items 1 to 5, wherein the skin bulge is performed to repair breast augmentation, bulge nose, or wrinkles or sagging.
8). A transplant material obtained by introducing mesenchymal stem cells in a gel material or cells differentiable therefrom into a biocompatible material.
9. The biocompatible material is
(1) hydroxyapatite, tricalcium phosphate, calcium deficient apatite,
Ceramic materials including amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, inorganic compounds capable of adsorbing calcium phosphates in vivo;
(2) Chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylated products and cross-links Natural polymers including the body; and
(3) A homopolymer or copolymer of a bioabsorbable polymer such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylate ester polymer, silicone resin, polylactic acid, polyglycolic acid, polyε-caprolactone, etc. Including synthetic polymers;
Item 9. The transplant material according to Item 8, which is at least one selected from the group consisting of:
10. Item 10. The transplant material according to Item 8 or 9, wherein the biocompatible material has a lump shape, a nonwoven fabric shape, a sheet shape, a bead shape, a disk shape, a porous shape, or a sponge shape.
11. Item 11. The transplant material according to any one of Items 8 to 10, wherein the mesenchymal stem cells are differentiated into various tissues in a polymer gel.
12 Item 12. The transplant material according to any one of Items 8 to 11, wherein the differentiated tissue is bone, cartilage, nerve, or liver.

  Cells are mixed with a precursor of gel material and gelled as necessary to retain the cells. By using a differentiation-inducing culture method, three-dimensional cultured tissues can be created in vitro and in vivo. did. These can be regenerated and reconstructed in various ways by transplanting directly to the affected area or a site requiring repair. As the transplantation method, the cultured tissue can be directly implanted into the affected area after the incision, but means such as injection using a syringe or a catheter without performing an operation such as incision can also be used.

  In the present invention, osteoblasts and chondrocytes are not particularly limited, and for example, those induced to differentiate from various mesenchymal stem cells can be used. Such differentiation induction from mesenchymal stem cells can be performed according to a conventional method.

  When the transplant material of the present invention containing osteoblasts and / or chondrocytes can be treated by partial bulging of the skin surface, such as breast reconstruction or taking breast enlargement, nose and wrinkles It can be preferably used for repair. The transplant material of the present invention forms a three-dimensional gel in vivo, and produces bone or cartilage tissue. Although this tissue has a certain strength, there is no sense of incongruity even when the skin surface is touched from the outside, and the raised state of the skin can be maintained permanently. The bone or cartilage tissue produced by the transplant material of the present invention does not require high strength as long as it can maintain the skin bulge.

When the transplanted material is in the form of a gel, it can be implanted subcutaneously by a surgical operation and is not gelled but is implanted in the living body (for example, body fluid such as blood or tissue fluid). In the case of containing a gel precursor material and an osteoblast / chondrocyte that can be gelled by reacting with each other, it may be injected under the site where the skin is to be raised by injection. The amount of cells to be injected can be a small amount of cells that can be placed into the body if there is only a slight skin bulge in a relatively limited area, such as an injury or scar, or a wrinkle or sagging. If large bulges such as breast reconstruction or breast augmentation are required, high-density cells will be used in large quantities. Since the transplant material of the present invention gels in vivo and can retain chondrocytes / osteoblasts inside, it can produce cartilage / bone tissue in the gel and keep the skin raised for a long time. it can.

For example, by injecting mesenchymal cells encapsulated in a gel directly under a wrinkle or skin dent that causes a cosmetic problem, wrinkles or dents are eliminated. The number of cells used in this case is 1 per applicable site.
X10 ⁶ to 1 × 10 ⁸ are exemplified. Furthermore, in the case of breast reconstruction or breast augmentation, a large amount of cells (1 × 10 ⁸ to 1 × 10 ⁹ cells can be used, and more cells can be used.

  As the gel material or gel precursor material used in the present invention, natural, synthetic or semi-synthetic proteins, peptides, polysaccharides, hydrogels and the like can be used.

  Specific gel materials or gel precursor materials include polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch and pectin, proteins such as gelatin, collagen, fibrin and albumin, poly-γ-glutamic acid, poly-L -Polyamino acids such as lysine and polyarginine, synthetic peptides having a repeating structure of arginine / alanine / aspartic acid, and derivatives thereof (for example, those crosslinked by a crosslinking agent or heat).

The volume of cells occupying the volume of the gel material or gel precursor material is usually 1 to 95%, preferably 5 to 90%, more preferably 30 to 60%.

The method for introducing cells into the gel material or the method for introducing the gel material containing cells into the biocompatible material is not particularly limited. For example, the size is larger than 1 G (G is gravitational acceleration) and is 200 G or less. It is preferable to apply an external force. Examples of the external force include centrifugal force, pressurization, decompression, and vibration.

  In the transplant material of another embodiment of the present invention, mesenchymal stem cells or cells differentiable therefrom are present in the gel material, and the gel material (including cells inside) is further introduced into the biocompatible material. It has the structure made. The transplanted material is obtained by culturing mesenchymal stem cells in the gel material or gel precursor material, and further introducing it into a biocompatible material to induce gelation or stem cell differentiation as necessary, and continue the culture. Can be manufactured. Such a transplant material can be obtained by three-dimensionally culturing in a biocompatible material, and can function as a regenerative tissue that functions three-dimensionally in a living body, unlike planar cultured cells. . When the cells are inside the gel, the three-dimensional structure of the cells can be easily maintained, and the functionality after transplantation can be enhanced.

Examples of the biocompatible material include the following (1) to (3):
(1) Hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, calcium phosphate in vivo Ceramic-based materials including inorganic compounds capable of adsorbing species;
(2) chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate,
Natural polymers including alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylates and cross-linked products; and
(3) A homopolymer or copolymer of a bioabsorbable polymer such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylate ester polymer, silicone resin, polylactic acid, polyglycolic acid, polyε-caprolactone, etc. Including synthetic polymers.

Preferred biocompatible materials include hydroxyapatite, triphosphate calcium,
Examples thereof include calcium carbonate, polylactic acid, and polyglycolic acid.

In one specific embodiment, mesenchymal stem cells can be encapsulated in a gel and used to induce differentiation as needed, but by performing differentiation-inducing culture for 1 to 28 days, Stem cells are differentiated into chondrocytes or osteoblasts and then encapsulated in gel, or mesenchymal stem cells encapsulated in gel are cultured for 1 to 28 days to differentiate into chondrocytes or osteoblasts. It is also possible to transplant after transplantation. In this case, since the cartilage matrix or the bone matrix is formed in the gel, a certain degree of hardness can be maintained, and transplantation is facilitated.

The transplant material of the present invention is, for example, adjusted to a relatively high cell concentration of 1 × 10 ⁶ to 1 × 10 ⁸ cells / ml for bone / cartilage regeneration and transplanted directly into a bone defect or cartilage defect, Alternatively, it is transplanted in combination with a biocompatible material, that is, a gel material encapsulating cells or a precursor material thereof is seeded on various biocompatible materials and cultured for a short period of 1 to 24 hours. After adhering at a density, it is transplanted into a bone defect or cartilage defect. Thereby, it differentiates into bone tissue or cartilage tissue at the site where the mesenchymal cells are transplanted. Furthermore, in order to produce cartilage matrix and bone matrix sufficiently, it is possible to extend the culture period for 1 to 3 weeks.

Treatment of ischemic myocardium, dilated cardiomyopathy, or chronic peripheral arterial occlusive disease (diabetic necrosis, arteriosclerotic occlusion, Buerger disease, etc.) affected area with mesenchymal cells encapsulated in gel Inject. In that case, 1 × 10 ⁷ to 1 × 10 ⁹ cells can be used per affected area.

  Further, mesenchymal cells can be induced to differentiate into various cells such as hepatocytes and nerve cells by addition of various differentiation-inducing factors, as well as bone and chondrocytes. It is also possible to regenerate it by transplanting it to a repaired part of various tissues / organs.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1
Example 1 of the present invention describes a method for producing a rat bone marrow-derived mesenchymal cell / synthetic polymer gel complex, and the results of an experiment for inducing bone differentiation of rat bone marrow-derived mesenchymal cells in the synthetic polymer gel. To do.

1. Cell Culture Rat bone marrow-derived mesenchymal cells (hereinafter referred to as MSC) were collected from the male Fisher344 rat femur. The collected MSCs are cultured in MEM medium supplemented with 15% fetal bovine serum (hereinafter referred to as FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (hereinafter referred to as basic medium). And allowed to grow. MSCs that adhered to the bottom of the culture dish and proliferated were collected by trypsin treatment, and a cell suspension was prepared using a 10% sucrose solution so that the cell concentration became 1 × 10 ⁶ cells / mL.

The above cell suspension is mixed with a liquid containing 1% synthetic polymer, brought into contact with a basic medium, the liquid containing the synthetic polymer is gelled, and the MSC / synthetic polymer gel complex encapsulating MSC It was created. The polymer is formed from a synthetic peptide, and the amino acid sequence constituting the polymer is as follows.
Ac-RADARADARADA-CONH ₂ (Ac: amino terminal, R: arginine, A: alanine,
D: Aspartic acid, CONH ₂ : carboxyl terminal)
From the next day, induction of bone differentiation of MSC was started. Bone differentiation induction medium (10 nM dexamethasone, 10 mM β-glycerophosphate, 0.28 mM ascorbic acid was added to the basic medium) was used for induction of bone differentiation. As a control, a medium obtained by removing dexamethasone from a bone differentiation-inducing medium was used. The culture was performed at 37 ° C. in the presence of 5% CO ₂ , and the medium was changed every 48 hours.

2. Morphological evaluation
In order to confirm that MSC differentiated into osteoblasts by induction of bone differentiation and bone matrix was produced, fluorescence with high affinity to bone matrix at 2 weeks, 3 weeks and 4 weeks after differentiation induction Staining was performed using a reagent (calcein), and observation was performed with a fluorescence microscope and a confocal laser microscope. In the bone differentiation-inducing group, a fluorescent region showing bone matrix deposition could be observed, but in the control group, no obvious fluorescent region was observed.

Using a confocal laser microscope, an image in which the fluorescent region diffuses not only in the XY plane but also in the Z-axis direction was captured in the bone differentiation-inducing group. Moreover, the tendency which the fluorescence area | region expanded was seen with time passage (FIGS. 1-3).

3. Biochemical evaluation (1)
In order to confirm that MSC differentiated into osteoblasts by induction of bone differentiation, cells were collected from the MSC / synthetic polymer gel complex at 2 weeks, 3 weeks, and 4 weeks after induction of differentiation. Phosphatase activity was measured. Alkaline phosphatase activity is measured using PNP in the presence of cells and PNPP.
This was carried out by quantifying the production amount.

In the evaluation, the amount of PNP produced in 30 minutes was converted per cell amount. The bone differentiation induction group showed significantly higher alkaline phosphatase activity than the control group. (Fig. 4)

4). Biochemical evaluation (2)
After the above measurement, the bone matrix (calcified bone matrix) was extracted from the MSC / synthetic polymer gel complex using an organic acid, and the amount of calcium was determined by the ICP method and the amount of osteocalcin was determined by the EIA method. In the differentiation induction group, significantly higher calcium and osteocalcin levels were quantified than in the control group (FIGS. 5 and 6).

From the above, it was shown that MSC proliferates in a synthetic polymer gel, differentiates into osteoblasts by induction of bone differentiation, and produces bone matrix in three dimensions.

Example 2
As Example 2 of the present invention, the results of an experiment of transplanting a synthetic polymer gel encapsulating rat bone marrow-derived mesenchymal cells into a syngeneic rat seeded with a porous ceramic will be described.

1. Cell Culture Rat bone marrow-derived mesenchymal cells (hereinafter referred to as MSC) were collected from the male Fisher344 rat femur. The collected MSCs are cultured in MEM medium supplemented with 15% fetal bovine serum (hereinafter referred to as FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (hereinafter referred to as basic medium). And allowed to grow. MSCs that adhered to the bottom of the culture dish and proliferated were collected by trypsin treatment, and a cell suspension was prepared using a 10% sucrose solution so that the cell concentration became 1 × 10 ⁶ cells / mL.

After the above cell suspension is mixed with a liquid containing 1% synthetic polymer, this mixed liquid is seeded on a porous ceramic and immediately contacted with a basic medium to gel the liquid containing the synthetic polymer. An MSC / synthetic polymer gel / ceramic composite containing MSC was prepared. The polymer is formed from a synthetic peptide, and the amino acid sequence constituting the polymer is as follows.
Ac-RADARADARADA-CONH ₂ (Ac: amino terminal, R: arginine, A: alanine, D
: Aspartic acid, CONH ₂ : carboxyl terminal)
From the next day, induction of bone differentiation of MSC was started. Bone differentiation induction medium (10 nM dexamethasone, 10 mM β-glycerophosphate, 0.28 mM ascorbic acid was added to the basic medium) was used for induction of bone differentiation. The culture was performed at 37 ° C. in the presence of 5% CO ₂ for 2 weeks, and the medium was changed every 48 hours.

2. Implantation experiment The MSC / synthetic polymer gel / ceramic composite prepared in 1 above was implanted subcutaneously in the back of male Fisher344 rats. As a control, a synthetic polymer solution containing no cells was gelled on a ceramic, and a synthetic polymer gel / ceramic composite was transplanted.

3. Histological evaluation
After 4 weeks, the implanted MSC / synthetic polymer gel / ceramic composite was recovered from the rat. For histological evaluation, the recovered MSC / synthetic polymer gel / ceramic composite is fixed in formalin, embedded in paraffin after decalcification, sliced, and stained with hematoxylin-eosin to prepare a tissue specimen did.

When the tissue specimen was observed with an optical microscope, deposition of bone matrix was observed along the pore wall of the porous ceramic only in the bone differentiation-inducing group. Bone matrix deposition was observed not only in the ceramic surface, but also in the internal pores (Figure 7-8).

From the above, by using the inside of a synthetic polymer gel encapsulating MSC, MSC is efficiently seeded on the porous ceramic, differentiated into osteoblasts by induction of bone differentiation, and three-dimensionally in vivo It has been shown to produce bone matrix.

Example 3
As Example 3 of the present invention, a method for producing a rat bone marrow-derived mesenchymal cell / polymer gel complex,
The results of the bone differentiation induction experiment of rat bone marrow-derived mesenchymal cells in a polymer gel are described.

1. Cell Culture Rat bone marrow-derived mesenchymal cells (hereinafter referred to as MSC) were collected from the male Fisher344 rat femur. The collected MSCs are cultured in MEM medium supplemented with 15% fetal bovine serum (hereinafter referred to as FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (hereinafter referred to as basic medium). And allowed to grow. MSCs that adhered to the bottom of the culture dish and proliferated were collected by trypsin treatment, and a cell suspension was prepared using a 10% sucrose solution so that the cell concentration became 1 × 10 ⁶ cells / mL.

The above cell suspension is mixed with a liquid containing 1% synthetic polymer, brought into contact with a basic medium, the liquid containing the synthetic polymer is gelled, and the MSC / synthetic polymer gel complex encapsulating MSC It was created. The polymer is formed from a synthetic peptide, and the amino acid sequence constituting the polymer is as follows.
Ac-RADARADARADA-CONH ₂ (Ac: amino terminal, R: arginine, A: alanine, D: aspartic acid, CONH ₂ : carboxyl terminal)
As a control, the above cell suspension was encapsulated in a 1% pig skin-derived type I collagen solution,
The gel was formed by neutralization and heating to prepare an MSC / collagen gel complex containing MSC.

From the next day, induction of bone differentiation of MSC was started. Bone differentiation induction medium (10 nM dexamethasone, 10 mM β-glycerophosphate, 0.28 mM ascorbic acid was added to the basic medium) was used for induction of bone differentiation. As a control, a medium obtained by removing dexamethasone from a bone differentiation-inducing medium was used. The culture was performed at 37 ° C. in the presence of 5% CO ₂ , the medium was changed every 48 hours, and the culture was continued for 4 weeks.

2． Biochemical evaluation At 2 weeks, 3 weeks, and 4 weeks after differentiation induction, bone matrix (calcified bone matrix) from MSC / polymer gel complex
Was extracted with an organic acid, and the amount of calcium was quantified by ICP method. In the differentiation induction group, MSC /
Both the synthetic polymer gel complex and the MSC / collagen gel complex were quantified with significantly higher calcium values than the control group. In addition, the calcium values between the MSC / synthetic polymer gel complex and the MSC / collagen gel complex in the differentiation induction group were comparable (FIG. 9).

From the above, it was shown that MSC proliferates to the same extent in synthetic polymer gels and collagen gels, and differentiates into osteoblasts to the same extent by inducing bone differentiation to produce bone matrix.

Example 4
As Example 4 of the present invention, the result of a transplantation experiment of a synthetic polymer gel encapsulating rat bone marrow-derived mesenchymal cells after induction of bone differentiation into syngeneic rats will be described.

1. Cell Culture Rat bone marrow-derived mesenchymal cells (hereinafter referred to as MSC) were collected from the male Fisher344 rat femur. The collected MSCs are cultured in MEM medium supplemented with 15% fetal bovine serum (hereinafter referred to as FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (hereinafter referred to as basic medium). And allowed to grow. Further, the cells were recovered after bone differentiation of MSC using a bone differentiation induction medium for 7 days, and the cell suspension was adjusted using a 10% sucrose solution to a cell concentration of 1 × 10 ⁶ cells / mL.

2. Transplantation experiment The above cell suspension was mixed with a liquid containing 1% synthetic polymer subcutaneously in the back of male Fisher344 rats, and this mixture was then injected subcutaneously into the back of syngeneic rats using a syringe. . The polymer is formed from a synthetic peptide, and the amino acid sequence constituting the polymer is as follows.
Ac-RADARADARADA-CONH ₂ (Ac: amino terminal, R: arginine, A: alanine, D: aspartic acid, CONH ₂ : carboxyl terminal)
As a control, 1% synthetic polymer solution containing no cells was injected subcutaneously into the back of syngeneic rats.

3. Morphological evaluation Synthetic polymer solution containing cells and synthetic polymer solution containing no cells can be injected subcutaneously into the back of syngeneic rats using a syringe. Resulting in skin bulges of similar size.

One week after transplantation, the injection site was observed. The bulge of the skin transplanted with the synthetic polymer solution containing cells maintained the size at the time of injection, while the bulge of the skin transplanted with the synthetic polymer solution containing no cells was It was smaller than. (Fig. 10, 11)

From the above, it was shown that the shape of the bulge of the skin can be maintained over a long period of time by using the cells after induction of bone differentiation. This retention effect is likely due to bone matrix production in the gel of mesenchymal cells. In this regard, we have confirmed that mesenchymal cells also differentiate into chondrocytes. That is, the mesenchymal cells can be differentiated into chondrocytes, and the skin bulging effect by cartilage matrix production can also be expected. That is, so-called wrinkle repair can be expected by using a bone matrix or a cartilage matrix and a gel. In particular, since it is in a gel state, wrinkles can be removed by injection. In view of the results of Example 3, it is expected that similar results will be shown even if animal-derived polymers such as collagen are used. Since collagen is said to have a cosmetic effect on the skin, a small amount of collagen gel and mesenchymal cells, especially a complex of mesenchymal cells and bone or cartilage-differentiated cells and collagen gel, is scraped. It becomes possible to use for. However, under the situation where a large amount of polymer solution is injected, the influence of animal-derived antigens is unknown, and it is preferable to use this method using a synthetic polymer.

Example 5
As Example 5 of the present invention, the results of an experiment for inducing differentiation of rat bone marrow-derived mesenchymal cells into hepatocytes and nerve cells on a culture dish coated with a synthetic polymer solution will be described.

1. Cell Culture Rat bone marrow-derived mesenchymal cells (hereinafter referred to as MSC) were collected from the male Fisher344 rat femur. The collected MSCs are cultured in MEM medium supplemented with 15% fetal bovine serum (hereinafter referred to as FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (hereinafter referred to as basic medium). And allowed to grow. MSCs that adhered to the bottom of the culture dish and proliferated were collected by trypsin treatment, and a cell suspension was prepared using a 10% sucrose solution so that the cell concentration became 1 × 10 ⁶ cells / mL.

A liquid containing 1% synthetic polymer was brought into contact with the following differentiation-inducing medium on a culture dish to gel, and the culture dish was coated with the synthetic polymer gel. The polymer is formed from a synthetic peptide, and the amino acid sequence constituting the polymer is as follows.
Ac-RADARADARADA-CONH ₂ (Ac: amino terminal, R: arginine, A: alanine, D: aspartic acid, CONH ₂ : carboxyl terminal)
The cell suspension was seeded on a culture dish coated with a synthetic polymer gel, and MSC hepatocyte differentiation induction and nerve cell differentiation induction were started the next day. For induction of hepatocyte differentiation, hepatocyte differentiation induction medium (DMEM medium supplemented with 10 nM dexamethasone, ITS, 20 ng / ml hepatocyte growth factor, 10% FBS) was used. ITS is a mixture of insulin, transferrin and selenium. As a control, a medium obtained by removing hepatocyte growth factor from a hepatocyte differentiation-inducing medium was used.

To induce neuronal differentiation, first cultivate in a neuronal differentiation induction medium (10 ng / ml epidermal growth factor, 10 ng / ml platelet-derived growth factor, 1% FBS added to DMEM medium), and after a few days add 50 ng / ml was changed to basic fibroblast growth factor. The culture was performed at 37 ° C. in the presence of 5% CO ₂ for 2 weeks, and the medium was changed every 48 hours.

2． Molecular Biology and Morphological Evaluation Differentiation into hepatocytes is achieved by collecting cultured cells, extracting RNA, synthesizing cDNA, and then expressing the gene expression of β-actin, a housekeeping gene, and albumin, a hepatocyte-specific protein. Confirmed by PCR.

  In both the hepatocyte differentiation-inducing group and the control group, β-actin was expressed at almost the same level, but the albumin gene expression was clearly higher in the differentiation-inducing group.

Differentiation into nerve cells is based on anti-neuron cell-specific enolase (NSE), a neuronal differentiation marker.
) Confirmed by immunostaining method using antibody. Axon-like processes were extended from the cell body, and the morphology similar to that of differentiated neurons was observed.

From the above, it was shown that mesenchymal cells differentiate into hepatocytes and neurons by induction of differentiation using a synthetic polymer gel as a carrier.

FIG. 1 is a fluorescence micrograph 2 weeks after induction of bone differentiation. A shows a differentiation induction group, B shows a control group. The green part is the deposited part of the bone matrix stained with calcein. FIG. 2 is a confocal laser scanning micrograph of the bone differentiation-inducing group 2 weeks after the induction of bone differentiation. A is an XY plane image, and B is a tomographic image in the Z-axis direction. The green part is the deposited part of the bone matrix stained with calcein. FIG. 3 is a confocal laser micrograph of the bone differentiation induction group 4 weeks after the induction of bone differentiation. A is an XY plane image, and B is a tomographic image in the Z-axis direction. The green part is the deposited part of the bone matrix stained with calcein. FIG. 4 shows the alkaline phosphatase activity of the cells collected from the MSC / synthetic polymer gel complex. Dex (+) indicates a bone differentiation induction group, and Dex (−) indicates a control group. Values are mean ± SD (standard deviation) of 6 experiments. Asterisks indicate significant differences by statistical methods. FIG. 5 shows the amount of calcium quantified by extracting the bone matrix (calcified bone matrix) from the MSC / synthetic polymer gel complex. Dex (+) indicates a bone differentiation induction group, and Dex (−) indicates a control group. Values are mean ± SD (standard deviation) of 6 experiments. Asterisk indicates significant difference by statistical method (p <0.01). FIG. 6 shows the amount of osteocalcin extracted from the bone matrix (calcified bone matrix) from the MSC / synthetic polymer gel complex and quantified. Dex (+) indicates a bone differentiation induction group, and Dex (−) indicates a control group. Values are mean ± SD (standard deviation) of 6 experiments. Asterisk indicates significant difference by statistical method (p <0.01). FIG. 7 is a tissue specimen image of MSC / synthetic polymer gel / ceramic composite recovered from the bone differentiation-inducing group. A bone matrix (arrow) produced by osteoblasts is deposited in the pores of the porous ceramic. FIG. 8 is a tissue specimen image of the synthetic polymer gel / ceramic composite recovered from the control group. Bone matrix deposition is not observed in the pores of the porous ceramic, and only the invasion (arrows) of fibrous tissue is observed. FIG. 9 shows the amount of calcium quantified by extracting the bone matrix (calcified bone matrix) from the MSC / polymer gel complex. Values are mean ± SD (standard deviation) of 6 experiments. An asterisk indicates a statistically significant difference (p <0.01), and N.S indicates no significant difference. FIG. 10 is a photograph immediately after a synthetic polymer solution and a synthetic polymer solution containing cells are injected subcutaneously into the back of syngeneic rats. A shows a site where a synthetic polymer solution containing cells is injected, and B shows a site where only a synthetic polymer solution is injected. FIG. 11 is a photograph one week after the synthetic polymer solution and the synthetic polymer solution containing cells were injected subcutaneously into the back of syngeneic rats. A shows a site where a synthetic polymer solution containing cells is injected, and B shows a site where only a synthetic polymer solution is injected. FIG. 12 is a photograph of the expression of a gene amplified by the PCR method observed by electrophoresis. The upper row shows the β-actin gene, the lower row shows the albumin gene, the left lane is the control group, and the right lane is the hepatocyte differentiation induction group. FIG. 13 is an immunostaining photograph after neural cell differentiation induction. The left side is a weakly enlarged photo, and the right side is a strongly enlarged photo. Arrowheads indicate neuronal cell bodies, and arrows indicate axon-like neurite outgrowth.

Claims (12)

A transplant material for treatment for raising skin, comprising at least one cell selected from the group consisting of osteoblasts and chondrocytes and a gel material or a gel precursor material that can be gelled in vivo. 2. The implant material according to claim 1, wherein the gel material or gel precursor material is a natural or synthetic or semi-synthetic protein, peptide, polysaccharide or hydrogel. The method according to claim 1 or 2, wherein the gel material is at least one selected from the group consisting of various synthetic peptides. The method according to any one of claims 1 to 3, wherein the volume of cells occupying within the volume of the gel material or gel precursor material is 5-95%. The method according to claim 1, wherein the gel has a gel viscosity of 1 to 1 × 10 ⁶ mPa · s (cP). The transplant material according to any one of claims 1 to 5, wherein the skin bulge is performed to reconstruct tissue lost due to surgery, disease or accident. The transplant material according to any one of claims 1 to 5, wherein the skin bulge is performed to repair breast augmentation, nose nose, or wrinkles or sagging. A transplant material obtained by introducing mesenchymal stem cells in a gel material or cells differentiable therefrom into a biocompatible material. The biocompatible material is
(1) hydroxyapatite, tricalcium phosphate, calcium deficient apatite,
Ceramic materials including amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, inorganic compounds capable of adsorbing calcium phosphates in vivo;
(2) Chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylated products and cross-links Natural polymers including the body; and
(3) A homopolymer or copolymer of a bioabsorbable polymer such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylate ester polymer, silicone resin, polylactic acid, polyglycolic acid, polyε-caprolactone, etc. Including synthetic polymers;
The transplant material according to claim 8, which is at least one selected from the group consisting of: The transplant material according to claim 8 or 9, wherein the biocompatible material has a lump shape, a nonwoven fabric shape, a sheet shape, a bead shape, a disk shape, a porous shape, or a sponge shape. The transplant material according to any one of claims 8 to 10, wherein the mesenchymal stem cells are differentiated into various tissues in a polymer gel. The transplanted material according to any one of claims 8 to 11, wherein the differentiated tissue is bone, cartilage, nerve, or liver.

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