KR101377558B1 - Tissue or Organ repair kit composed of growth factors, barriers and scaffolds - Google Patents

Abstract

The present invention provides a kit for regenerating tissues or organs consisting of three elements, a growth factor, a shield, and a scaffold, wherein the tissues and organs of humans and animals are replaced with the three elements of the growth factor, a scaffold, and a shield of the present invention. Reconstituted kits can be used to regenerate into normal tissues or organs. It is particularly preferred in the case of multiple or multiple defects as well as deletion of one site. Therefore, in the regeneration of tissues or organ defects due to physical or chemical tissues or organ damage or diseases such as inflammation or tumors, synthetic tissues or organs, heterogeneous tissues without side effects of tissue defects or movement and functional constraints of other normal areas Or an innovative kit that can replace transplantation of organs and autologous tissue or organs.

Description

Tissue or Organ repair kit composed of growth factors, barriers and scaffolds}

The present invention relates to a kit for tissue or organ regeneration comprising a growth factor, a shielding membrane, and a scaffold, and more particularly, to remarkably improve the completeness and integrity of tissue or organ regeneration.

Recently, in medicine, the regeneration of tissue or organ defects due to diseases such as tissue or organ damage due to physical, chemical and burn or diseases such as inflammation or tumors has been dealt with importantly. Defects in tissues or organs cause fluids to escape into the air or are easily infected with bacteria. In addition, if the skin, muscle, bone tissue and nerve tissue is damaged in a complex, it is difficult to regenerate into normal tissues or organs of each layer, it is replaced by scar tissue, accompanied by mobility and functional constraints.

Conventionally, in order to solve this problem, by implanting the tissues of other parts of the patient himself or pulling the surrounding tissue to fill or cover the tissue defects, the side effects of tissue defects or mobility and functional constraints of other normal regions have been caused.

Recently, to prevent this drawback, a shield is used to block the growth of other tissues before they are healed or to prevent foreign bacteria from entering the tissue. In addition, it uses a porous scaffold that provides a space for cells of damaged tissue to grow at the defect site. In addition, attempts to grow cells by using growth factors to move stem cells necessary for healing to the defect site by chemotaxis or by manipulating or culturing stem cells necessary for healing or regeneration and transplanting them into the defective site have been increasing.

Shielding membranes have been currently used for shielding membranes made of nanofibers with pores of less than a few micrometers, or for compressing collagen sponges. For physical strength, barrier films reinforced with titanium or stainless steel mesh have also been used. However, when exposed to the open window of the nanofiber shielding membrane, some bacteria had a problem that induces inflammation by invading the micropores while changing the shape. In the present invention, the sheet or film having no pores in the shielding membrane shows better clinical results, and the nanofibers or compressed sponges are returned to their original unfolded shape even when deformed to conform to the curved shape, but the sheet or film is maintained in the deformed shape. Since the advantages are very clinically beneficial, the shielding membrane using the same is included in the product composition.

Scaffolds are used to maintain the shape and space of cells in damaged tissues for growth. The scaffold is composed of an absorbent polymer, calcium phosphate ceramic, or the like. However, the side effect is that cells from other adjacent tissues grow inside the scaffold even before the cells of damaged tissue are fully grown. In the present invention, the scaffold and the shielding film are configured in one combination to sufficiently complement the function of the shielding film and the role of the scaffolding.

Growth factors such as BMP-2 allow mesenchymal stem cells, which are necessary for healing, to migrate to the defect site with chemotaxis and differentiate into various tissues. BMP-2 grows mesenchymal stem cells into bone tissue in patent WO 1993/000050, BMP-2 induces growth of neural tissue in patent WO 1995/005846, and BMP-2 tendons or ligaments in patent WO 1996/039169 It is said to induce regeneration. Based on the report that BMP-2 acts on the development of skin tissue in the embryo (Scaal et al .; Development growth differentiation, 2008 Volume: 50, Issue: 9, Pages: 743-754), the present invention provides a cell test and a clinical case. Skin tissue regeneration was confirmed through.

The method of inducing tissue regeneration by applying a growth factor to the shielding membrane, such as patent registration KR10-564366, may have insufficient tissue regeneration volume and shape because there is no space for cells of damaged tissue to grow.

Stem cells may be applied by injection method as in Patent Publications KR10-2008-0075959 and Patent Publications KR 10-2009-0078304 and Patent Publication 10-2011-0037907, but the probability of the cells being settled and growing into damaged tissue may be low. As in WO 2009/154840 A2, stem cells may be applied to a defect site with a fibrin sealant, but there is no shielding function so that stem cells may grow to defect areas of other tissues.

Previous patents have researched and developed scaffolds or shields for a state in which one type of tissue is missing. In clinical practice, however, the defects are deep and wide due to cancer, diabetes, and inflammation, and at least one or more types of multiple tissues are damaged, and multiple tissues must be regenerated at the same time. In this patent, a scaffold and a shield are combined to regenerate various growth factors or mesenchymal stem cells to various tissues by using appropriate growth factors or stem cells. The specific method is to arrange the combination of scaffold and shielding layer by layer for each damaged tissue or to regenerate them sequentially by changing the pore size or material of the scaffold in consideration of the regeneration order of each tissue. Combined to be regenerated.

Extensive defects can easily be deformed by external forces and the tissue regeneration can weaken the scaffolds, causing the space and shape of the cells to grow to be distorted, thus maintaining skeletal morphology in each layer in a combination of shielding and scaffolds. Added layer. In addition, in the case of coronary tissues such as blood vessels and neural tissues, a combination of coronary shielding membrane and scaffold was constructed.

Tissue or organ damage due to physical, chemical and burns, or tissue or organ defects caused by diseases such as inflammation or tumors are being addressed.

When fluid or fluid is leaked into the air due to a tissue or organ defect or bacterial infection is easily performed, and complex damage to skin, muscle, bone tissue and nerve tissue is difficult, it is difficult to regenerate into normal tissues or organs in each layer. Mobility and functional constraints. Conventionally, in order to solve this problem, tissues of other parts of the patient himself have been transplanted. Recently, a barrier is used to block the growth of other tissues before they are healed, or to prevent foreign bacteria from entering the tissue. It also uses a porous scaffold that provides space for cells in damaged tissue to grow in the defect. Not only that, there is a growing tendency to grow cells by using growth factors to move stem cells necessary for healing to the defect site by chemotaxis, or by manipulating or culturing stem cells necessary for healing or regeneration and transplanting them into the defective areas. to be. Until now, however, the individual introduction of these technologies alone had a deep and wide area of defects such as cancer, diabetes, inflammation, and extractions, which damaged at least one or more types of multiple tissues and could not produce satisfactory results when multiple tissues had to be regenerated at the same time. In addition, there was a problem that the side effects of tissue defects or mobility and functional constraints of other normal areas.

The present inventors have conducted a long study to solve the problems of the prior art, and as a result of experiments in the clinical use of the three factors of the growth factor, shielding membrane and scaffold in a kit, cancer, diabetes, inflammation, extraction, etc. As the defects are deep and wide, at least one or more types of tissues are damaged, and when the multiple tissues must be regenerated at the same time, satisfactory results are obtained. Finding the facts completed the present invention.

Human and animal missing tissues or organs can be regenerated into normal tissues or organs using a kit consisting of three elements of the growth factor, scaffold and barrier of the invention. Particularly preferred is the case of multiple or multiple defects as well as deletion of one site. Therefore, in the regeneration of tissues or organ defects due to physical or chemical tissues or organ damage or diseases such as inflammation or tumors, synthetic tissues or organs, heterogeneous tissues without side effects of tissue defects or movement and functional constraints of other normal areas Or an innovative kit that can replace transplantation of organs and autologous tissue or organs.

Figure 1a shows the dimerization and purification of rhBMP-2 and SDS-PAGE analysis, U is non-reducing rhBMP-2, R is reducing rhBMP-2, Dimer is dimer fraction, Monomer is monomer fraction,
1B is a graph showing that pure rhBMP-2 was purified in HPLC profile,
1c is a result of enzymatic experiment showing osteoblast activity by injecting purified rhBMP-2 dimer into C2C12 cells, which are myoblasts, and alkaline phosphatase in concentration-dependent manner in 3 days culture,
Figure 1d is a cell experiment showing the change in osteoblasts after the injection of rhBMP-2 into myoblasts C2C12 cells,
1E is a lyophilized electron micrograph (500-fold) containing rhBMP-2 in a calcium phosphate scaffold,
1f is a lyophilized electron micrograph (3000-fold) containing rhBMP-2 in a calcium phosphate scaffold,
FIG. 2A shows two kinds of shielding membranes of the collagen membrane 1 pressed and crosslinked with the collagen sponge, the PTFE sheet 3 and the PTFE sheet 2 held in the molded state;
Figure 2b is a two-layer shield consisting of a PTFE sheet (5) for the protection of external bacteria and physicochemical and a collagen shield (4) having affinity for growth factors or stem cells,
FIG. 2C is an example of a shield 6 composed of a metal or synthetic mesh 7 reinforced to maintain the shape of the shield. An example of a multilayer shielding film in which a mesh is fixed between different shielding film layers 8 and 9, or fixed to a lower layer of a shielding film, and the mesh is formed so as to extend outwardly from the center of the shielding film.
3a shows a two-layer scaffold composed of a PLGA sponge 10 and a collagen sponge 11,
3b is a scaffold 12 composed of calcium phosphate synthetic bone particles and a collagen solution in a gel phase,
Figure 3c is a lattice layer structure scaffold 13 in PCL to maintain the shape of the wide scaffold,
3d shows a tubular scaffold 14 made of PLGA sponge for nerve or blood vessel regeneration,
FIG. 3E illustrates a scaffold 16 consisting of a collagen scaffold 15 that is biodegraded first for tissue that is regenerated first, and a PLGA sponge that is biodegraded more slowly for tissue that is regenerated later when more than one tissue is missing,
FIG. 3F shows a scaffold 18 consisting of a large pore sponge 17 that is biodegraded first for tissue that is regenerated first, and a small pore sponge that is biodegraded more slowly for tissue that is regenerated later when two or more tissues are missing. ,
FIG. 3G shows a block-like scaffold layer of calcium phosphate and collagen for the PLGA sponge layer 19 for the later regenerated site with a layer of shield 23 and four scaffolds. 20, a scaffold consisting of a lattice layer scaffold 21 of PCL for maintaining the shape of a wide scaffold and a collagen sponge 22 for tissue that is regenerated first,
Figure 3h is a scaffold composed of collagen gel 25 contained in the lattice solids of the PCL 26 and the collagen sponge 24 for shock absorption to maintain the shape when the external pressure is severe,
4A shows a lyophilized gel-like scaffold filled in a syringe,
4B shows a scaffold gelled with water for injection in a syringe,
5A is a kit consisting of rhBMP-2 (26), a block shaped scaffold 27 of calcium phosphate and collagen, and a non-absorbent PTFE sheet shielding membrane 28,
FIG. 5B shows a structure consisting of a rhBMP-2 (26), a block-shaped scaffold (27) of calcium phosphate and collagen, and a non-absorbent PTFE sheet shield (28).
Figure 6a is a kit consisting of a lyophilized scaffold (29) and a non-absorbent PTFE sheet shielding film 30, rhBMP-2 is added to the collagen sponge,
6B is a structure in which the rhBMP-2 is a kit composed of a freeze-dried scaffold 29 and a non-absorbent PTFE sheet shielding film 30 which is injected into a collagen sponge, and the scaffold 29 and the shielding film 30 are configured.
7A shows a kit including a PLGA scaffold 31 and a rhBMP-2 contained in a collagen shielding film 32 and lyophilized shielding film 5a;
7B is a kit including a PLGA scaffold 31 and a rhBMP-2 contained in the collagen shielding film 32 and lyophilized shielding film 5a, in which the scaffold 31 and the shielding film 32 are configured;
8A is a freeze dried kit containing rhBMP-2 in both the tubular scaffold 33 and the collagen shield 34 composed of tubular PLGA sponge,
8B shows a structure in which the scaffold 34 and the shielding membrane 33 are tubular in a freeze-dried kit containing rhBMP-2 in both the tubular scaffold 34 and the collagen shielding membrane 33 composed of tubular PLGA sponges,
9 is a histoscopic microscopical measurement that both bone area, fat area and chalky area increase in the test group in which rhBMP-2 and hPDLSC are present.
10 is a PLGA scaffold containing rhBMP-2 and tetracycline antibiotic dissolved in water for injection,
11 is a manufacturing procedure of the tissue regeneration shape retention shielding film,
12A is a group implanted with only bone morphogenic protein (concentration 0.025 mg / ml rhBMP-2) using a scaffold composed of TCP / HA. Two weeks later, a tissue photo showing healing results,
12B is a group implanted with only bone morphogenic protein (concentration 0.025 mg / ml rhBMP-2) using a scaffold composed of TCP / HA. Tissue pictures showing healing results after 8 weeks,
12C is a group implanted with only bone morphogenic protein (concentration 0.025 mg / ml rhBMP-2) using a shielding membrane in a scaffold composed of TCP / HA. Two weeks later, a tissue photo showing healing results,
12D is a group implanted with only bone morphogenic protein (concentration 0.025 mg / ml rhBMP-2) using a shielding membrane on a scaffold composed of TCP / HA. Tissue pictures showing healing results after 8 weeks,
13A is a photograph of two molar teeth immediately before extraction with inflammation,
Figure 13b is a picture of two soft tooth formed by soft tissue defects,
Figure 13c is a photograph of the rhBMP-2 and the scaffold and the shielding film,
Figure 14a is a depressed gingival photograph at 3 weeks of healing period of the group implanted with PTFE shielding membrane and rhBMP-2 (concentration 1.5 mg / ml rhBMP-2) solution,
FIG. 14B is a photograph showing that the scaffold composed of collagen sponge and bone forming protein (concentration 1.5 mg / ml rhBMP-2) are stabilized horizontally at 3 weeks of healing period, but do not grow vertically.
FIG. 14C is a photograph reproduced into the dermal layer both vertically and horizontally at 3 weeks of the healing period of the group implanted with the osteogenic protein (concentration 1.5 mg / ml rhBMP-2) using a scaffold composed of TCP / HA and a PTFE shielding membrane.
FIG. 14D shows the dermal layer and epithelium in both vertical and horizontal phases at 3 weeks of healing period of the bone graft protein (concentration 1.5mg / ml rhBMP-2) transplanted using a scaffold and PTFE shielding membrane composed of TCP / HA and collagen sponge. The reproduced picture.

The present invention provides a kit for tissue or organ regeneration comprising three elements, a growth factor, a shielding membrane, and a scaffold. The present invention has improved tissue or organ regeneration completeness and integrity.

As growth factors that can be used in the present invention, recombinant human Bone Morphogenetic Protein (hereinafter referred to as "BMP") has been developed and utilized to date, and these BMPs have almost equivalent bone formation functions. Such BMPs include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, and BMP-13. TGF-β supergroups, which are co-duplexes of amino acids constituting BMP (combination of amino acids of B2 and amino acids of B4), fibroblast growth factor or proteins such as GDF , Growth factor selected from one or two of platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and transformative growth factor, keratinocyte growth factor 2 (KGF2) and MP52 protein and the recombinant protein of the growth factor. to be.

In the present invention, a growth factor configured for tissue or organ regeneration kit is contained in a gel-like scaffold, filled in a syringe or a vial, and then lyophilized to be stored in a vial or a syringe to provide a kit. Water can be injected into the defects and gelled.

The material of the shielding film that can be used in the present invention is PCL, PLA, PLGA, PGA synthetic absorbent polymer material; Natural absorbent polymers of alginic acid, chitosan, collagen, hyaluronic acid, cellulose and mixtures thereof; poly (vinylidene fluoride), poly (tetrafluoroethylene), poly (vinyl alcohol), poly (hydroxyalkanoate), poly (ethylene terephthalate), poly (butylene terephthalate), poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N- isopropylacrylamide), poly (dimethyl siloxane), polydioxanone, and polypyrrole, poly (glycolic acid), poly (lactic acids), poly (ethylene oxides), poly (lactide-co-glycolides), poly (s-caprolactone), polyanhydrides, It is selected from the group consisting of polyphosphazenes, poly (ortho-esters and polyimides synthetic polymer materials; metal thin films of titanium and stainless steel).

The shielding membrane may have pores of less than 0.01 ~ 30㎛ prevent neighboring bacteria or cells from passing through or growing inside. Externally exposed areas prevent the invasion of bacteria and the escape of body fluids from defective tissues or organs. On the other hand, the shielding film installed inside the tissue or organ shields other adjacent tissues or organs from interfering with each other. This allows the tissue or organ to be regenerated to be integral with the surrounding normal tissue or organ.

The thickness of the shielding film is 0.01-0.5 mm. If it is thinner than 0.01mm, it is easily deformed to the external magnetic pole, and if it is thicker than 0.5mm, it is difficult to mold.

In the present invention, the shielding film configured for the tissue or organ regeneration kit may be composed of a multilayer of 2 to 5 layers. In order to contain the growth factor in the shielding film, a plurality of shielding film layers may be formed by adding a shielding film layer of a hydrophilic material. The outer layer may be composed of a layer containing physicochemical stability to block external stimuli and a growth factor inside.

In the present invention, the shielding film configured for the tissue or organ regeneration kit may have non-porous sheets or pores. Preferably, the pores of the shielding film may be used as a shielding film as well as a shielding film made by compressing a collagen sponge or a shielding film made of nanofibers having pores of 0.01-30 μm or more. Bacteria do not penetrate the sheet or film that has no pores in the shielding membrane to prevent infection, and nanofibers or compressed sponges return to their original unfolded shape even if they are deformed to conform to the curved shape, but the sheet or film is deformed. It has the advantage of being maintained.

In the present invention, the shielding film configured for the tissue or organ regeneration kit may be reinforced with a reinforcing layer to increase the strength of the shielding film.

To this end, the material of the reinforcing layer may be a metal selected from polycaprolactone or polylactic acid, polyglycolic acid and copolymers of lactic acid and glycoic acid, titanium and stainless steel. Preferably, in order to maintain the shape of the shielding film, a synthetic polymer layer or a metal layer may be formed between the plurality of shielding film layers or under a single layer. This layer may consist of plastic polycaprolactone or polylactic acid, polyglycolic acid and copolymers of the three materials or metals of titanium or stainless steel. The shape may be a mesh layer, and the thickness may be 0.01 to 0.5 mm, and may be formed to be extended in the outer direction from the center of the shielding film.

In the tissue or organ regeneration kit of the present invention, the shielding membrane may be variously configured. For example,

1) two types of shielding membranes (FIG. 2A), which are a collagen membrane (1) pressed and crosslinked with a collagen sponge and a polytetrafluoroethylene (PTFE) sheet (2) held in a molded state (3). have.

2) A double shielding membrane (FIG. 2B) consisting of a polytetrafluoroethylene (PTFE) sheet (5) for external bacteria and physicochemical defense and a collagen shielding membrane (4) having affinity for growth factors or stem cells Can be configured.

3) Shielding film 6 consisting of reinforced metal or synthetic mesh 7 for maintaining the shape of the shielding film. A shielding film (FIG. 2C) in which a mesh is fixed between different shielding layers 8 and 9 or a mesh is fixed to a lower layer of the shielding layer (FIG. 2C), and the mesh may be configured to be extended in the outer direction from the center of the shielding layer. have. The synthetic polymer mesh may be molded by injection molding or sheet and bonded to the shielding film 31 by thermocompression bonding.

The material of the scaffold that can be used in the present invention is a biodegradable polymer material of PCL, PLA, PLGA, PGA; Natural polymers of chitosan, alginic acid, collagen, hyaluronic acid, chondroitin, glucosamine, cellulose; Complexes thereof; And a material selected from the group consisting of synthetic bone, allogeneic bone, xenograft, demineralized bone or cadaveric derived dermis.

The shape of the scaffold that can be used in the present invention is a sheet, porous sheet, sponge, jelly, gel, lyophilized, microspheres, tubular (tube), lattice or lattice structure containing collagen It may be a grid.

In the present invention, a single layer of the scaffold may be used, and if necessary, the scaffold may be composed of a multilayer of two or more layers and five or less layers.

In the present invention, when using a multi-layer scaffold, it can be used in the form of being separated from each other, bonded or glued.

In the present invention, the growth factor may also be prepared by incorporating a growth factor into a gel-like scaffold and filling the syringe or vial with lyophilization.

In the present invention, it is also possible to use a scaffold added with a reinforcing layer for maintaining the shape of the scaffold. At this time, the material of the reinforcing layer may be a material selected from biodegradable polymer materials of PCL, PLA, PLGA, PGA.

In the present invention, it is also possible to use a scaffold having a lattice solid shape and a collagen sponge filled inside the lattice. As the lattice-like material, it is possible to use PCL, PLA, PLGA, PGA synthetic polymer material, a mixture of synthetic polymer material and calcium phosphate ceramic, or a material selected from titanium or stainless steel metal.

More preferably, the tissue cells have different growth rates, so that they grow or regenerate in the order of epithelial cells, dermal cells, bone cells, ligament cells and neurons. Therefore, when the skin consisting of the epidermis and the dermis becomes defective, the epidermis regenerates first rather than the dermis, and the epithelium grows first before the regenerated dermis is reproduced, resulting in a dent scar. Alternatively, when the bone tissue and the skin are damaged at the same time, the skin tissue first occupies a bone tissue defect, and thus the bone tissue cannot be completely regenerated. At the same time, the skin that is being regenerated has a property to maintain a constant thickness, so that an inner groove is formed in accordance with the appearance of the missing bone tissue. Therefore, when two or more tissues or organs are damaged, the growth rate is faster so that the scaffold of the first healing tissue or organ is larger and the scaffold of the later healing tissue or organ is less porous and growing first. The tissue or organ is regenerated first to a certain thickness. In addition, the extinction or absorption of the material should be controlled as much as the rate at which new tissues or organs are completed, and the slow regeneration site will allow the material to retain its shape until regeneration is complete. The materials used as scaffolds have different shape retention periods in the body, so that the collagen sponge is maintained for less than 3 weeks, the PGA polymer for less than 6 months, the PLA polymer for less than 12 months, and the PCL polymer for less than 24 months. The retention period can be controlled by synthesizing a polymer having various retention periods into a copolymer. As such, various shapes suitable for the shape of the defect area are essential for the shielding film constituted in the tissue or organ regeneration kit of the present invention.

As various configurations of the scaffold, a double scaffold (FIG. 3A) consisting of a polymer (PLGA) sponge 10 and a collagen sponge 11 of lactic acid and glycolic acid may be constructed.

1) Calcium phosphate synthetic bone particles and collagen solution may be composed of a scaffold 12 (Fig. 3b).

2) A scaffold (FIG. 3C) injected into the lattice layer 13 by PCL may be constructed to maintain shape in a wide range of scaffolds.

(1) A tubular scaffold (FIG. 3D) made of a polymer (PLGA) sponge 14 of lactic acid and glycolic acid for nerve or blood vessel regeneration can be constructed.

(2) If two or more tissues or organs are missing, the first biodegradable collagen scaffold 15 for the first regenerated tissue or organ and the slower biodegradable PLGA sponge 16 for the later regenerated tissue or organ 16 A scaffold consisting of (FIG. 3E) may be configured.

(3) If two or more tissues or organs are missing, a larger pore sponge 17 which is biodegraded earlier for the tissue or organ that is first regenerated and a smaller pore sponge that is biodegraded more slowly for the tissue or organ that is later regenerated A scaffold consisting of 18 can be constructed (FIG. 3F).

(4) a PLGA sponge layer 19 for the last reclaimed site with a layer of shield and a scaffold consisting of 1 to 5 species, and a block of calcium phosphate and collagen 20 for the next reclaimed site, a wide range of A scaffold may be constructed that consists of a lattice layer 21 of PCL that maintains the shape of the scaffold at the site and a collagen sponge 21 for the tissue or organ that is regenerated fastest (FIG. 3G). In addition, the damaged tissue or organ may be up to five skin, muscles, ligaments, bones, nerves, etc. The scaffold may also be composed of five layers accordingly.

(5) A scaffold composed of collagen 25 filled inside the lattice solids of the PCL 26 and the collagen sponge 26 for shock absorption may be configured to maintain the shape when the external pressure is severe (FIG. 3H). .

In the present invention, a growth factor configured for tissue or organ regeneration kit is contained in a gel-like scaffold, filled in a syringe or a vial, and then lyophilized to be stored in a vial or a syringe to provide a kit. Water can be injected into the defects and gelled.

In the present invention, as an example for the tissue or organ regeneration kit, in preparing a gel scaffold, hyaluronic acid solution (0.1 ~ 10wt / wt%) containing a growth factor of 1mcg ~ 2 mg / ml is preferred Preferably, 0.5 mg / ml is filled in a syringe or vial and lyophilized (FIG. 4A). Immediately before use in the clinic, water for injection or saline is inhaled or injected into a syringe or vial to gel and inject into the defect (FIG. 4B).

Tissue or organ cells have different growth rates and grow or regenerate in the order of epithelial cells, dermal cells, bone cells, ligament cells and nerve cells. Therefore, when the skin consisting of the epidermis and the dermis becomes defective, the epidermis regenerates first rather than the dermis, and the epithelium grows first before the regenerated dermis is reproduced, resulting in a dent scar. Alternatively, when the bone tissue and the skin are damaged at the same time, the skin tissue first occupies a bone tissue defect, and thus the bone tissue cannot be completely regenerated. At the same time, the skin that is being regenerated has a property to maintain a constant thickness, so that an inner groove is formed in accordance with the appearance of the missing bone tissue. Therefore, when two or more tissues or organs are damaged, the growth rate is faster, so that the scaffold of the first healing tissue or organ is more porous, and the scaffold of the later healing tissue or organ is less porous and growing first. Allow the tissue or organ to regenerate first to a constant thickness. In addition, the extinction or absorption of the material should be controlled as much as the rate at which new tissues or organs are completed, and the slow regeneration site will allow the material to retain its shape until regeneration is complete. The materials used as scaffolds have different shape retention periods in the body, so that the collagen sponge is maintained for less than 3 weeks, the PGA polymer for less than 6 months, the PLA polymer for less than 12 months, and the PCL polymer for less than 24 months. The retention period can be controlled by synthesizing a polymer having various retention periods into a copolymer.

The tissue or organ regeneration kit provided by the present invention may have multiple scaffolds separated from each other, bonded or adhered to each other. Preferably, in the case where two or more tissues or organs are to be regenerated, the scaffold is multilayered. Each layer can be a different polymeric material. The layer corresponding to the tissue or organ that is regenerated first may be first degraded and replaced with a neoplastic tissue or organ. The layers are divided according to the order in which they are reproduced, so that the scaffold layer of tissue or organ to be regenerated later is maintained in shape.

In the case where two or more kinds of tissues or organs are to be regenerated at the same time, even through the same polymer material, the distribution of the through pores may be different from each other. The more penetrating pores, the more likely it is to take advantage of the properties that are first created by the new tissue or organ. The layer corresponding to the tissue or organ that is regenerated first has a lot of through-pores so that it can be regenerated first, and the scaffold layer of the cell layer that is regenerated in a lower order can be configured to have less through-pores, thereby increasing the shape retention of the scaffold. .

If a defect in one type of tissue or organ (e.g., dermis or bone tissue or organ alone) is so large that the volume of the scaffold is too large to maintain shape, an additional layer is provided to facilitate shape deformation and maintain the deformed shape. May be

In the present invention, a reinforcing layer for maintaining the shape of the scaffold configured for the tissue or organ regeneration kit may be added, and the material may be selected from biodegradable polymer materials of PCL, PLA, PLGA, and PGA.

In the present invention, the scaffold configured for the tissue or organ regeneration kit may be a lattice shape and a collagen sponge may be filled in the lattice.

In the present invention, the lattice solid material configured for the tissue or organ regeneration kit may be selected from a synthetic polymer material, a mixture of a synthetic polymer material and a calcium phosphate ceramic, or a metal. Preferably, when damaged tissues or organs are exposed to strong external forces, a mixture of PCL, PLA, PLGA, PGA synthetic polymers or synthetic polymers and calcium phosphate ceramics, or lattice-shaped scaffolds made of titanium or stainless steel metal, A polymeric sponge filled inside the lattice can be constructed with other scaffold layers.

In the present invention, the growth factor is composed of the scaffold contained in the shielding membrane, the growth factor is composed of the shielding membrane contained in the scaffold or the tissue or organ regeneration kit consisting of the growth factor is contained in the scaffold and the shielding membrane The growth factor, the scaffold and the shield may be configured together so that the growth factor of the growth factor, the function of the shield and the role of the scaffold may be complemented. In the existing publications, studies on scaffolds that maintain the space and shape in which tissues of damaged tissues or organs are grown at the defect site in tissue or organ regeneration using growth factors have been mainly conducted, and recently, growth factors have been scaffolded. Studies using only barriers are starting, and their studies have a laboratory approach rather than studies based on clinical efficacy or side effect prevention. The present invention is based on the results of a case using a growth factor in the clinic, the conditions to be achieved for tissue or organ regeneration that is integral with the surrounding normal tissue or organ of the defect site should be used at the same time growth factor, scaffold and shielding membrane Is that.

The scaffold layer serves as a growth factor such as BMP-2 or a carrier for stem cells, and maintains a three-dimensional space having penetrating pores to allow the surrounding normal blood vessels to grow inside.

In order to apply growth factors to scaffolds or barriers, these materials must be hydrophilic. Therefore, in order to use the growth factor directly on the scaffold or the shielding film or to include it in advance in these materials, there are matters to be considered in the material composition and composition.

In applying the shielding membrane to a defective tissue or organ, it is preferable that the shielding membrane positioned at the open site uses a hydrophobic polymer to prevent bacterial invasion. To apply the growth factor, a hydrophilic shielding layer must be added to the lower layer.

In addition, in the application or inclusion of growth factors in the scaffold, the material for maintaining the form is a hydrophobic synthetic polymer or calcium phosphate salt crystals that chelate with the growth factor protein to inhibit their release, thus scaffolding hydrophilic synthetic or natural polymers. It must be filled internally or contained outside its scaffold.

There are four ways to apply growth factors to scaffolds and shields.

In general, a method of hydrating the growth factor solution directly on the scaffold and the shield layer is used. However, due to poor operability, growth factors may be contained in the scaffold, the shielding film, or the scaffold and the shielding film. As a method of containing a growth factor, a method of directly hydrating a solution of an effective drug concentration of a growth factor in a target scaffold or a shielding film and then freeze-drying is used.

In order to apply growth factors to scaffolds or barriers, these materials must be hydrophilic. Therefore, in order to use the growth factor directly on the scaffold or the shielding film or to include it in advance in these materials, there are matters to be considered in the material composition and composition.

In applying the shielding film to the defective tissues, it is preferable that the shielding film placed at the open site uses a hydrophobic polymer to prevent bacterial invasion. To apply the growth factor, a hydrophilic shielding layer must be added to the lower layer.

In addition, in the application or inclusion of growth factors in the scaffold, the material for maintaining the form is a hydrophobic synthetic polymer or calcium phosphate salt crystals that chelate with the growth factor protein to inhibit their release, thus scaffolding hydrophilic synthetic or natural polymers. It must be filled internally or contained outside its scaffold.

There are four ways to apply growth factors to scaffolds and shields. In general, a method of hydrating the growth factor solution directly on the scaffold and the shield layer is used. However, due to poor operability, growth factors may be contained in the scaffold, the shielding film, or the scaffold and the shielding film. As a method of containing a growth factor, a method of directly hydrating a solution of an effective drug concentration of a growth factor in a target scaffold or a shielding film and then freeze-drying is used.

In the present invention, in addition to the growth factor in the tissue or organ regeneration kit, stem cells differentiate into pluripotent mesenchymal stem cells, such as pluripotent mesenchymal stem cells, astrocytic cells, adipocytes, chondrogenic cells, osteogenic cells, or insulin-secreting pancreatic beta cells. Contains at least one stem cell selected from the group consisting of capable stem cells, pluripotent hematopoietic stem cells, pluripotent stem cells, pluripotent neural stem cells, pluripotent pancreatic stem cells, extracted tooth pulp cells and periodontal ligament cells You may.

In the present invention, in addition to the growth factor in the tissue or organ regeneration kit, it may further contain one or more drugs selected from anti-inflammatory analgesics, antibacterial agents and corticosteroids. Preferably, phenylpropionate derivatives including pyroxhamm, ketoprofen, flurbiprofen, fenoprofen, and ibuprofen in addition to growth factors in tissue or organ regeneration kits. Series of amides; Oxycam derivatives such as pyroxicam, tenoxicam and meloxicam; Diclofenac; And anti-inflammatory drugs selected from indomethacin (indomethacin), amoxicillin, erythromycin, metronidazole, Amoxicillin, Amoxacillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, Other Lincosphaporin ), Tetracycline hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride, may be contained at least one corticosteroid drug selected from methylprednisolone, hydrocortisone acetate.

When applying growth factors to antibiotics on scaffolds, when applying growth factors to scaffolds and barriers in tissue or organ defects with osteomyelitis or skin inflammation, use tetracycline and water for injection to reduce infection Can be. This result is shown in FIG. Instead of the tetracycline of this example, the enumerated drugs and the like can be used.

When tissue or organ damage is extensive, a three-dimensional image of a damaged tissue or organ is acquired by computer tomography (CT) or a three-dimensional optical analyzer, and a three-dimensional shape in which the tissue or organ is reproduced is acquired by a simulation program. This three-dimensional shape and simulation program is used to design shielding membranes and scaffolds to maintain shape during regeneration of new tissues or organs, and to produce laser photopolymerization using polymers used as shielding materials, polymer solution evaporation, crosslinking, A shielding film is manufactured by a thermoplastic manufacturing method and a mechanical processing method using a computer milling center, and a custom shielding film and a scaffold with a polymer used as a scaffolding material are prepared inside the shielding film. This result is shown in FIG.

Next, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

Example 1

Kit consisting of growth factor, scaffold and shield:

RhBMP-2 lyophilized in vials in FIG. 5A (FIG. 5B) kit consisting of a block-shaped scaffold 27 of collagen containing calcium phosphate synthetic bone and a non-absorbent PTFE sheet shielding membrane 28 in FIG. 5A. It is a method of hydrating a growth factor in saline solution and using it in a scaffold composed of calcium phosphate synthetic bone-containing collagen. The collagen component of this scaffold contains a growth factor and is gradually released to surrounding stem cells.

Preparation of collagen block scaffold containing calcium phosphate synthetic bone is as follows.

Bovine ligaments and pig skin subcutaneous tissues are removed, ground to small size, and the collagen component is extracted using a surfactant, a defatting agent and a bactericide. Pepsin treatment is performed at pH 3 to separate the collagen fiber bundles to obtain fine collagen fibers. It is lyophilized to purify pure collagen to make a collagen solution with a weight ratio of 3 to 5%. When the calcium phosphate salt powder is mixed with a binder and naphthalene, and solidified by applying mechanical stirring or compression, porous synthetic bone particles are produced while naphthalene is released. Put it in a mold and lyophilize 3 ~ 5% collagen solution and crosslinker at the same time.

Example 2

Kits consisting of scaffolds and shields containing growth factors:

In FIG. 6A, the rhBMP-2 growth factor is lyophilized on a scaffold 29 composed of a collagen sponge and a non-absorbent PTFE sheet shielding film 30 is constructed (FIG. 6B). This is because collagen sponges are widely used as carriers of growth factors, and are excellent in releasing growth factors. On the other hand, by using PTFE sheet as a shielding membrane, not only the growth factor does not pass through the shielding membrane, but also the hydrophobic material does not bind the growth factor, so that the growth factor is released to the surrounding stem cells. have. In the collagen sponge manufacturing method, a sponge is prepared by adding 3 to 5% (w / w) of the collagen solution to the mold by adding a cross-linking agent to the mold and freeze drying. In the method of manufacturing the PTFE shielding membrane, the polymer material is dissolved in each solvent in an electrospinning machine to spin nanofibers to cross-link at the moment of making the film, and micropores are formed between the fibers and cut into a convenient form.

PTFE shielding film and collagen sponge is a material that is not bonded to each other can also be bonded using a butylcyanoacrylic binder.

Example 3

Kits consisting of shields and scaffolds containing growth factors:

In FIG. 7A, rhBMP-2 is a kit composed of a scaffold 31 composed of a freeze-dried shielding membrane and a PLGA sponge in which rhBMP-2 is injected into the collagen shielding membrane 32, and has a structure composed of the scaffold 31 and the shielding membrane 32 (FIG. 7B). )to be. This applies when the PLGA copolymer has adequate strength to be inserted into a small diameter and recessed scar tissue so as to have continuity and integrity with surrounding tissues and to apply a small amount of growth factor to small defects. In the method of manufacturing a collagen shielding membrane, a collagen solution having a weight ratio of 3 to 5% is placed in a mold having a depth of 3 mm, lyophilized to make a sponge, and then put into a compressor to be thinned to a thickness of less than 0.3 mm, and then crosslinked. In addition, the copolymer (PLGA) sponge of lactic acid and glycolic acid is dissolved in a PLGA synthetic polymer in a solvent such as chloroform, poured into a layer of salt particles, lyophilized, and washed with salt to prepare a sponge.

Example 4

Kits consisting of scaffolds and shields containing growth factors:

In FIG. 8A, a growth factor is embedded in both the tubular PLGA copolymer sponge scaffold 33 and the collagen shielding membrane 34 to freeze-dried kits, and blood vessels of terminal tissues or organs are damaged and blood is easily eluted. If it is difficult to express the growth factor in both the scaffold and the shielding film. In the method of manufacturing a tubular scaffold, salt particles are filled in a mold having a tubular inner groove, and then a freeze-dried PLGA copolymer solution dissolved in a chloroform solvent is prepared. The tubular collagen shielding membrane was also freeze-dried by filling a 3-5% collagen solution into a mold having a tubular width of 3 mm internal grooves to form a sponge and pressed to a thickness of 0.3 mm in a tubular compactor to cross-link (FIG. 8B).

Example 5

Production Example of Skin Regeneration Shape Retaining Shielding Film:

In the case of extensive skin damage, a three-dimensional image of skin tissue is acquired by CT (computer tomography) or a three-dimensional optical analyzer, and a three-dimensional shape in which the skin tissue is reproduced is acquired by a simulation program. This 3D shape and simulation program is used to design a shielding film that maintains shape during skin tissue regeneration, and a laser photopolymerization shielding film using a polyepoxy polymer, a thermoplastic shielding film using a polycaprolactone (PCL) polymer, or a polyether ether ketone ( Engineering membrane blocks, such as polyether ether ketone (PEEK) resins or polytetrafluoroethylene (PTFE) resins, are fabricated in computer milling centers to produce shielding films. Collagen scaffolds and rhBMP-2 are added to the defective skin tissue volume, and the skin regeneration shape maintenance membrane is fixed. The results are shown in Fig.

Example 6

Various configurations of shields:

1. There are two kinds of shielding membranes: a collagen membrane (1) pressed and crosslinked with a collagen sponge and a polytetrafluoroethylene (PTFE) sheet (2) held in a molded state (3). This is shown in Figure 2a.

2. A double shielding membrane may be composed of a polytetrafluoroethylene (PTFE) sheet (5) for external bacterial and physicochemical defense and a collagen shield (4) having affinity for growth factors or stem cells. . This is shown in Figure 2b.

3. A shielding film (6) consisting of a reinforced metal or synthetic mesh (7) for maintaining the shape of the shielding film. A mesh is fixed between the different shielding layers 8 and 9, or a mesh is fixed to a lower layer of the shielding layer, and the mesh may be configured to be extended in the outer direction from the center of the shielding layer. The synthetic polymer mesh 7 was molded by injection molding or sheet and bonded to the shielding film 6 by thermocompression bonding. This is shown in Figure 2c.

Example 7

Various configurations of scaffolds:

As various configurations of the scaffold, a double scaffold composed of a polymer (PLGA) sponge 10 and a collagen sponge 11 of lactic acid and glycolic acid may be configured. This is shown in Figure 3a.

1. Calcium phosphate synthetic bone particles and collagen solution may be composed of a scaffold composed of a gel (12) (Fig. 3b).

2. A scaffold injected into the lattice layer 13 in PCL can be constructed for maintaining shape over a wide range of scaffolds (FIG. 3C).

3. A tubular scaffold made of a polymer (PLGA) sponge 14 of lactic acid and glycolic acid for nerve or blood vessel regeneration can be constructed (FIG. 3D).

4. If two or more tissues are missing, a scaffold consisting of a collagen scaffold 15 that is biodegraded earlier for the first regenerated tissue and a PLGA sponge 16 that is slower biodegraded for the later regenerated tissue will be constructed. May be (FIG. 3E).

5. If two or more tissues are missing, a scaffold consisting of a larger, firstly biodegradable pore sponge (17) for the first regenerating tissue and a slower, more biodegradable sponge (18) for later regeneration tissue. May be configured (FIG. 3F).

6. PLGA sponge layer 19 for the last reclaimed site with a layer of shield and a scaffold consisting of 1 to 5 species, blocks of calcium phosphate and collagen 20 for the next reclaimed site, a wide range of sites A scaffold consisting of a lattice layer 21 of PCL to maintain the shape of the scaffold and a collagen sponge 21 for the fastest regenerating tissue may be constructed (FIG. 3G). In addition, the damaged tissue may be up to five skin, muscles, ligaments, bones, nerves, etc. The scaffold may be composed of five layers accordingly.

7. A scaffold consisting of collagen 25 filled inside the lattice geometry of PCL 26 and collagen sponge 26 for shock absorption can be constructed to maintain shape in the event of severe external pressure (FIG. 3H).

Example 8

Preparation of Gel Scaffolds:

The growth factor in the hyaluronic acid solution (0.1 ~ 10wt / wt%) to 0.5 mg / ml, filled in a syringe or vial and lyophilized (Fig. 4a). Immediately before use in the clinic, water for injection or saline is inhaled or injected into a syringe or vial and gelated and injected into the defect (FIG. 4b).

Example 9

Kits consisting of scaffolds and shields containing growth factors:

In FIG. 8A, a growth factor is embedded in both the tubular PLGA copolymer sponge scaffold 33 and the collagen shielding membrane 34 to freeze-dried kit, and the blood vessels of the terminal tissue are damaged so that the blood easily elutes the growth factor. In difficult cases, growth factors are included in both the scaffold and the shield. In the method of manufacturing a tubular scaffold, salt particles are filled in a mold having a tubular inner groove, and then a freeze-dried PLGA copolymer solution dissolved in a chloroform solvent is prepared. The tubular collagen shielding membrane was also freeze-dried by filling a 3-5% collagen solution into a mold having a tubular width of 3 mm internal grooves to form a sponge and pressed to a thickness of 0.3 mm in a tubular compactor to cross-link (FIG. 8B).

Example 10

Application of antibiotics to growth factors along with scaffolds:

When applying growth factors to scaffolds and barriers in tissue defects with osteomyelitis or skin inflammation, tetracycline and water for injection can be used to reduce infection. This is shown in FIG. 10.

Instead of the tetracycline of this example, the enumerated drugs and the like can be used.

Example 11

These examples illustrate a method for producing BMP-2 (high BMP-2) by genetic recombination techniques, which illustrate known methods. All other growth factors used in the present invention are also known, and in the present invention, a three-element kit is prepared using these known growth factors.

1. Production of rhBMP-2:

1) Cloning of Human BMP-2 gene:

To obtain human BMP-2 gene, total cellular RNA was extracted from U2OS cells using Trizol (Gibco BRL, NY, USA) solution and subjected to reverse transcription. Polymerase chain reaction (PCR) was performed using 5'-AATTTACAGCTTCTAGCGACACCCACAACCCT-3 '. PCR products were isolated and inserted into pGEM-T vector (Promega, USA) and cloned using E. coli (DH5α) cells.

2) High-density cell culture:

The fermentation tank (KoBioTec, Incheon, Korea) was used for the cultivation at a high density using the method of Fatemeh et al. Sterilized nutrient medium (Glucose 33.3 g / L, peptone 10 g / L, yeast extract 5 g / L, MgSO ₄ 1 g / L, CaCl ₂ 0.048 g / L, ZnSO ₄ 0.0176 g / L, CuSO ₄ 0.008 g / Incubate for 24 hours with the addition of L).

3) Protein purification:

The suspension was stored in -80 ° C deep freezer (Nihon Freezer, Japan). After thawing the frozen suspension at refrigeration temperature, the cells were crushed by pressing and centrifuged for 45 minutes at 5,500xg, 4 ℃. When the renatured feature rhBMP-2 has the original tertiary structure, the N-terminus has a heparin-binding site.

2. Biochemical properties of purified rhBMP-2:

1) Purity and identification test of rhBMP-2 stock solution: SDS-PAGE:

As a result, the rhBMP-2 stock solution showed the same moving distance as the standard solution, and showed a purity of 95% or more (FIG. 1A).

2) HPLC (High performance Liquid Chromatography) analysis:

Purified rhBMP-2 dimer was dissolved in 0.1% Trifluoroacetic acid (TFA) at a concentration of 1 µg / µl, and each fraction of a C4 reversed-phase HPLC column (4.6 mm x 50 mm, 300 µm, 5 µm particle size; Gracevydac, CA, USA) Protein was detected and monitored. The test results of the rhBMP-2 stock solution showed the same retention time as that of the standard solution, and the rhBMP-2 stock solution should show the same retention time as the standard solution.

3. Results of rhBMP-2 protein production and purification:

After dimerization, affinity chromatography was performed on a heparin column. As a result, as shown in FIG. 1A, most monomers and a small amount of dimer were eluted in the 0.3 M NaCl fraction and dimers were eluted in the 0.5 M NaCl fraction. Purified rhBMP-2 monomer and monomer were confirmed by analysis by DS-PAGE. The size of the produced monomer was calculated to be about 114 amino acid residues, and the molecular weight of the monomer was found to be about 14 kDa, and the dimer appeared as a band having the same size as the monomer under non-reducing conditions since the two monomers were linked by disulfide bonds. The dimer size was about 28 kDa.

4. In vitro test of purified rhBMP-2:

As a result of rhBMP-2 application to myoblasts, alkaline phosphatase, a representative protein of osteoblasts, was secreted after 3 days of culturing (Fig. 1c), and the cell morphology was transformed into osteoblasts under microscope (Fig. 1d). Therefore, rhBMP-2 was confirmed as a function of bone formation protein.

5. A sterile rhBMP-2 solution was mixed in a gamma-ray sterilized calcium phosphate scaffold in a sterile environment at a ratio of 1000: 1, and lyophilized under the temperature conditions of Table 1 under a pressure of 5 mTorr, as shown in FIGS. 1E and 1F. And it was found. As a result, it was confirmed that the rhBMP-2 growth factor penetrated into the porous scaffold.

Temperature
(° C) -30 -20 -10 0 5 10 15 20 time
(time) 3 One One One One One One 9

Experimental Example 1

Periodontal tissue regeneration effect using the kit comprising the three elements of the present invention, growth factor and collagen sponge scaffold and human periodontal ligament stem cells:

In order to identify and culture the stem cells (hPDLSC) by separating periodontal ligaments from the extracted tooth of humans, and to examine the tissue regeneration response according to the bone morphogenetic protein type 2 (BMP-2), the subcutaneous subcutaneous of rats with immunodeficiency experiments The regeneration of periodontal tissue consisting of cementum area, alveolar bone and adipocytes was observed.

The control group transplanted only collagen sponge, the test group transplanted BMP-2 with collagen sponge without hPDLSC, the hPDLSC alone transplanted with collagen sponge without BMP-2, and finally the BMP-2 and hPDLSC transplanted with collagen sponge. One group was tested.

As a result, all of the chalky, alveolar bone and adipocytes were differentiated in the group containing both BMP-2 and hPDLSC collagen sponges. The group containing only BMP-2 did not regenerate chalky, and the group containing only hPDLSC only chalky. Regenerated, nothing in the control group. The results are shown in Fig.

Therefore, it was concluded that both BMP-2 growth factor and periodontal ligament-derived stem cells must be present for the periodontal tissue regeneration.

Experimental Example 2

Comparison of the test group using both the scaffold and the shield and the control group using the scaffold alone in applying the growth factor:

1. Animal test method:

Forty male rats formed critically sized cranial defects with a diameter of 8 mm. A group of 20 rats divided into two groups and implanted with bone formation protein (concentration 0.025mg / ml rhBMP-2) using a scaffold composed of TCP / HA, and bone formation protein (concentration 0.025mg) on a scaffold composed of TCP / HA. / ml rhBMP-2) was divided into upper non-absorbent membranes (e-PTFE membrane), and the results were compared histologically at 2 and 8 weeks postoperatively.

2. Experimental Results:

In the histological comparison, the two-week (Fig. 12C) and eight-week (Fig. 12D) healing of the shielding group showed continuous bone formation above the scaffold. Healing of weeks (Fig. 12a) and week 8 (Fig. 12b) showed irregular bone formation.

Therefore, by applying the shielding film to the scaffold having a three-dimensional structure for tissue or organ regeneration, it was possible to regenerate the complete form of tissues or organs and components by preventing the surrounding heterogeneous tissues from growing inside.

Experimental Example 3

Tissue or organ regeneration effects of kit consisting of growth factor, scaffold and shield:

1. Test method:

In 16 adults, four patients were divided into four groups (Fig. 13A), and the growth factors and scaffolds or shields or scaffolds and shields were applied to soft tissue defects (Fig. 13B) caused by extraction. The procedure was divided into groups (FIG. 13C).

Group implanted with PTFE shielding membrane and rhBMP-2 (concentration 1.5 mg / ml rhBMP-2) solution (FIG. 14A), scaffold and bone morphogenetic protein (concentration 1.5 mg / ml rhBMP-2) consisting of calcium phosphate ceramic particles and collagen sponge ) Transplanted group (FIG. 14B), a bone grafted protein (concentration 1.5 mg / ml rhBMP-2) transplanted using a scaffold composed of TCP / HA and a PTFE shield (FIG. 14C), TCP / HA and collagen sponge Using the scaffold and PTFE shielding membrane together with each other, the bone-forming protein (concentration 1.5mg / ml rhBMP-2) was divided into groups transplanted (FIG. 14D).

2. Test result:

In the visual comparison, the soft tissues of the scaffolds and shields did not have continuity as the soft tissues healed inward compared to the surrounding soft tissues. Especially in scaffold group composed of shielding membrane and TCP and collagen sponge, soft tissue was thickest and some epithelial cells were regenerated. Therefore, by applying the shielding membrane together with the scaffold with the three-dimensional structure for soft tissue regeneration, it prevents the invasion of external bacteria and prevents the infection and prevents the surrounding epithelial cells from growing inside, thereby regenerating the soft tissue and its composition. have.

3. Conclusion:

The growth group of the present invention, a test group consisting of a three-element kit consisting of a scaffold and a shielding membrane was regenerated into a tissue integrating with the surrounding tissue.

Experimental Example 4

Periodontal tissue regeneration effect using growth factor, collagen sponge scaffold and human periodontal ligament stem cells:

In order to identify and culture the stem cells (hPDLSC) by separating periodontal ligaments from the extracted tooth of humans, and to examine the tissue regeneration response according to the bone morphogenetic protein type 2 (BMP-2), the subcutaneous subcutaneous of rats with immunodeficiency experiments The regeneration of periodontal tissue consisting of cementum area, alveolar bone and adipocytes was observed.

The control group transplanted only collagen sponge, the test group transplanted BMP-2 with collagen sponge without hPDLSC, the hPDLSC alone transplanted with collagen sponge without BMP-2, and finally the BMP-2 and hPDLSC transplanted with collagen sponge. One group was tested.

As a result, all of the chalky, alveolar bone and adipocytes were differentiated in the group containing both BMP-2 and hPDLSC collagen sponges. Regenerated and nothing was reproduced in the control group (FIG. 9).

Therefore, it was concluded that both BMP-2 growth factor and periodontal ligament-derived stem cells must be present for the periodontal tissue regeneration.

Experimental Example 5

Comparison of the test group using both the scaffold and the shield and the control group using the scaffold alone in applying the growth factor:

1. Animal testing method:

Forty male rats formed critically sized cranial defects with a diameter of 8 mm. A group of 20 rats divided into two groups and implanted with bone forming protein (concentration 0.025 mg / ml rhBMP-2) using a scaffold composed of TCP / HA, and a bone forming protein (concentration 0.025 mg) into a scaffold composed of TCP / HA. / ml rhBMP-2) was divided into upper non-absorbent membranes (e-PTFE membrane), and the results were compared histologically at 2 and 8 weeks postoperatively.

2. Experimental results:

In the histological comparison, the two-week (Fig. 12C) and eight-week (Fig. 12D) healing of the shielding group showed continuous bone formation above the scaffold. Healing of weeks (Fig. 12a) and week 8 (Fig. 12b) showed irregular bone formation.

Therefore, by applying the shielding film to the scaffold having a three-dimensional structure for tissue regeneration, it was possible to regenerate the complete structure and composition by preventing the surrounding heterogeneous tissue from growing inside.

Claims (20)

Kit for tissue or organ regeneration consisting of growth factors, shields and scaffolds.
The method according to claim 1, wherein the growth factor is composed of the scaffold with lyophilization on the shield, the growth factor is composed of the shield with lyophilization on the scaffold, or the growth factor is lyophilized on the scaffold and the shield, respectively. Kit for tissue or organ regeneration.
The method according to claim 1 or 2, wherein the growth factor is BMP-2 (Bone Morphogenetic Protein 2), BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP- Bone Morphogenetic Proteins (BMP) selected from the group consisting of 9, BMP-10, BMP-11, BMP-12, and BMP-13, and the co-duplex of amino acids constituting them (amino acids of B2 and amino acids of B4) Transforming growth factor beta superfamily (TGF-β), platelet-derived growth factor, protein such as fibroblast growth factor, fibroblast growth factor or growth differentiation factor (GDF), Tissues or organs selected from one or more selected from the group consisting of insulin-like growth factor, epidermal growth factor and transformative growth factor, keratinocyte growth factor 2 (KGF2) and MP52 protein and genetically engineered proteins of said growth factors Regeneration Kit.
According to claim 1 or 2, the material of the shielding film is a bioabsorbable synthetic polymer material of polycaprolactone (PCL) or polylactic acid (PL), polyglycolic acid (PGL) and a copolymer of lactic acid and glycoic acid (PLGL); Bioabsorbable natural polymers of alginic acid, chitosan, collagen, hyaluronic acid and cellulose and mixtures thereof; Polyvinylidene fluoride (polyvinylidene fluoride), poly (tetrafluoroethylene), polyvinyl alcohol (PVA), polyhydroxyalkanoate, polyethylen terephthalate ( polyethylene terephthalate, polybutylene terephthalate, polymethyl methacrylate, polyhydroxyethylmethacrylate, polyN-isopropylacrylamide, Polydimethylsiloxane, polydioxanone, polypyrrole, polyglycolic acid (PGA), polylactic acids (PA), polyethylene oxides, polyethylene and glycol acids PLGL, polycaprolactone (PCL), polyanhydrides, polyphosphazenes, Lee O- ester (polyortho-esters), polyimide (polyimides), titanium, and the material selected tissue or organ regeneration kit for from the group consisting of a thin metal film of stainless steel.
The kit for tissue or organ regeneration according to claim 1 or 2, wherein the shielding film is composed of a single layer or a multilayer of 2 to 5 layers.
The kit for tissue or organ regeneration according to claim 1 or 2, wherein the shielding membrane is a non-porous sheet or a membrane having pores.
The kit for regenerating tissues or organs according to claim 1 or 2, wherein the shielding film has a high strength as a reinforcing layer.
8. The tissue or organ regeneration kit according to claim 7, wherein the material of the reinforcing layer is a metal selected from polycaprolactone, polylactic acid, polyglycolic acid and copolymers of lactic acid and glycoic acid, titanium and stainless steel.
3. The method according to claim 1 or 2,
The material of the scaffold is a bioabsorbable polymer material of polycaprolactone (PCL), polylactic acid (PL), polyglycolic acid (PGA), and a copolymer of lactic acid and glycoic acid (PLGL); Natural polymers of chitosan, alginic acid, collagen, hyaluronic acid, chondroitin, glucosamine, cellulose; Complexes thereof; And a tissue or organ regeneration kit selected from the group consisting of synthetic bone, allogeneic bone, xenograft, demineralized bone or cadaveric derived dermis.
3. The collagen according to claim 1 or 2, wherein the shape of the scaffold is in the form of a sheet, a porous sheet, a sponge, a jelly, a gel, a lyophilized phase, a microsphere, a tubular (tube), lattice or lattice structure. Kit for regenerating tissue or organ which is lattice containing this.
11. The tissue or organ regeneration kit according to claim 10, wherein the growth factor is contained in a gel-like scaffold and filled in a syringe or vial and then lyophilized.
The tissue or organ regeneration kit according to claim 1 or 2, wherein the scaffold is composed of a single layer or a multilayer of two or more layers and five or less layers.
The kit for tissue or organ regeneration according to claim 12, wherein the multiple scaffolds are separated from each other, adhered or glued together.
The kit for tissue or organ regeneration according to claim 1 or 2, wherein a reinforcing layer is added to maintain the shape of the scaffold.
15. The bioabsorbable polymer according to claim 14, wherein the material of the reinforcing layer is polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polyglycolic acid, and a copolymer of lactic acid and glycoic acid (PLGA). Kit for regenerating selected tissues or organs.
The kit for tissue or organ regeneration according to claim 1 or 2, wherein the scaffold is lattice solid and the collagen sponge is filled in the lattice.
The material of claim 16, wherein the lattice-like material is polycaprolactone or polylactic acid, polyglycolic acid, a synthetic high molecular material of lactic acid and glycoic acid, a mixture of synthetic high molecular material and calcium phosphate ceramic, titanium or stainless steel metal. Kit for tissue or organ regeneration selected from.
Pluripotent mesenchymal stem cells, pluripotent hematopoietic stem cells, pluripotent stem cells, pluripotent neural stem cells, pluripotent pancreatic stem cells, harvested tooth pulp cells according to claim 1 or 2. And tissue or organ regeneration kit containing at least one stem cell selected from the group consisting of periodontal ligament cells.
The phenylpropionic acid derivative family according to claim 1 or 2, wherein pyrocampum, ketoprofen, flurbiprofen, fenoprofen, and ibuprofen are used in addition to the growth factor. Nonsteroidal anti-inflammatory agents (enides, NSAIDs); Nonsteroidal anti-inflammatory agents (oxydes, NSAIDs) of the oxycam derivatives including pyroxicam, tenoxicam and meloxicam; Diclofenac; And anti-inflammatory drugs selected from indomethacin (indomethacin), amoxicillin, erythromycin, metronidazole, Amoxicillin, Amoxacillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, Other Lincosphaporin ), A kit for tissue or organ regeneration comprising at least one corticosteroid agent selected from tetracycline hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride, methylprednisolone, and hydrocortisone acetate.
The tissue or organ regeneration kit according to claim 1 or 2, comprising a shield membrane designed and manufactured to obtain a three-dimensional image of the damaged tissue or organ and retaining the shape to be reproduced by a simulation program, and a scaffold filled therein.

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