JP2004254759A - Composite material for promoting hair growth - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composite material for promoting hair growth which achieves an excellent affinity to the cells along with an improvement in mechanical strength. <P>SOLUTION: The composite material for promoting hair growth comprises a sponge-like biodegradable absorbent polymer containing a fibrous biodegradable absorbent polymer. The sponge-like biodegradable absorbent polymer is preferably a collagen sponge. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a composite material for promoting hair growth useful as a scaffold for hair regeneration.
[0002]
[Prior art]
In biological tissue engineering, it is important to construct an environment (field) for inducing tissue regeneration in cells. Scaffolds for cell growth and differentiation required for this field construction have been made from various polymeric materials. In addition, these scaffold materials can also be used as a culture substrate for various cells. The conditions of the scaffold material include: 1) having an affinity for cells, 2) maintaining a sufficient mechanical strength for a period required for inducing regeneration of a living tissue, and 3) having a biodegradable and absorbing property. It can be rapidly degraded and eliminated as the regeneration progresses so as not to hinder the progress of the regeneration. 4) It must be porous so that cells can easily enter and oxygen and nutrients can be supplied to the entered cells. , Etc. are required. As a scaffold material, collagen (a major component of the extracellular matrix), which is particularly excellent in affinity for cells, is widely used in the form of a sponge.
[0003]
However, the collagen sponge has been disadvantageous in that the strength of the collagen sponge decreases when it is touched or immersed in a cell culture solution or a body fluid, and the collagen sponge cannot maintain a matrix-like shape for a long time. In order to solve the problem of the mechanical strength of the collagen sponge, a composite material in which a collagen sponge is coated with a bioabsorbable plastic (Japanese Patent Application Laid-Open No. Hei 7-236688) or a polymer material for a skeleton made of a bioabsorbable material is used. Is molded into a sponge shape or the like, and a medical material (Japanese Patent Laid-Open No. 2001-204807) or the like in which a gel such as collagen is contained therein has been proposed. However, these composite materials have drawbacks such as the inability of cells to directly contact collagen and the slow decomposition of the material, and thus require improvement as a scaffold for effective regeneration induction of living tissues. In addition, satisfactory affinity as a scaffold for hair regeneration could not be obtained because the affinity with cells was not sufficiently high.
[0004]
Prior art document information related to the present invention is as follows.
[0005]
[Patent Document 1]
JP-A-7-236688
[Patent Document 2]
JP-A-2001-204807
[Non-patent document 1]
[Problems to be solved by the invention]
An object of the present invention is to provide a hair growth-promoting composite material having excellent affinity with cells and improved mechanical strength.
[0006]
[Means for Solving the Problems]
The present invention provides a hair growth promoting composite material comprising a sponge-like biodegradable absorbent polymer containing a fibrous biodegradable absorbent polymer. Preferably, the sponge-like biodegradable absorbent polymer is a collagen sponge. Since the composite material of the present invention has a higher ability to retain its structure when a cell suspension is added than a conventional collagen sponge, it is excellent as a scaffold for cell culture and tissue engineering in regenerative medicine such as hair growth. ing.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The composite material of the present invention is characterized by comprising a sponge-like biodegradable absorbent polymer containing a fibrous biodegradable absorbent polymer.
[0008]
Examples of the sponge-like biodegradable absorbent polymer used in the present invention include sponge-shaped proteins such as collagen, gelatin, fibrin, gluten, and fibroin and derivatives thereof; polyamino acids and derivatives thereof; chitin and chitosan. , Hyaluronic acid, alginic acid, starch, dextran and the like and derivatives thereof; mixtures and complexes of two or more of these. Further, a complex of a natural polymer and a synthetic polymer is also preferably used in the present invention.
[0009]
The sponge shape means a porous shape capable of providing a space suitable for cell growth.
[0010]
In a particularly preferred embodiment of the present invention, the sponge-like biodegradable and absorbable polymer is collagen. The preferred collagen used in the present invention is not particularly limited, and is a known acid-solubilized collagen, enzyme-solubilized collagen, alkali-solubilized collagen, or a solubilized collagen obtained from animal bone or skin as a raw material. A collagen solution such as chemically modified collagen or regenerated collagen obtained by regenerating collagen fibers from solubilized collagen can be used. Specific examples include type I collagen derived from pig skin, type I collagen derived from pig tendon, type II collagen derived from bovine nose cartilage, type I collagen extracted from fish, and the like. Alternatively, collagen may be obtained by genetic recombination, or may be produced by chemical synthesis.
[0011]
As the biodegradable polymer, gelatin is preferably used. Gelatin is derived from collagen that can be collected from all parts of the body such as skin, bones and tendons of various animal species including cows, pigs, fish, etc. It can be obtained by denaturation by various treatments such as decomposition and enzymatic decomposition. Denatured gelatin of recombinant collagen may be used.
The concentration of the collagen solution is not particularly limited, but is preferably 0.1-4% by weight, more preferably 0.3-2% by weight.
[0012]
A collagen sponge can be easily formed by freeze-drying a collagen solution. In order to change the sponge hole diameter, a known technique such as changing the freezing temperature can be used. Further, for the purpose of adjusting the pore size of the collagen sponge to an optimum range, an organic solvent having a property of not mixing with water may be added to the collagen solution and freeze-dried. Examples of such an organic solvent include halogenated hydrocarbons such as chloroform, carbon tetrachloride, and methylene chloride; esters such as ethyl acetate and ethyl propionate; aromatic hydrocarbons such as benzene and toluene; hexane and cyclohexane. And the like; ethers such as diethyl ether and diisopropyl ether; and the like, and these may be used alone or in combination of two or more. Further, a method of adding various additives to the collagen solution, a method of bubbling the collagen solution before freeze-drying, and the like are also known.
[0013]
As the fibrous biodegradable polymer used in the present invention, a natural or synthetic polymer having biodegradability can be used, for example, polyglycolic acid, polylactic acid, poly-ε-caprolactone, polydioxanone. , Poly-β-malic acid, polyorthoesters, polydiaminophosphazenes, and copolymers thereof. Among these, polyglycolic acid, polylactic acid, poly-ε-caprolactone, and copolymers thereof are particularly preferable. Only one kind of biodegradable absorbent polymer may be used, or two or more kinds may be used in combination.
[0014]
According to the present invention, the spongy biodegradable absorbent polymer contains a fibrous biodegradable absorbent polymer, thereby maintaining excellent affinity with cells and at the same time obtaining sufficient mechanical strength. Can be. The fiber may be a long fiber or a short fiber, and may be a nonwoven fabric suitably loosened. The diameter of the fiber is not particularly limited, but a fiber having a diameter of 1 to 100 μm can be preferably used. It is preferable that the content of the fibrous biodegradable absorbent polymer is such that the weight ratio of fibrous biodegradable absorbent polymer / sponge-shaped biodegradable absorbent polymer is 0.2 to 10.0. When collagen is used as the sponge-like biodegradable polymer, the fibrous biodegradable polymer is 0.01 to 10% by weight based on the collagen solution (before adding the organic solvent and various additives). And more preferably 0.4-1% by weight.
[0015]
A sponge-like biodegradable absorbent polymer containing a fibrous biodegradable absorbent polymer can be freeze-dried and then subjected to a crosslinking treatment to further increase the mechanical strength. By adjusting the degree of crosslinking, the rate of biodegradation of the sponge-like biodegradable and absorbable polymer can be controlled. The method of the crosslinking treatment is not particularly limited, and for example, a chemical crosslinking method, a thermal dehydration crosslinking method under vacuum, or a crosslinking method by irradiation with ultraviolet rays, gamma rays, electron beams, or the like can be used. Also, these methods can be used in combination. For example, when performing chemical cross-linking, a cross-linking agent such as glutaraldehyde, carbodiimide, carbonylimidazole, diepoxy compound, dianhydride, epichlorohydrin and the like can be used. When performing thermal dehydration crosslinking under vacuum, for example, the crosslinking can be performed at a crosslinking temperature of 100 to 170 ° C. for 2 to 120 hours.
[0016]
The composite material for promoting hair growth of the present invention thus obtained has a fibrous biodegradable polymer randomly embedded in a crosslinked porous sponge made of a biodegradable polymer. It has a structure. That is, the sponge substance is collagen having a high cell affinity, and fibers are incorporated to mechanically reinforce it. For this reason, a sponge having excellent affinity with cells and maintaining sufficient mechanical strength for a period required for skin tissue regeneration and hair growth can be obtained. Furthermore, the sponge can be degraded and absorbed in vivo over time without negatively affecting hair growth. Therefore, it is useful as a scaffolding material for hair growth.
[0017]
As shown in the following examples, when a hair growth experiment was performed using the hair growth promoting composite material of the present invention and a conventional collagen sponge, in the conventional collagen sponge, epidermal cysts and ectopic hair were found. While it is often formed and interferes with normal hair regeneration, when a collagen sponge reinforced with polyglycolic acid (PGA) fiber is used, normal hair growth is observed at the implantation site and the number of hair growth is also reduced. Significantly higher. This is because collagen does not have high mechanical strength, which reduces pore structure size, impedes cell migration and results in leakage of cell suspension, which results in epidermal cysts and ectopic hair formation. It is considered to be. In contrast, a collagen sponge incorporating PGA fibers according to the present invention is mechanically reinforced, so that the size of the pore structure in the sponge can be maintained during cell seeding. Therefore, it is considered that ectopic hair formation was hardly observed, the formation of the epidermal follicle was suppressed, and a place where the hair follicle could be normally regenerated was provided. Therefore, the composite material for promoting hair growth of the present invention is useful as a scaffold material in regenerative medicine such as hair growth.
[0018]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
Preparation of Cultured Cells C57BL / 6J mouse and athymic nude mouse BALA / cAJcl-nu were purchased from CLEA Japan. Fetal mice were removed from the uterus of C57BL / 6J mice on days 16-17 of gestation and washed in Dulbecco's phosphate buffered saline (PBS: Sigma-Aldrich, St. Louis, MO). At this point, skin cells isolated from fetal mice retain the ability to form hair follicles in a hair regeneration assay. The fetal back skin was harvested with scissors and the skin was separated from the subcutaneous tissue by centrifuge. The skin was placed in KGM-2 (purchased from Clonetic) supplemented with 0.05% collagenase IV at 4 ° C. for 16 hours and then separated into epidermis and dermis. The epidermis was dissociated by pipetting in KGM-2, and the dermis was dissociated by pipetting in KGM-2 without addition of insulin, epidermal growth factor and bovine pituitary extract. The dissociated cells were each filtered through a 70 μm nylon filter, and the number of cells was measured. The cells obtained from the epidermis and the dermis were adjusted to a total of about 1 × 10 ⁷ cells, and centrifuged at 1000 rpm for 5 minutes to give a suspension of about 60 μl of KGM-2. Cells were stored on ice until use.
[0019]
Preparation of collagen sponge Two types of collagen sponges, one with and without PGA fibers, were prepared. A 3 mg / ml collagen solution (manufactured by Nitta Gelatin Co., Ltd.) is poured into a mold in which polyglycolic acid (PGA) fibers (manufactured by Gunze) having a diameter of 20 μm are arranged, and then lyophilized to obtain a dry weight ratio. A collagen sponge (PGA (+)) containing 1.33 wt% of PGA fiber / collagen and containing polyglycolic acid fiber was obtained. This was subjected to thermal dehydration crosslinking at 140 ° C. for 12 hours. A collagen sponge containing no PGA fiber was similarly prepared using only the collagen solution (PGA (-)). Next, these collagen sponges were gas-sterilized. Since the collagen sponge containing the polyglycolic acid fiber is slightly hydrophobic as compared with the collagen sponge, the collagen sponge was washed once with 70% ethanol immediately before use, and then washed once with a phosphate buffer (PBS) and once with KGM-2.
[0020]
Insertion of silicon chamber A sterile silicon chamber was implanted into the back of athymic nude mice (BALA / cAJcl-nu) to prevent migration of cells from adjacent skin (Fig. 1). Anesthetized by injecting 0.02 ml of 13% pentobarbital (Dainippon Pharmaceutical Co., Ltd.) into the peritoneal cavity of 8-week-old athymic nude mice (purchased from BALA / cA Jcl-nu CLEA Japan) per gram of body weight A 1 cm diameter wound right above the fascia was made on the back. Of the pair of upper and lower silicon chambers, the lower one was inserted between the skin and the chest wall, sewed with nylon thread, and fixed. Next, a collagen sponge having a diameter of 1 cm and a thickness of 3 mm was mounted in the chamber, and the previously prepared cell suspension was uniformly dropped on the sponge. An upper chamber was covered to prevent drying of the culture. One week after implantation, the upper chamber was removed and 10-12 days later the lower chamber was removed.
[0021]
Grafts were observed daily for the first 10 days after transplantation and every other day thereafter for up to 3-4 weeks. After a lapse of 3-4 weeks, all of the hairs that grew were shaved and the number was counted. The area of hair growth was also measured to compare the density of hair growth (three per group). In order to histologically examine the hair-grown skin, it was left in a 10% formalin solution (Wako Pure Chemical Industries, Ltd.) at room temperature for 2 hours, embedded in paraffin, and 8 μm thick sections were prepared. Eosin staining was performed.
[0022]
Macroscopic observations of the transplantation site To investigate the effect of collagen PGA (-) sponge and PGA (+) sponge on hair formation, the transplantation site is usually used until 3-4 weeks when hair regeneration is completed. Was observed. Hair follicles were observed within one week in the skin grafted with PGA (-) sponge. However, as shown in FIGS. 2A and 2B, hair growth was slow and sparse. The size of the fixed implantation site extended beyond the size of the original sponge (FIG. 2A). In addition, ectopic hair growth was visible under the skin around the site of implantation (FIG. 2C).
[0023]
In contrast, when the implant was applied on a PGA (+) sponge, hair formation was induced slightly later than the PGA (-) sponge 10-12 days after implantation. However, the hair on the PGA (+) sponge was very thick and uniformly growing normally compared to the hair on the PGA (-) sponge (FIGS. 2C and 2D). The size and shape of the implantation site on the PGA (+) sponge did not change from the original sponge size. Ectopic hair growth was hardly observed (FIG. 2E).
[0024]
Was quantitatively analyzed hair density on the strip each transplant, PGA (-) and mean hair growth density of PGA (+) on the sponge, it was respectively 300 and 2,500 / cm ² (Figure 4). This indicates that PGA (+) sponge is more advantageous for hair growth.
[0025]
Histological findings Next, the grafts were examined histologically. Although the basic structure of the skin, that is, the epidermis and the dermis were regenerated, all transplanted sites of the PGA (-) sponge contained epidermal cysts in the dermis and ectopic hair formation was observed under the skin (FIG. 3A). , FIG. 5). Even when the hair grew out of the skin, the arrangement was somewhat disordered. In some cases, so many epidermal cysts were formed that completely disrupted normal hair formation (FIG. 3B). With the PGA (+) sponge, few epidermal cysts were observed and ectopic hair formation was much less than with PGA (-) (FIGS. 3C and 3D). Further, on the PGA (+) sponge, the hair arrangement was regularly arranged from the head to the tail in an inclined manner. Sebaceous glands were formed normally. That is, in this assay, PGA (+) collagen sponge did not interfere with normal hair formation and growth as a scaffold.
[0026]
From the above results, in the conventional collagen sponge, epidermal cysts and ectopic hairs are often formed, and normal hair regeneration is hindered, whereas when the collagen sponge of the present invention reinforced with PGA fibers is used, Showed that the above-mentioned phenomenon was not observed, and normal hair growth was observed at the transplant site. In addition, no change in the size of the transplanted site was observed, indicating that the material was a scaffold for hair regeneration with excellent cell affinity and mechanical properties.
[Brief description of the drawings]
FIG. 1 is an illustration of a chamber used in a hair regeneration experiment.
FIG. 2 shows hair growth on an implant.
FIG. 3 shows hematoxylin and eosin staining of graft sections.
FIG. 4 is a graph showing the hair growth density at a transplant site.
FIG. 5 is a graph showing the number of epidermal cysts generated at a transplant site.

Claims (2)

A composite material for promoting hair growth, comprising a sponge-like biodegradable absorbent polymer containing a fibrous biodegradable absorbent polymer. The composite material for promoting hair growth according to claim 1, wherein the sponge-like biodegradable and absorbable polymer is a collagen sponge.

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