KR20190101319A - An transplantation implant for promoting hair growth - Google Patents

Abstract

The present invention relates to an implant for bio-transplantation containing a stem cell having hair growth functional cytokine secretion ability. It is an object of the present invention to provide the implant for bio-transplantation for promoting hair growth and/or alleviating and treating hair loss.

Description

Biotransplantation implant for promoting hair growth {AN TRANSPLANTATION IMPLANT FOR PROMOTING HAIR GROWTH}

 The present invention relates to an implant for promoting hair growth.

Hair plays a variety of roles in the human body, including head protection, outward effects, and maintaining the temperature of the head. It is not an important organ for life, but it is a measure of health and an important part of the body that determines appearance. Hair loss has been recognized as a series of aging phenomena, but recently, it is revealed that hair loss progresses due to various genetic factors such as stress, westernized eating habits, nutritional imbalance, and changes in social activities.

Hair is produced in the hair follicle, a structure in which the skin is embedded by the continuous proliferation of stromal cells at the base of the scalp, and has a hair cycle including various stages of growth. Hair is the anagen (growing phase) that makes up 90% of normal hair, the catagen (transitional phase) where hair growth and hair roots are atrophy, the telogen (a resting phase) and the dropper (the hairball becomes dry and hairy). Exogenous growth is repeated according to the four stages of growth cycle.

The causes of hair loss include genetic factors, stress, and aging. One of the typical mechanisms is that testosterone (T), a male hormone, is converted to dihydrotestosterone (DHT) by 5α-reductase (RD) to shrink hair follicles of the scalp. It causes hair loss. As the age increases, DHT is increased, which slows down the protein synthesis of hair follicle cells, which increases the proportion of resting hair follicles, leading to the rapid progression of hair loss. In addition, as aging progresses, keratin-secreting stem cells are destroyed in the papillary portion of hair follicles, thereby reducing the supply of stem cells. This promotes the progress of hair loss.

Hair formation has a pattern that circulates periodically. That is, the anagen, gowing phase, which accounts for 90% of normal hair, the catagen, transitional phase, which stops growth and hair root atrophy, the telogen, resting phase, where the hairballs become dry and hairy, and dropouts. Hair undergoes growth and hair loss through four stages of hair cycle (exogen). In particular, as hair follicles undergo apoptosis during the degenerative phase, hair follicles are reduced in size during the resting phase.

The methods developed to improve and treat the symptoms of hair loss include applying a material that promotes hair growth (synthetic material, natural product, cell culture solution or extract thereof), transplanting hair, injecting stem cells, etc. There is this. For example, a minoxidil coating agent is widely used as a hair loss treatment agent, and Korean Patent No. 10-1498201 describes a hair loss preventing or hair growth improving composition including an Mongwoo auditory extract as an active ingredient. No. 1484033 describes a hair loss preventing or hair growth composition comprising peanut shell extract.

In recent years, interest in treatment with stem cell cultures has been increasing, which is based on the abundance of growth factors that promote hair growth in cultures using mesenchymal stem cells.

However, these methods have limited or repeated application and injection when hair follicles disappear and need to be newly formed. In addition, continuous supply of growth factors is important for hair re-formation. Injection or application is only a one-time supply that is difficult to sustain. Mesenchymal stem cells also have limited growth.

Therefore, the present inventors studied a method of continuously supplying growth factors necessary for hair growth in vivo, and when stem cells cultured under specific conditions were implanted using a bio-insertion implant, stem cell growth-promoting factors such as bFGF were used. By continuously supplying the body, by inducing the growth of stem cells in the papillary portion of the hair follicles confirmed that long-term effective action of stem cells is possible and completed the present invention.

It is an object of the present invention to provide an implant for living body for promoting hair growth and / or improving and treating hair loss.

For this purpose, the present invention provides an implant for living body comprising a stem cell having a hair growth functional cytokine secretion ability.

The implant of the present invention can be promoted to long-term hair growth and hair loss improvement / treatment by releasing components having a hair growth function in the living body.

1 shows a method of culturing AF-N using adherent cell culture (FIG. 1A) and a method of injecting AF-N into terasite and culturing (FIG. 1B).
Figure 2 shows the concentration of bFGF in the control medium according to the culture method (1: AF-N 10 × 10 ⁵ cultured adherent cells, 2: AF-N 4 × 10 ⁵ cultured adherent cells, 3: adherent cells AF-N 1.0 × 10 ⁶ cultured, 4: Terrasite injection AF-N 10 × 10 ⁵ cultured, 5: Terrasite injection Cultured AF-N 4 × 10 ⁵ , 6: Terrasite injection cultured AF-N 1.0 × 10 ⁶ pcs.)
3 to 6 show the concentrations of bFGF (FIG. 3), PDGF-AA (FIG. 4), IGF (FIG. 5) and Wnt7a (FIG. 6) in conditioned media according to the culture method, respectively. -N 10 X 10 ⁵ , 2: AF-N 4 × 10 ⁵ with adherent cell culture, 3: AF-N 1.0 × 10 ⁶ with adherent cell culture, 4: AF-N 10 × with terasite injection culture 10 ⁵ , 5: AF-N 4 × 10 ⁵ cultured with terasite injection, and AF-N 1.0 × 10 ⁶ cultured with terasite injection).
Figure 7 shows the cell viability when injecting the stem cells of Examples 1 to 4 into terasite.
8 is a schematic diagram showing a process for confirming the biofunctionality of terasite into which stem cells are injected using a mouse.
9 is a photograph confirming the hair growth induction effect by placing the terasite after the first plucking.
10 is a photograph confirming the hair growth induction effect by placing the terasite after the second plucking. At this time, only the hypoxic cultured stem cells whose hair growth effects were confirmed were compared.
11 is a photograph confirming the hair growth induction effect by placing terasite after the third plucking. At this time, only the hypoxic cultured stem cells whose hair growth effects were confirmed were compared.
12 is a photograph confirming the hair growth induction effect by placing the terasite after the first plucking by adding the control group.
Figure 13 is a photograph comparing the hair growth phase induction by placing the terasite after the first plucking in the aging mouse model.
14 is a photograph comparing the induction of the hair growth phase by placing the terasite after the second plucking in the aging mouse model.
FIG. 15 shows the morphology of dedifferentiated amniotic stem cells inside terasite 9 days after tissue placement.
16 is a result of analyzing the hair follicle formation difference of the skin tissue in each group.
17 shows the results of measuring HFDP cell activity through AP staining of skin tissue of each group.

The present invention

The present invention relates to an implant for a living body including a stem cell having a hair growth functional cytokine secretion ability.

In addition, the present invention

Injecting stem cells having a hair growth functional cytokine secretion ability in the implant for implantation,

The present invention relates to a method for preparing a living implant for improving hair loss.

In addition, the present invention

Injecting stem cells having a hair growth functional cytokine secretion ability in the implant for implantation,

The present invention relates to a method for preparing a living implant for treatment of hair loss.

In addition, the present invention

Injecting stem cells having a hair growth functional cytokine secretion ability in the implant for implantation,

The present invention relates to a method for preparing a living implant for promoting hair growth.

In addition, the present invention

It relates to a method for improving hair loss comprising the step of placing the implant of the present invention in a living body.

In addition, the present invention

It relates to a hair loss treatment method comprising the step of implanting the implant of the present invention in a living body.

In addition, the present invention

It relates to a method for promoting hair growth comprising the step of placing the implant of the present invention in a living body.

In addition, the present invention

The present invention relates to the use of an implant for biological implantation comprising stem cells having a hair growth functional cytokine secretion ability for hair loss treatment, hair loss improvement, or hair growth promotion.

Hereinafter, the present invention will be described in detail.

Stem Cells Having a Functional Hairy Cytokine Secretion

Stem cells having a hair growth functional cytokine secretion ability of the present invention is preferably a stem cell derived from amniotic fluid. This is because the cells in the amniotic fluid secrete growth hormone. In addition, the stem cells having a hair growth functional cytokine secretion ability of the present invention is preferably a reverse differentiated amniotic stem cells. In addition, it is preferable that the stem cells having a hair growth functional cytokine secretion ability of the present invention are fetal-derived mesenchymal stem cells. More preferably, the stem cells having a hair growth functional cytokine secretion ability of the present invention are fetal-derived dedifferentiated mesenchymal stem cells.

The dedifferentiated stem cells may be made using a conventionally known dedifferentiation method, but is not particularly limited. For example, dedifferentiated stem cells of the present invention can be made by introducing dedifferentiation factors into mesenchymal stem cells. Such dedifferentiation factors are also not particularly limited, but it is preferable for safety and efficiency to use the verified ones. For example, the dedifferentiated stem cells of the present invention can be prepared using Oct4, Klf, Myc, Sox, Nanog, etc. as dedifferentiation factors, preferably the dedifferentiated stem cells of the present invention can be made using the Nanog gene. have. The reverse differentiated stem cells of the present invention are characterized in that the growth period is increased, the number of growth divisions is increased, and growth factor secretion is promoted as compared with non-differentiated stem cells.

The hair growth functional cytokine refers to cytokines having hair growth promoting effect, hair loss treatment effect, hair loss inhibiting effect, and hair loss improving effect. The cytokine may be selected from the group consisting of bFGF, PDGF-AA, IGF and Wnt7a.

The stem cells are preferably stem cells cultured under hypoxic conditions. In this case, the low oxygen condition means an oxygen condition lower than an oxygen concentration under atmospheric pressure, preferably an oxygen concentration is 0.01% to 8%, more preferably 0.05% to 5%, and still more preferably 0.10% to Indicates a condition of 3%.

At this time, the culture may be a culture in a living implant, or may be a general adherent cell culture, preferably a general adherent cell culture.

Implants for Bio Placement

The implant for biotransplantation of the present invention is a thin film polymer chamber made of a biocompatible membrane. The implant for living implantation is a container for implantation in vivo, that is, a living transplant container, and has a space for injecting stem cells, and secretes substances produced by stem cells, particularly cytokines, out of the implant through a thin film. The implant preferably has angiogenic function that protects stem cells from rejection by the implanted subject and induces the formation of capillaries close to the membrane when implanted subcutaneously. Through the angiogenic function, the implant provides nutrition and blood to stem cells in the membrane. The implant is preferably made of a material that is biocompatible, that is, does not induce an immune response. Such materials are already well known, for example polyurethane and the like. The implant preferably has a porous surface in which a plurality of micropores are located on the surface, wherein the pores do not pass through a large material such as cells, but are preferably sized to allow access to proteins or nutrients. Therefore, stem cells injected into the implant through the hole can not come out of the implant, preventing cancer caused by stem cells, while growth promoters, stem cell secretions, are released into the blood to form blood vessels around the oxygen and nutrients Is fed into the implant. This significantly increases the survival of stem cells. The implant can also be easily removed after a long time after implantation. The implant may be used by purchasing or manufacturing a commercially available product satisfying the above conditions. For example, the implant is preferably TheraCyte ™.

The implant for implantation of the present invention is for treating or improving hair loss. In addition, the implant for living body of the present invention is for promoting hair growth. At this time, the living implant implant of the present invention may be for treating or improving hair loss due to aging. In addition, the living implant implant of the present invention may be for promoting hair growth in a patient suffering from hair loss due to aging.

The implant for living body implantation of the present invention may include 1.0 × 10 ³ to 1.0 × 10 ¹⁰ stem cells of the present invention, in which the cultured stem cells themselves are injected into the implant. However, the present invention allows stem cells to operate for a long time in vivo, and does not inject a culture of stem cells into an implant. The stem cells of the present invention are injected and implanted in an implant having a permeable membrane, because oxygen and nutrients necessary for survival can be supplied through the blood of the implanted subject. It is also because injecting more stem cells than that of injecting the culture solution into the implant is much more effective for promoting hair growth and preventing / improving / treating hair loss.

The present invention can be planted by injecting the stem cells of the present invention of the implant for implantation of the present invention to plant hair without the risk of immune rejection or cancer cell development.

The implant for living body implantation of the present invention is preferably implanted in vivo, preferably subcutaneously (subcutaneously). At this time, the subject receiving the implant for a living body of the present invention is an animal including a human, especially a person suffering from hair loss.

Injecting stem cells having a hair growth functional cytokine secretion ability into a living implant

The present invention relates to a method for preparing an implant for a biological implant for promoting hair growth / hair loss treatment / alopecia improvement / hair loss including injecting a stem cell having a hair growth functional cytokine secretion ability into a bio implant. The injection method itself may be a conventional method of cell injection in the implant is not particularly limited.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, only the present embodiments to make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to fully inform the person having the scope of the invention, which is defined only by the scope of the claims.

<Materials and Methods>

Experimental animals

Six-week-old C57BL / 6 mice were used as experimental animals. Meanwhile, for the aging model test, an aged C57BL6 mouse model of 13 weeks or older was used.

Nanog-introduced Amniotic-derived De-differentiation Rehabilitation Mesenchymal Stem Cells

Reprogrammed amniotic fluid-derived mesenchymal stem cells with nanog (AF-N) established by introducing the Nanog gene are retroviral vectors used in mesenchymal stem cells of amniotic embryos. Nanog gene was introduced and induced by overexpression of Nanog gene. Hereinafter, the amniotic fluid-derived re-differentiation mesenchymal stem cells established by introducing the Nanog gene are referred to as "AF-N".

Stem Cell Culture

Amniotic stem cells were cultured in DMEM, 10% FBS, 1% P / S, 1% L-glutamine, 4ng / ml bFGF, 5ng / ml selenium, and 50ug / ml vitamin C medium.

Injecting Amniotic Stem Cells into Therasite

Incubate each amniotic stem cell group in a 100 mm cell culture dish until it covers 70-80% of the surface of the attached culture vessel, count the number of cells, and suspend 1.0 × 10 ⁷ cells in 20 μl low-glucose DMEM, and 22G needle Inject into the terasite using. After that, the inlet is sealed with a strong adhesive.

Terrasite Transplantation

After anesthetizing the mouse, make a hole about 10mm on the right side of the back and insert terasite into it. After that, sew with silk thread for suture or use a wound clip to suture the wound.

CCK (Cell Counting Kit)

Sacrifice the mice contained in the experimental group to remove the placed terasite and then wash the outside clean with PBS. After mixing AF growth medium and CCK 100 μl together and incubate for 30 minutes. After harvesting the cultured medium (harvest), the wavelength absorbance of 450nm is measured.

<Example 1>

Amniotic stem cells without the Nanog gene were cultured under normal oxygen conditions (Normoxia) to obtain amniotic stem cells.

<Example 2>

AF-N (re-differentiated amniotic stem cells established by introducing the Nanog gene) was cultured under normal oxygen conditions (Normoxia) to obtain amniotic stem cells.

<Example 3>

Amniotic stem cells without the Nanog gene were cultured under Hypoxia (hypoxia) conditions at 1% oxygen concentration to obtain amniotic stem cells.

<Example 4>

AF-N (reversely differentiated amniotic stem cells established by introducing the Nanog gene) was cultured under Hypoxia (hypoxia) conditions at 1% oxygen concentration to obtain amniotic stem cells.

Experimental Example 1 Confirmation of Cytokine bFGF Secretion of AF-N Injected into Terasite

The concentration difference of the hair growth functional cytokine bFGF in the conditioned medium obtained according to the culture method of AF-N was evaluated. Specifically, the number of AF-N stem cells was changed to 10 × 10 ⁵ , 4 × 10 ⁵ , and 1.0 × 10 ⁶ , respectively, and these were cultured using adherent cell culture, which is a general stem cell culture method ( 1A) and AF-N were injected into terasite and cultured (FIG. 1B), the concentration of bFGF in each of the conditioned media was measured using an ELISA assay.

As a result, the concentration of bFGF increased with the increase in the number of cells when cultured using an attached cell culture, but when AF-N was incubated with terasite, the number of cells injected increased. The secretion of bFGF did not increase and the concentration of bFGF in conditioned media remained constant (FIG. 2).

This is similar to the case of AF-N injection into terasite, which is similar to general cell culture using adherent cell culture. It is secreted out of the terrasite through the membrane. Therefore, it was confirmed that terrasite does not limit hair growth functionality and can be used as a means of delivery of hair growth agents.

On the other hand, when AF-N is injected into terasite and cultured, bFGF secretion is kept constant without significant change. 1) As stem cells grow in a limited space inside the terasite, the metabolism of cells is increased. This may be the case, or 2) because terasite is not only spatially limited, but also the pathway through which cytokines can be released, the amount of passage that can pass within a given time due to bottlenecks Two reasons have been suggested that they may have been limited.

Therefore, when AF-N was injected into terasite and cultured, it was confirmed through additional experiments whether the concentration of bFGF in the control medium was constant because the amount of cytokine that could pass through the terasite membrane was saturated.

<Experiment 2> Confirmation of hair growth functional cytokine secretion of AF-N injected in therasite

In Experimental Example 1, when AF-N was injected into terasite and cultured, it was confirmed whether the concentration of bFGF in the control medium was constant because the amount of cytokine that could pass through the terasite membrane was saturated and placed in the mouse. In order to determine the number of stem cells to be injected into the terasite, AF-N was cultured in the same manner as in Experimental Example 1, and the concentration of the hair growth functional cytokines in the obtained adjustment medium was measured. The cytokines measured at this time are bFGF, PDGF-AA, IGF and Wnt7a, which are known as growth factors that promote hair growth.

As a result, in the case of PDGF-AA and Wnt7a, the amount of cytokines released increased in the case of adherent cell culture as in the case of bFGF, but increased in the case of culture in terasite. Although an increase in the cytokine amount was observed, the increase was relatively small compared to the adherent cell culture.

On the other hand, in the case of IGF, unlike bFGF, PDGF-AA, and Wnt7a, the concentration was constant and then decreased in adherent cell culture conditions regardless of the increase in the number of cells injected. It was confirmed that the concentration of IGF in the conditioned medium was steadily increased with the increase in the number of cells (Fig. 3: bFGF, Fig. 4: PDGF-AA, Fig. 5: IGF, Fig. 6: Wnt7a) (1: adherent cell culture) AF-N 10 × 10 ⁵ , 2: AF-N 4 × 10 ⁵ with adherent cell culture, 3: AF-N 1.0 × 10 ⁶ with adherent cell culture, 4: AF-N 10 with terasite injection culture ⁵ × 10 ⁵ , 5: AF-N cultured with terasite injection culture 4 × 10 ⁵ , 6: AF-N 1.0 × 10 ⁶ with terasite injection culture).

Therefore, it was determined that the pathway through which cytokines could be released in terasite did not reach saturation. Therefore, the reason that the concentration of cytokines in the conditioned medium did not increase with the increase in the number of cells injected into the terasite was thought to be because the spatial limitation of terasite affects the metabolism of stem cells.

Taken together, the results of some hair functional cytokines increased as the number of cells injected into terasite under in vitro environmental conditions increased. In addition, in vivo, serum passed through the terasite membrane. Since it is possible to supply stem cells to activate the metabolism of stem cells and release more cytokines, the experiment was performed by setting the number of cells to be injected into the terasite to 1.0 × 10 ⁷ which is the highest of the recommended amount. That is, in the subsequent in vivo test, 1.0 × 10 ⁷ stem cells were injected into the terasite, and the mice were placed and tested.

Experimental Example 3 Evaluation of Cell Viability in Terrasite

Stem cells of Examples 1 to 4 were injected into the terasite by 1.0 × 10 ⁷ cells, respectively, and then placed in the skin of the C57BL / 6 mouse model. And after 14 days of placement, the collected terasite was collected and evaluated for individual stem cells injected using a cell counting kit.

The cell counting kit (CCK) can sensitively measure the viability of a cell by changing its color due to the formazan dye released by the highly water-soluble tetrazolium salt (WST-8) reduced by NADP / NADPH dehydrogenase.

In addition, (1) TB sites as measured by a (biological graft vessel) (indicated by TB site) and (2) a positive-derived MSCs (AF) to the TB site into pieces 1.0 × 10 ⁷ when the directly measured ( As a positive control, labeled Positive). The above (1) is a stem cell viability was measured as soon as the AF is put in the terasite and not planted in the mouse, the AF into the terasite, (2) is not cultured under normal oxygen conditions without introducing the Nanog gene One amniotic stem cell was put into terasite. That is, (1) and (2) are groups not planted in mice.

As a result, all of the amniotic stem cells of Examples 1 to 4 showed higher cell viability measurements than those measured by terasite alone without injection of cells, which means that the stem cells survived 14 days after implantation. This also means that the injected stem cells were protected by the terasite membrane and were not affected by the immune rejection response.

On the other hand, comparing the results of Examples 1 to 4, the dedifferentiated amniotic stem cells showed higher viability than the amniotic stem cells, and stem cells grown at low oxygen conditions than the normal oxygen concentration showed higher viability.

Amniotic stem cells of Example 3, although measured after 14 days of implantation, was not significantly different from Example 1 grown at normal oxygen concentration. Therefore, it was determined that dedifferentiated amniotic stem cells cultured under hypoxic conditions were most suitable for developing hair loss treatment (FIG. 7).

Experimental Example 4

C57BL / 6 mice, which are mainly used to confirm hair functionality, have a characteristic that the skin color is determined by the amount of melanin in the hair follicles because melanocytes that make pigments are present only in the hair follicles, not in the epidermis. Since the melanin synthesis in hair follicles is done only in the growing season, the skin color becomes black in the growing season, and the skin color becomes pink in the degenerative and resting periods when the melanin pigment is not synthesized. There is an advantage.

In the present invention, the amniotic stem cells of Examples 1 to 4 were injected into terasite by 1.0 × 10 ⁷ cells, and after the first plucking, C57BL / 6 mice in which a second hair growth phase occurred. This was planted and hair growth functionality was evaluated. At this time, the growth pattern of the hair was observed using the mice (untreated group) which did not process any sample as a control. 8 is a schematic diagram of such a mouse body functional verification process.

As a result, when the amniotic stem cells of Examples 3 and 4 were injected into terasite and placed therein, the hair growth functional effect was greater than that of other experimental groups (Examples 1 and 2, the control group, which was not treated). 9).

This may be interpreted as an increase in the secretion of cytokines under hypoxic conditions in combination affects hair growth functionality. On the other hand, the placement of terasite resulted in a large wound in the animal model, which may affect the hair growth function, and after the wound was healed, plucking was performed, and the hair growth function was evaluated again.

After the wound was healed as previously determined and observed after plucking, when the amniotic stem cells of Examples 3 and 4 were injected into terasite from the 7th day after the second plucking, as in the case of the first plucking Compared with the control group, anagen induction was clearly seen (FIG. 10).

In addition, the 3rd plucking and hair induction effect were observed, and similar results were observed after the 1st and 2nd plucking. In other words, when the implanted Example 3 and Example 4 were injected into the terasite, the difference in anagen induction appeared from the 8th day of the implantation compared to the control group, and after 12 days, it became more clear. In addition, it was observed that the area where the anagen progresses was wider than that when it was placed after the first and second plucking. Especially, the anagen area was larger when the dedifferentiated amniotic stem cells of Example 4 were injected than Example 3 An increase was confirmed (FIG. 11).

Taken together these results, it was confirmed that by injecting stem cells into the terrasite implants, hair growth functional effects can be maintained continuously. In particular, it was confirmed that the effect of hair growth functional is the best when the injection of dedifferentiated amniotic stem cells cultured under hypoxic conditions of Example 4.

Experimental Example 5

The experiment was performed in the same manner by adding a few control groups to the experimental groups of Experimental Example 4. Specifically, in order to confirm the effect of the therapies on the hair growth function of the wound caused by the placement of Theracyte was set as a negative negative control to implant only Theracyte without stem cell injection (Theracyte only). In addition, the case of daily dedifferentiation amniotic fluid stem cell culture (stem cell number: 1.0 × 10 ⁷ ) to the skin of the mouse was used as a control (smear). In addition, an effective wound closure is achieved by using a wound clip suitable for large wound closure. Otherwise, the experiment was performed under the same conditions and methods as in Experimental Example 5. Stem cells of Examples 1 to 4 were also calculated to be about 1.0 × 10 ⁷ cells to be injected into Theracyte as in Experimental Example 4. The placements were performed on C57BL / 6 mice in which a second hair growth phase occurred after primary plucking.

As a result, when using terasite as in Experimental Example 4, the hair growth effect of stem cells can be maintained continuously. Especially, when the inverted amniotic fluid stem cells of Example 4 were injected into terasite and implanted, the hair growth effect was the best. It was confirmed (FIG. 12).

Experimental Example 6

Based on the above experimental results, it was confirmed that the dedifferentiated amniotic stem cells injected into terasite had a hair growth function. Therefore, the hair growth ability was evaluated using an aged mouse hair loss model having similar characteristics to the elderly hair loss patients who actually suffer from hair loss.

To this end, an aged C57BL6 mouse model of at least 13 weeks of age was prepared. The mouse model was characterized as having spontaneous hair loss progressing without any treatment, and easy hair growth cycle and determination. (McMahon WM, Sundberg JP.Animal Models and Biomedical Tools, ed.Sundberg JP, pp. 493-497. CRC Press, Boca Raton, FL, 1994.).

Specific experimental method was performed in the same manner as in Experimental Examples 4 and 5. In this experiment, the case where only terasite was implanted without stem cell injection was set as an additional negative control so as to confirm the effect of the terasite implantation on the hair growth function (Theracyte only). In addition, the case where the stem cell culture solution (stem cell number: 1.0 × 10 ⁷ cells) of Example 4 was smeared on the skin of the mouse was used as a control (Example 4-smear). And the stem cells of Examples 2 and 4 as in Experimental Example 4, the number of cells injected into the terasite was calculated to be about 1.0 × 10 ⁷ . The placements were performed on C57BL / 6 mice in which a second hair growth phase occurred after primary plucking.

 As a result, as in the results of the previous experiments, it was confirmed that the induction of the growth stage (anagen induction) occurs faster in the case of Examples 2 and 4 injected into the terasite than the group that smeared theracyte only and Example 4 (Fig. 13). ). This means that the hair growth of dedifferentiated amniotic stem cells is higher than that of amniotic stem cells.

Experimental Example 7

As a result of Experimental Example 6, it was confirmed that the teratite containing the reverse differentiated amniotic stem cells of the present invention in the aging model. Therefore, in order to investigate the functional difference of anagen induction in the aging model, the aging model in which Experiment 6 was conducted was 19 weeks old after 6 weeks (6 weeks after the anagen phase started again after the 13-week old model test). 19 weeks old) Secondary plucking experiments were performed using mouse models.

As a result, the results were more pronounced than in the case of Experimental Example 6, in which the hair loss progressed in the control group due to the aging of the mouse model, and the partial growth of anagen induction was observed, whereas the hair growth in the terasite-planted group occurred. It was judged that there was a big difference with time due to the continuous secretion of cytokines and the expression of functionality. This, in turn, suggests that the secretome effect of cells present inside the terrasite is greater than applying conditioned medium to the skin. As for the hair growth effect, the group of Example 3 (terasite) and Example 4 (terasite) had the best effect, and the following was the Example 4 smear group, and Theracyte only. The group had the worst effect (FIG. 14).

Experimental Example 8

Previously, the survival confirmation of the amniotic fluid stem cell group was confirmed through CCK (cell counting kit). To confirm this once again, a test was conducted to determine what form of amniotic fluid stem cells survived inside the terasite. In this case, the terasite sample was used in Example 7, the 7th day sample, and the H & E staining was performed by cutting the inside of the terasite (section).

As a result, it was shown that there was nothing inside in theracyte only. On the other hand, it was confirmed that the stem cells are organized and formed inside the terasite into which the stem cells of Example 2 and Example 4 are injected (FIG. 15). This proved that the cells continued to function by growing and surviving for at least six weeks.

Experimental Example 9

Experimental examples 6 and 7 demonstrated anagen induction through the external portion of the aging mouse model. Therefore, histological analysis confirmed that hair follicles were actually formed. To this end, on the fifth day of the test progress of Experimental Example 7, skin tissue of the aging mouse model was sampled and analyzed.

As a result, a large amount of hair follicle tissue was identified in the skin tissues of the terasite group (Examples 2 and 4), and the anagen phase was found to have more hair follicles in the later anagen phase than the previous anagen phase. Confirmed. On the other hand, the smear group in Example 4 was found to have a large number of HFDP cell groups that may become hair follicles, but it was judged to be somewhat inferior to the terasite groups in terms of electrical function in the anagen phase (FIG. 16).

Experimental Example 10

In order to confirm the results of Experiment 9 again, each tissue was AP-stained to measure the activity of HFDP cells. In this test, the higher the activity on hair formation, the larger and clearer the positive areas for AP staining.

As a result, it was visually confirmed that the HFDP cell activity of the terasite group into which the stem cells of Example 2 and Example 4 were injected was significantly superior to that of theracyte only group and the group to which stem cells of Example 4 were smeared (FIG. 17). ).

Therefore, terrasite freed stem cells from immune response and increased its functionality through paracrine effect.

Claims (13)

Implants for living body containing stem cells having a hair growth functional cytokine secretion ability.
The method of claim 1,
Implants for biological placement for the treatment or improvement of hair loss.
The method of claim 1,
Implants for living body for promoting hair growth.
The method of claim 1,
The hair growth functional cytokine is implant for living body, characterized in that selected from the group consisting of bFGF, PDGF-AA, IGF and Wnt7a.
The method of claim 1,
The stem cells are implants for living body, characterized in that the stem cells cultured under hypoxic conditions.
The method of claim 1,
The stem cell is a living implant, characterized in that the stem cells derived from amniotic fluid.
The method of claim 1,
The stem cells are implants for living body, characterized in that the reverse differentiated amniotic stem cells.
The method of claim 1,
The stem cells are implants for living body, characterized in that the fetal-derived mesenchymal stem cells.
The method of claim 1,
The living implant is a living implant, characterized in that it comprises 1.0 × 10 ³ to 1.0 × 10 ¹⁰ stem cells.
The method of claim 1,
Implants for living body, characterized in that implanted under the scalp.
The method of claim 1,
The implant for living implants for implants for the treatment or improvement of hair loss due to aging.
Injecting stem cells having a hair growth functional cytokine secretion ability in the implant for implantation,
Method for producing a living implant for improving hair loss.
Injecting stem cells having a hair growth functional cytokine secretion ability in the implant for implantation,
Method for preparing a living implant for treatment of hair loss.

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