JPH11246420A - Wound curing accelerator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a wound curing accelerator exhibiting higher wound curing effect in a wound position or an ulcer position. SOLUTION: This wound curing accelerator is characterized by inserting or attaching a blood platelet into a matrix (e.g. collagen gel) and the blood platelet is characterized by a blood platelet of itself. Further, the wound curing accelerator contains cytokine, a cell growth factor (e.g. basic fibroblast growth factor) or a stimulating agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a wound healing promoter.

[0002]

2. Description of the Related Art Conventionally, in the treatment of skin ulcers and pressure sores, the formation of granulation and the regeneration of the epidermis are expected by the natural healing power of patients while removing necrotic tissue and suppressing the growth of bacteria with antibacterial agents. The main method was to do it. In other words, in many cases, an ointment containing an antibiotic is used, and a method of waiting for the formation of granulation and regeneration of the epidermis by the natural healing power of the patient while suppressing the growth of bacteria and the like has been adopted.

[0003] However, in recent years, along with the progress of basic research on wound healing, drugs and treatments that actively promote granulation and epidermis formation have been desired.
Substances that promote granulation and epidermal regeneration have come to be applied clinically. Recently, various cell growth factors and the like have been developed as wound healing agents. For example, in patients with various diseases such as diabetes and elderly people, there is a problem that ulcers and pressure sores tend to occur and are difficult to heal. In particular, in diseases such as diabetes, the production of various cell growth factors required in the wound is suppressed, and as a treatment method, administration of purified cell growth factors or genetically modified cell growth factors can be considered. ing. For example, genetically modified basic fibroblast growth factor and the like have been developed for clinical application.

[0004] It is known that platelets contain various cell growth factors such as platelet-derived growth factor (PDGF), vascular endothelial cell growth factor (VEGF), and epidermal growth factor (EGF). Platelets are known to play an important role in wound healing through these factors.

[0005] On the other hand, matrices such as collagen, chitin, chitosan, and fibrin have an effect of promoting healing in wounds, and are used as wound coatings and adhesives. For example, collagen plays an important role as a cell substrate as an extracellular matrix, and is used as a substrate for various cells in vitro. Various wound dressings such as collagen membranes have been developed for the treatment of shallow burns in which epidermal cells remain in burns and the like. Further, in the case of a deep burn in which epidermal cells are completely lost or a wound with complete loss of skin, autogenous skin transplantation or the like is required, and development of artificial skin that survives as it is to form skin has been desired. Therefore, as artificial skin with a structure equivalent to living skin,
Hybrid artificial skin in which fibroblasts and epidermal basal cells are cultured on collagen gel has been developed (E. Bel).
l, P. Ehrlich, D .; J. Butle, T .;
Nakatsuji: Living Tissue F
ormed in vitro and Accept
ed as Skin-Equivalent Tis
sue of Full Thickness. Sci
ence Vol. 211. p1052, 1981,
I. V. Yannas, J .; F. Burke, D .; P.
Orgill, E .; M. Skrabut: Wound
Tissue Can Utilize a Poly
meric Template to Synthes
size a Functional Extension
no of Skin. Science Vol. 21
5. 174, 1982). Thus, skin cells are used for artificial skin because they can be easily collected from a small amount of skin pieces of the patient themselves, are relatively easy to culture, and can proliferate to the required number of cells.

[0006] As described above, in the treatment of skin ulcers and pressure sores, drugs and treatments that actively promote granulation, epidermis formation, and angiogenesis are desired along with progress in basic research on wound healing. As a result, substances that promote granulation, epidermal regeneration, and angiogenesis have come to be applied clinically. Hybrid artificial skin in which fibroblasts and epidermal basal cells are cultured on collagen gel is also used as artificial skin.

[0007] However, since cytokines and cell growth factors form a complex network, administration of a single cytokine or cell growth factor to a living body in a high volume results in increased side effects. In addition, leukocytes, platelets, bone marrow cells, etc. are major cytokine-producing cells, but even if these cells are locally transplanted, they do not stay at the target affected area and spread, resulting in reduced effects and increased side effects. Can cause Administration of collagen or the like as an extracellular matrix does not exert a sufficient healing promoting action when the wound healing power of a living body such as an elderly person is deteriorated. Even with artificial skin, it is effective after leukocyte infiltration and granulation, which are early stages of wound healing, but does not exert a strong effect on promoting healing from the early stage of wounding.

[0008] As described above, in the treatment of wounds, a therapeutic agent that further promotes wound healing is desired. As a substance having such a wound healing action, activated platelet-rich plasma is mixed with a microcrystalline collagen carrier, and a platelet-derived angiogenic factor and a growth factor, and a composition for treating a wound containing the carrier (particularly, Table 1
No. 1628) and a substrate for cell culture in which a cell growth factor derived from platelets is carried on a substrate containing collagen (Japanese Patent Application Laid-Open No. 63-79588). However, these cells carry cell growth factors released from platelets on collagen, and there is a possibility that these cell growth factors may be lost or inactivated from the carrier or the substrate during the preparation process.

[0009]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to provide a wound healing promoting agent having a higher wound healing effect at a wound site or an ulcer site. .

[0010]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have made intensive studies and found that platelets are embedded in a matrix, for example, collagen gel, fibrin gel, etc., and implanted in a wound. We found that a strong wound healing promoting effect was exhibited, and that a strong wound healing promoting effect was exhibited by implanting platelets in a matrix and implanting it simultaneously with cytokines, cell growth factors or stimulants. The present invention has been completed.

[0011] That is, the invention of claim 1 is a wound healing promoter characterized in that platelets are embedded or adhered to a matrix. The invention according to claim 2 is the wound healing promoter according to claim 1, wherein the platelets are autologous platelets.

[0013] The invention according to claim 3 is the wound healing promoter according to claim 1 or 2, wherein the matrix is a biodegradable matrix. The invention according to claim 4 is the wound healing promoter according to claim 3, wherein the matrix is a collagen gel or a fibrin gel. Claim 5
The described invention is the wound healing promoter according to any one of claims 1 to 4, further comprising a cytokine, a cell growth factor, or a stimulant.

Hereinafter, the present invention will be described in detail. The wound healing promoting agent of the present invention is characterized in that platelets are embedded or adhered to a matrix.

The matrix is not particularly limited as long as it is capable of embedding or adhering platelets and is not dermatologically toxic. Examples of the natural organic polymer include cellulose and cellulose. Polysaccharides such as chitin, chitosan, agarose and calcium alginate; proteins such as collagen, gelatin, albumin and fibrin; and as synthetic organic polymers,
Examples include polymethyl methacrylate, polystyrene, polyethylene, polylactic acid, silicone resin, and polytetrafluoroethylene resin.

As the matrix, biodegradable ones are particularly preferable, for example, collagen, chitin, chitosan, fibrin and the like are preferable. When the matrix is a matrix having biodegradability, the wound healing promoting agent of the present invention is used in contact with the wound site, and after the wound healing has progressed, it is not necessary to peel off or wash off the wound. is there.

When the matrix itself has a wound healing promoting effect, such as collagen, chitin, chitosan, fibrin, etc., the healing promoting effect is synergistic with the wound healing promoting effect of platelets. It is particularly preferable because it is exhibited.

The matrix has biodegradability,
It has a wound healing promoting effect by itself and is easy to obtain,
In addition, collagen gel or fibrin gel is particularly preferable because it is easy to use.

The above-mentioned platelets refer to platelets of humans and animals, and particularly, platelets of autologous ones, that is, those of a person who is to treat a wound. This is because, in the case of autologous platelets, wound healing is promoted safely without host rejection to platelets after transplantation.

Platelets can be collected and separated by known methods. For example, a method of separating from blood collected with sodium citrate using a centrifugal separation method, a centrifugal separation method in a density gradient solution, a gel filtration method, or the like may be used. Moreover, what was cultured may be sufficient.

In the agent for promoting wound healing of the present invention, the number of platelets embedded or adhered to the matrix can be freely increased or decreased according to the required number of cells.

The method for obtaining a wound healing promoter having platelets embedded or adhered to a matrix is not particularly limited.
For example, the following method can be mentioned. First, platelets are separated from blood. This is mixed with the matrix in a solution state to gel the mixture. After the mixture is gelled, it may be cultured as necessary.

The agent for promoting wound healing of the present invention is used in contact with a wound site. The above-mentioned contact may be brought into contact with the wound site by inserting or covering the wound site, in addition to the case where the wound site is brought into contact with a bandage, tape, bandage, or the like.

The wound healing promoting agent according to claim 5 of the present invention further comprises a cytokine, a cell growth factor or a stimulant, and the wound healing promoting agent according to any one of claims 1 to 4, Agent.

[0024] The wound healing promoting agent according to claim 5 can be obtained, for example, by immersing a platelet embedded or adhered to a matrix in a solution containing cytokines, cell growth factors or stimulants. it can. Examples of the cytokine include interferon-
γ, tumor necrosis factor-α, interleukin-1α, interleukin-1β and the like. Examples of the cell growth factor include acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor. Examples of the stimulant include thrombin and platelet activating factor.

The method of using the wound healing promoter according to the fifth aspect is the same as that of the wound healing promoter according to the first aspect.

In the wound healing promoter of the present invention,
After the platelets have been activated with a stimulant, they may be implanted in the wound.

The shape of the wound healing promoter according to claims 1 to 5 of the present invention is not particularly limited, and can be adapted to the shape of the wound site. For example, pellets, membranes, microcapsules and the like can be used. Various shapes can be obtained by molding.

In the wound healing promoting agent of the present invention, fibroblasts and epidermal cells used in conventional artificial skin are mixed together with platelets and embedded or adhered to a matrix. Good.

[0029]

When the wound healing promoter of the present invention is brought into contact with a wound site, various healing promoters such as cell growth factors are produced from platelets at the wound site, thereby promoting wound healing.

In addition, the agent for promoting wound healing of the present invention can satisfactorily stay at the wound site because platelets are embedded or adhered to the matrix.

The agent for promoting wound healing of the present invention assists infiltration of wounds such as leukocytes and the like, and artificially activates leukocytes and the like even in patients with various diseases such as diabetes and the elderly. It can promote wound healing in such cases.

Particularly, the wound healing promoter according to claim 5 is
Since it contains cytokines, cell growth factors, or stimulants in addition to platelets, the effect of promoting wound healing is particularly high.

[0033]

Embodiments of the present invention will be described below.

Example 1 Heart blood was collected from 7-week-old Wistar rats using sodium citrate as an anticoagulant, and platelets were collected from the blood by centrifugation. This platelet is 1 × 10 ⁷
An acid-soluble type I collagen solution derived from calf skin (trade name: CELLGE, manufactured by Koken Co., Ltd.)
N) (final concentration of collagen: 0.25% by weight). This was mixed and stirred under ice cooling, and 100 μl was injected into a 96-well microplate (manufactured by Iwaki Glass Co., Ltd.).
The mixture was heated at 30 ° C. for 30 minutes to form a disk-shaped platelet-implanted collagen gel, which was used as a wound healing promoter. This was subjected to the following test.

Example 2 In the same manner as in Example 1, a disk-shaped collagen gel having platelets embedded therein was prepared. This is called Human Recombi
nant basic FGF (Genzyme)
Was immersed in a calcium / magnesium-free phosphate buffer containing 1 μg / ml for 2 hours to obtain a wound healing promoter.
This was subjected to the following test.

Comparative Example 1 An acid-soluble type I collagen solution derived from calf skin (trade name: CELLGEN, manufactured by Koken Co., Ltd.) was stirred under ice-cooling (collagen concentration: 0.25% by weight), and a 96-well microplate ( After injection of 100 µl into Iwaki Glass Co., Ltd., 37 ° C
For 30 minutes to produce a disc-shaped collagen gel,
This was subjected to the following test.

Comparative Example 2 Heart blood was collected from 7-week-old Wistar rats using sodium citrate as an anticoagulant, and platelets were collected from the blood by centrifugation. This platelet is 1 × 10 ⁷
The cells were suspended in a calcium / magnesium-free phosphate buffer so as to have a concentration of cells / ml to prepare a platelet suspension. 100 μl of this platelet suspension was subjected to the following test.

Comparative Example 3 A disk-shaped collagen gel was prepared in the same manner as in Comparative Example 1. This is called Human Recombinant b
1 μg / m of asic FGF (Genzyme)
Each of the samples was immersed in a calcium / magnesium-free phosphate buffer containing 2 g for 2 hours, and subjected to the following test.

(Test) Each of the wound healing promoters of Examples 1 and 2 and the samples of Comparative Examples 1 to 3 were separately subjected to the following tests.

Seven-week-old male Wistar rats were purchased from SLC Japan, preliminarily reared for one month, and weighed 2 weeks.
Twenty-five rats of 25-300 g were subjected to the experiment per test.

The skin of the back of the rat, which was shaved using a hair clipper and a shaver, was incised along the midline (about 3 cm) under ether anesthesia. As a control on the side,
Only collagen gel was inserted.

After the insertion of the sample, the incision was sutured at three places at equal intervals using a metal suture device (manufactured by Natsume Seisakusho). After inserting the sample, the granulation tissue formed on the seventh day was taken out and weighed. The ratio of the weight of the granulation tissue of the sample to the weight of the granulation tissue of the control was defined as the growth granulation tissue ratio (%), and the average value of the four rats subjected to the test is shown in Table 1.

[0043]

[Table 1]

Table 1 shows that the agent for promoting wound healing of the present invention has a higher wound healing effect than the transfer of collagen gel, platelets, cell growth factors and the like.

[0045]

According to the present invention, there is provided a wound healing promoting agent having a higher wound healing effect at a wound site or an ulcer site. Is done.

The composition of the agent for promoting wound healing according to claim 2 comprises:
As described above, according to the present invention, there is provided a wound healing promoting agent which does not cause host rejection to platelets after transplantation and has a higher wound treatment effect at a wound site or an ulcer site.

The composition of the agent for promoting wound healing according to claim 3 comprises:
As described above, according to the present invention, since the matrix has biodegradability, there is no need to peel or wash off after the progress of wound healing. An effective wound healing promoter is provided.

The composition of the agent for promoting wound healing according to claim 4 comprises:
As described above, according to the present invention, since the matrix is a collagen gel or a fibrin gel, it has biodegradability, itself has a wound healing promoting effect, and is easily available and easy to use. The present invention provides a wound healing promoting agent which has a higher wound treatment effect and is easy to use and easy to manufacture.

The composition of the agent for promoting wound healing according to claim 5 comprises:
As described above, according to the present invention, in addition to platelets,
Furthermore, since it contains a cytokine, a cell growth factor or a stimulant, a wound healing promoter having a particularly high wound treatment effect at a wound site or an ulcer site is provided.

Claims (5)

[Claims] An agent for promoting wound healing, wherein platelets are embedded or adhered to a matrix. 2. The agent for promoting wound healing according to claim 1, wherein the platelets are autologous platelets. 3. The wound healing promoter according to claim 1, wherein the matrix is a biodegradable matrix. 4. The wound healing promoter according to claim 3, wherein the matrix is a collagen gel or a fibrin gel. 5. The wound healing promoting agent according to claim 1, further comprising a cytokine, a cell growth factor, or a stimulant.