WO2024148571A1

WO2024148571A1 - New technical method for treating chloasma by means of stem cell transplantation operation

Info

Publication number: WO2024148571A1
Application number: PCT/CN2023/071927
Authority: WO
Inventors: 刘景卫; 刘诗雨
Original assignee: 南海仁术国际皮肤医院（海南）有限公司
Priority date: 2023-01-12
Filing date: 2023-01-12
Publication date: 2024-07-18

Abstract

Provided is a technical method for treating chloasma by means of a hair follicle stem cell transplantation operation. The technical method comprises the following steps: extraction of hair follicles; separation of hair follicles; inactivation of hair follicles; in-vitro culture of hair follicle stem cells; and transplantation of hair follicle stem cells. A portion of hair follicle outer root sheaths containing hair follicle stem cells is obtained by means of a precise extraction and separation method, and by means of in-vitro hair follicle separation and hair follicle inactivation and in-vitro culture of hair follicle stem cells, the thickening of postoperative skin is achieved, chloasma is removed, an affected part of the skin barrier is repaired, and therefore the aim of clinically curing chloasma is achieved.

Description

A new technical method for stem cell transplantation to treat melasma

Technical Field

The present invention relates to the technical field of hair follicle transplantation, and in particular to a technical method for treating chloasma by hair follicle transplantation containing hair follicle stem cells and hair follicle melanin stem cells.

Background technique

Why can't melasma be cured and keeps recurring? Perhaps there is something wrong with our research direction?
There are many studies on melasma. The latest research believes that the influence of estrogen/progesterone and testosterone, ultraviolet exposure, racial genetic factors, and thyroid dysfunction are important causes of melasma. Damage to the basement membrane, skin vascularization, skin barrier imbalance, neuropeptide regulation, oxidative stress, low expression of the H19 gene, and disorders of the wNT and iNOS signaling pathways are important mechanisms for the onset of melasma. These signaling pathway molecules are interrelated and affect each other. The research is getting deeper, more detailed, and more complex, but it is not very helpful for treatment.

At present, the treatment of melasma in academia still focuses on how to remove melanin. Such as hydroquinone, tranexamic acid, retinoic acid, fruit acid and laser treatment. Especially for laser treatment, many devices have been developed for pigmentation: IPL, DPL, OPT for intense pulsed light, Q-switched laser, picosecond laser or ruby, sapphire, ND:YAG laser, etc. It can be said that the technology is becoming more and more advanced and the price is getting more and more expensive, but it still can't cure melasma. If we follow this idea, we will not be able to cure melasma in the next 100 years.

So we have reason to think, is our thinking wrong?
Reason 1. Is melasma a pigmentary disease?
Chloasma is manifested as symmetrical yellow-brown pigmentation spots on the zygomatic face, cheeks, forehead and other parts. Under the microscope: the melanin in the basal layer and spinous layer of the epidermis increases, but there is no increase in melanocytes. Free melanin particles can be seen in the upper dermis or engulfed by melanophils. This is an article published in the 10th issue of the "Chinese Journal of Dermatology" in 2016. The author is Zhu Liping, etc. from Kunming Medical University. This is a rare article, because publicly published academic papers on the pathology of chloasma are relatively rare both at home and abroad. The author found that 8 patients with chloasma had varying degrees of epidermal thinning, flattening of the skin, accumulation of melanin in the spinous layer cells, and inflammatory infiltration around the dermal capillaries. But like most academic papers, the research focus of this paper is still on melanin and melanocytes, and there is no further discussion on the problem of epidermal thinning.

In fact, we have observed in clinical practice that many patients with melasma have evidence of damaged skin barrier. Therefore, melasma is not just a pigmentation problem, but also very common. If we only focus on the study of melanin and ignore the condition of damaged skin barrier, we may not grasp the essence of things.

Reason 2. What are the merits and demerits of melanin?
The pathogenesis of melasma: the number of melanocytes has not increased, but the volume of melanocytes has increased, and the secretion of melanin particles has increased. However, almost all drugs, products and equipment for the treatment of melasma are currently targeting melanin. It seems that melanin has become a public enemy that doctors and patients treat unanimously. We know that melanin is synthesized by melanocytes and then transported to keratinocytes to take effect . The main function of melanin is to scatter and absorb ultraviolet rays to protect the skin from damage. The risk of skin cancer in white people is 70 times higher than that in black people. Ultraviolet rays can also induce the synthesis of melanin. In other words, melanin is produced to protect the skin from damage! Why does the skin become darker after being exposed to the sun? That's because the skin synthesizes more melanin to resist damage from ultraviolet rays.

Figure 1 Pathological diagram of the formation of chloasma. Therefore, melanin is not only harmless but also beneficial to the human body. So why must melanin be removed?
Reason 3. Is melanin the only element that protects against UV rays?
Although the average thickness of the epidermis is only 0.1 mm, the mechanical barrier of the epidermis also has a certain protective effect against ultraviolet rays. The stratum corneum can scatter and reflect ultraviolet rays, and can also absorb short-wave ultraviolet rays. The spinous cell layer and basal layer can absorb long-wave ultraviolet rays. There is no literature to prove that the thicker the epidermis, the stronger the defense against ultraviolet rays, because there is no need to prove it, the fact itself is so. It is the same as wearing a thick piece of clothing in the sun is not easy to get sunburned or tanned.

Conclusion 1: Under physiological conditions, the physical barrier of the epidermis and the melanin barrier constitute a common defense line against ultraviolet radiation. If the ultraviolet rays become stronger, the common defense line will be strengthened accordingly; if the degree of strengthening of the common defense line is not enough to resist ultraviolet rays, the skin may cause damage or even tumors. Formula 1 Physical barrier + melanin barrier = common defense against ultraviolet rays = radiation power of ultraviolet rays. The physical barrier and melanin barrier of the epidermis are equivalent to the physical sunscreen and chemical sunscreen of the human body. The former requires a certain thickness to achieve the corresponding sunscreen function, and the latter achieves the sunscreen effect by absorbing ultraviolet rays, which requires a certain amount. Since the synthesis rate of melanin in the skin is relatively fast, the melanin barrier is the first to be strengthened. Therefore, after ultraviolet radiation, the skin first becomes darker, but the epidermis can also adapt to thickening after long-term exposure to ultraviolet rays.

Conclusion 2: If the physical barrier of the epidermis is damaged or thinned, and the ultraviolet rays remain unchanged or increase, the skin can only strengthen the melanin barrier by synthesizing more melanin to strengthen the common defense line and achieve the purpose of protecting against ultraviolet radiation.

Further conclusion 3: Chloasma is caused by the weakening of the physical barrier function of the epidermis, which leads to increased melanin synthesis and thus the formation of skin pigmentation. Therefore, the pigmentation in chloasma is only a secondary damage. If the above conclusion is true, all the pathological links of chloasma can be well explained: due to the damage of the physical barrier of the epidermis, the skin cannot tolerate the stimulation of physical, chemical and biological factors, which directly leads to capillary dilation or causes capillary dilation through inflammatory response. Inflammation itself can cause pigmentation; due to the weakening of the physical barrier, it cannot resist ultraviolet radiation, so it can only rely on increasing the pigment of the skin to resist ultraviolet rays.

Figure 2: Schematic diagram of the pathogenesis of melasma
Some people may ask, how to explain the chloasma caused by pregnancy? Is it also related to the barrier? In fact, most chloasma caused by pregnancy will recover on its own because the skin barrier is not damaged. And those chloasma caused by pregnancy that do not recover on their own must have problems with the skin barrier. Chloasma is divided into pigmentary, vascular and mixed types. The latter two can clearly observe the damage of the epidermal barrier, but what about pigmentary chloasma? If there is no problem with the physical barrier, then there is a problem with the melanin barrier, that is, there is a malfunction in the synthesis, transport and metabolism of melanin and keratinocytes.

Reason 4. Why is melasma prone to recurrence?
A major feature of melasma is recurrence. Why? The first reason is that many times the focus is on removing the spots instead of treating the patient. Her behavior habits are not corrected, so what happens after the treatment is over? So it is easy to relapse. The second reason is that the focus of removing spots is to reduce melanin, and not enough attention is paid to barrier repair. Even if melanin is reduced in the short term, because it cannot resist ultraviolet radiation, melanin will soon be synthesized in larger quantities. Sometimes, the process of removing melanin leads to further damage to the skin barrier. According to the principles of formula 1 and conclusion 2, more melanin will be produced. This is the reason why melasma rebounds and worsens.

Physical barrier↓+melanin barrier↑=combined defense against UV rays=radiation power of UV raysReason 5. How to treat melasma?
According to conclusion 1, if laser, photon or drug methods are used to inhibit melanin and reduce pigmentation, the essence is to reduce the melanin barrier function and the defense capability of the common defense line. Although the effect may be achieved in the short term, the physical barrier of the epidermis has not been enhanced. When the ultraviolet radiation remains unchanged, enhanced or even weakened, melasma is likely to recur or even worsen. Therefore, it is a rogue idea to only remove pigment without repairing it, no matter what kind of high-tech is used. The treatment of melasma should first repair the skin barrier. In the process of the skin barrier becoming stronger, it is correct to appropriately remove melanin.

Reason 6. How to repair the skin barrier?
6.1 Stop behavioral habits that damage the skin barrier. Many patients with melasma have bad skin care habits, such as frequent exfoliation, deep cleansing, rubbing the face with a towel, etc. Maybe it won’t be a problem once or twice, but if it becomes a habit, the skin barrier will be damaged if it is done every day. Many whitening and spot-lightening products are harmful to the skin barrier, so it is recommended to use them less. Pay attention to rest and don’t stay up late. Adequate sleep helps pigment metabolism and also helps the recovery of the skin barrier. Ultraviolet rays can promote the synthesis of melanin, and overexposure can also cause damage to the skin barrier, so sun protection is very important.

6.2 Promote the proliferation of epidermal cells The basal layer of the epidermis contains stem cells, which account for about 1%-10%. The cells are small and dense, and the ratio of nucleus to cytoplasm is large. Hair follicles also contain stem cells. The vast majority of epidermal stem cells are in a dormant state. Only when the skin is damaged, the remaining epidermal stem cells differentiate and proliferate, and quickly migrate to the upper layer of the epidermis; on the other hand, after the hair follicle stem cells near the wound edge receive the damage repair instruction, they can also activate the hair follicle stem cell-epidermal channel to participate in the repair. According to this principle, we can artificially create damage, which can achieve the purpose of "awakening" epidermal stem cells and make them proliferate and differentiate, but will not cause obvious trauma and inflammation, making the skin worse. Is there such a treatment method?
⑴ The needle of skin rolling needle is very thin, with a diameter of about 0.2 mm and a needle length of 0.3-2 mm. After the rolling needle treatment, hundreds of thousands of needle holes will be left on the skin. Many people think that the focus of the rolling needle is transdermal drug delivery, which is actually incorrect. Skin rolling needle is very consistent with the requirement of stimulating epidermal stem cells without excessively damaging the skin. Of course, applying growth factors after surgery is beneficial to promoting the proliferation and differentiation of stem cells, which is supported by literature. But how long the needle is, how much force is needed, what is the end point of treatment, and the strongest "awakening" effect of stem cells requires further research. Although there is not much publicly published data to confirm, there is no doubt that moderate skin rolling needles can improve the skin barrier and melasma.

⑵ Plum blossom needles are also used by doctors in the clinic to treat alopecia areata and skin barrier damage. In essence, they can "awaken" skin stem cells. However, plum blossom needles are too long and thick, which causes more damage to the skin. In addition, plum blossom needles create fewer needle holes than skin rolling needles, and the stimulation density is not enough.

⑶ After the fractional laser acts on the skin, it will artificially create many focal thermal injuries on the skin. This stimulation can awaken skin stem cells, which is also the basis for fractional laser to repair scars. There have been related reports on the treatment of melasma, but the author attributed it to the damage of laser to melanin. Since the fractional laser itself can cause damage to the skin barrier, the energy and density are too high, and the damage caused may be greater than the repair effect after the epidermal stem cells are "awakened". Therefore, choosing the right parameters can repair the skin barrier of melasma, thereby achieving the effect of lightening the spots. Otherwise, it may be counterproductive.

6.3 Inhibit the shedding of keratinocytes Although the stratum corneum is composed of 4-20 layers of dead cells, it constitutes the main part of the physical barrier of the epidermis. We know that keratinocytes fall off from the epidermis every day, and those with damaged skin barriers fall off faster. According to the conclusions drawn in this article, the thicker the physical barrier of the epidermis, the less melanin the skin produces. Therefore, in addition to promoting the proliferation of epidermal stem cells, we can also thicken the stratum corneum by inhibiting the shedding of keratinocytes. The stratum corneum cells use the intercellular matrix like "mortar" to glue the dead keratinocytes together to form a structure similar to a brick wall, which is the skin barrier we are familiar with. The outer layer of the stratum corneum may have less "mortar" or lack of effective ingredients. Therefore, if these "mortar" components are appropriately supplemented to make the keratinocytes that are about to fall off "stick" together, the physical barrier of the epidermis will gradually become thicker. Many medical skin care products contain these physiological ingredients or ingredients with similar functions, such as ceramide, hyaluronic acid, collagen, fatty acids, prickle oil, natural moisturizers, etc., which help to restore the skin barrier. Some instruments and equipment such as ion introduction and ultrasound introduction can promote the absorption of effective ingredients and also help to restore the skin barrier. However, patients with melasma prefer to use some whitening and spot-lightening products, and many manufacturers are willing to promote them in this way, although many whitening and spot-lightening ingredients are not conducive to the keratin barrier. In fact, there are almost no whitening and spot-lightening skin care products that can truly improve melasma. Even if there is some improvement in a short period of time, it will soon relapse or even become more serious because the skin barrier has not recovered or has even been damaged. Therefore, for patients with melasma, it is best to use repair skin care products, and after the skin barrier gradually improves, whitening and spot-lightening skin care products can be appropriately used.

Reason 7. Do you need dark spot removal treatment?

By changing bad skin care habits, promoting the proliferation of epidermal stem cells, and inhibiting the shedding of stratum corneum cells, the skin barrier can be gradually restored. After the physical barrier's defense against ultraviolet rays is enhanced, according to the principle of formula 1, the content of melanin in the skin will naturally decrease gradually. This is why many melasmas are improved through barrier repair. Physical barrier ↑ + melanin barrier ↓ = joint defense against ultraviolet rays = ultraviolet radiation. Of course, under the premise that the skin barrier is gradually restored, the degradation of melanin can be promoted and the synthesis of melanin can be inhibited in a gentle way. These methods include but are not limited to: low-energy large-spot Q-switched laser, gentle intense pulsed laser treatment, gentle picosecond laser treatment, oral tranexamic acid, and topical pigment-lightening products.

The above reasons can be used to analyze the treatment methods of melasma: 1. Repairing the skin barrier of melasma is the prerequisite for curing melasma. 2. Removing skin inflammation and abnormal blood vessel proliferation is the reason for inhibiting the abnormal increase in the volume of melanocytes and the production of melanin particles. 3. Physical barrier ↑ + melanin barrier ↓ = joint defense against ultraviolet rays = ultraviolet radiation. 4. Keratinocytes can not only repair the epidermis but also reduce ultraviolet radiation. Can a kind of autologous tissue that can promote the proliferation of keratinocytes, thereby repairing the skin barrier, inhibiting blood vessel proliferation, removing skin inflammation and inhibiting the production of melanin particles by pathological melanocytes be implanted through surgery? How to realize the above plan? Through practice, we found that hair follicle stem cells fully have this function, and transplanting hair follicle stem cells can treat melasma.

Hair follicle stem cells are a type of stem cell in the outer root sheath of human hair follicles. Hair follicle stem cells are adult stem cells that are in a quiescent state in the body and show amazing proliferation ability under in vitro culture. Studies have found that hair follicle stem cells have multidirectional differentiation potential. They can differentiate into epidermis, hair follicles, sebaceous glands, and participate in the process of skin wound healing. The epidermis is located in the superficial layer of the skin and is composed of keratinized stratified flat cells. From the base to the surface, it can be divided into five layers: basal layer, spinous layer, granular layer, transparent layer and stratum corneum. The basal cells in the basal layer are undifferentiated cells with certain differentiation potential. The new cells can gradually move to the shallow layer and differentiate into other layers of epidermal cells. However, in actual clinical experiments, hair follicle stem cells are mainly used as seed cells to rebuild the epidermal structure when the skin is damaged. When the epidermis is severely damaged, hair follicle stem cells can migrate to the wound site and form new epidermal cells to promote wound healing. Heidari et al. used morphological analysis and histological analysis to prove that skin wound tissue with hair follicle stem cells is more likely to form blood vessels and differentiate into epidermal cells, forming an epidermal structure with multiple functions.

After culturing hair follicle stem cells in a cell collagen gel mixture for 25 days, well-differentiated epidermis, dermis, and hair structures appeared, and the epidermal structure had a distinct basal layer, spinous layer, and incompletely keratinized stratum corneum. In the process of hair follicle stem cells forming epidermis, fibroblasts, as the main matrix component, promoted the proliferation, differentiation, and migration of epidermal cells, regulated the morphology and interaction of epidermal cells, and finally successfully induced hair follicle stem cells to differentiate into epidermal cells. In addition, many molecular substances such as integrin-linked kinase and Pygo2 also play an important regulatory role in the activation of hair follicle stem cell repair after skin damage.

Ideal tissue-engineered skin should have the functions of rapid and lasting adhesion, good tissue compatibility, low infection rate, not easy to be damaged again, and can promote rapid proliferation of subcutaneous tissue, and can restore or improve some skin functions after transplantation. At present, Dong Zhiyong et al. have induced human hair follicle stem cells to differentiate into epidermal keratinocytes, and constructed an engineered epidermis with a multi-layer cell structure and relatively good functions. Although the constructed engineered epidermis is composed of only a few layers of cells and has not yet reached a complete epidermal structure, the role of hair follicle stem cells in skin reconstruction, damaged skin repair, and tissue engineering skin construction is unquestionable. In actual clinical experiments, hair follicle outer root sheath cells have been successfully used in the treatment of lower limb ulcers, which strongly confirms the ability of hair follicle stem cells to promote epidermal reconstruction and their actual clinical utility.

Hair follicle stem cells are a type of adult stem cell that has strong in vitro differentiation ability, is easy to obtain, has strong proliferation ability, is relatively safe to use, and has multidirectional differentiation potential. They play an important role in tissue regeneration after skin damage, angiogenesis, superficial nerve repair, and reconstruction of the epidermis and skin appendages during the recovery of body functions. Therefore, transplanting hair follicle stem cells can not only rebuild the skin barrier of melasma, but also inhibit the proliferation of blood vessels in melasma, eliminate inflammation, restore the normal function of melanocytes, promote epidermal thickening, and enhance the ability to defend against ultraviolet rays.

technical problem

The purpose of the present invention is to provide an outer hair root sheath containing hair follicle stem cells to treat chloasma by minimally invasive surgery. The outer hair root sheath containing hair follicle stem cells is extracted and separated by FUE technology and cultured to promote the differentiation and proliferation of hair follicle stem cells. The hair follicle stem cells are activated and the activity is improved by cultivation to promote the rapid transformation and proliferation of hair follicle stem cells. Then, the hair follicle stem cells and their outer hair root sheath are precisely transplanted to a specific layer of the epidermis of chloasma, thereby inhibiting the inflammation of melanocytes in chloasma and reducing the secretion of melanin particles. At the same time, it can promote the thickening of the epidermis, enhance the defense against ultraviolet rays, and rebuild the skin barrier of chloasma to solve the problems in the above-mentioned background technology.

Technical Solutions

To achieve the above object, the present invention provides the following technical solutions:
Methods and steps of hair follicle stem cell transplantation for the treatment of melasma:
The technical method of hair follicle stem cell transplantation for treating melasma includes the following steps:
Step S1, extraction of hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extract a hair follicle unit, and preferably extract single or double follicles, preferably white hair follicles.

Step S2, separation of hair follicles, separation of the hair follicle unit under a 2-15 times magnifying glass or electron microscope to obtain a single hair follicle outer root sheath containing hair follicle stem cells after removing the epidermal tissue.

Step S3, inactivation of hair follicles. Whether to inactivate hair follicles is determined according to the severity of chloasma. For patients with small area, white skin and mild symptoms, inactivation treatment is adopted. The hair follicles before transplantation are inactivated. The method is: using a hair follicle inactivation needle under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle; for patients with large area, darker pigment and severe disease, the hair follicles before transplantation may not be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required.

Step S4, in vitro culture of hair follicle stem cells, placing the extracted and separated hair follicles containing the outer hair root sheath of hair follicle stem cells into culture medium for culture, so as to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells.

Step S5, transplantation of hair follicle stem cells, using hair follicles as support to transplant the outer hair root sheath containing hair follicle stem cells, under the positioning of the hair follicle implantation positioning needle, the hair follicle implantation needle is used to implant the above outer hair root sheath into a specific layer of the chloasma area, thereby solving the inflammation of chloasma melanocytes and reducing the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, rebuild the skin barrier of chloasma, and achieve the purpose of treating chloasma.

Furthermore, the specific method of step S1 is to extract hair follicles, use FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extract a hair follicle unit, and preferably extract single or double hair follicles, preferably white hair follicles.

Furthermore, in step S2, the hair follicles are separated by removing the epidermal tissue from the hair follicle unit under a 2-15 times magnifying glass or electron microscope to obtain a single hair follicle outer root sheath containing hair follicle stem cells, with an accuracy rate of up to 100%, thus avoiding damage to the hair follicle stem cells and outer root sheath during the separation process.

Furthermore, in step S3, the inactivation of hair follicles is selected according to the severity of chloasma. For patients with small area, white skin and mild symptoms, inactivation treatment is adopted to inactivate the hair follicles before transplantation. The method is: using a hair follicle inactivation needle under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle; for patients with large area, darker pigment and severe disease, the hair follicles before transplantation may not be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required.

Furthermore, in step S4, the hair follicle stem cells are cultured in vitro by placing the extracted and separated hair follicles of the outer hair root sheath containing the hair follicle stem cells into a culture medium for culture, so as to enhance the activity of the hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of the hair follicle stem cells.

Furthermore, the main components of the special culture medium are tranexamic acid injection, compound glycyrrhizin injection, vitamin C injection and normal saline.

Furthermore, in step S5, the transplantation of hair follicle stem cells is performed by transplanting the outer hair root sheath containing hair follicle stem cells based on the hair follicles. Under the positioning of the implantation positioning needle of the hair follicle transplantation, the hair follicle implantation needle is used to implant the above-mentioned outer hair root sheath into a specific layer of the chloasma area, thereby solving the inflammation of chloasma melanocytes and reducing the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, rebuild the skin barrier of chloasma, and achieve the purpose of curing chloasma.

Beneficial Effects

Compared with the prior art, the present invention has the following beneficial effects:
1. The present invention obtains the outer hair root sheath containing hair follicle stem cells through a unique outer hair root sheath in vitro separation technology and treats chloasma through transplantation surgery.

2. The present invention invents an in vitro culture technology for hair follicle stem cells. The obtained hair follicle stem cells are cultivated in vitro with a special culture medium, which promotes the rapid multiplication of hair follicle stem cells and inhibits the production of melanocytes.

3. The present invention uses hair follicle inactivation technology to inactivate hair follicles before transplantation with the help of a magnifying glass or a microscope and a hair follicle inactivation needle, thereby achieving hair follicle inactivation and inhibiting the secretion of melanin particles without causing hair growth.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the specific embodiments of the present invention, but do not constitute a limitation of the present invention.

FIG1 is a schematic diagram of the pathological formation of chloasma. FIG2 is a schematic diagram of the pathogenesis of chloasma. FIG3 is a flow chart of the technical method of treating chloasma with hair follicle stem cell transplantation of the present invention.

Best Mode for Carrying Out the Invention

The preferred embodiment of the present invention is described in detail below in conjunction with FIG. 3 . It should be understood that the preferred embodiment described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

The key technical points of the present invention are as follows: 1. In vitro separation technology of the outer hair root sheath is used to obtain the outer hair root sheath of the hair follicle containing hair follicle stem cells and treat chloasma through transplantation surgery. 2. In vitro culture technology of hair follicle stem cells, the obtained hair follicle stem cells are cultured in vitro, and the main components of the special culture fluid are tranexamic acid injection, compound glycyrrhizin injection, and vitamin C injection; 3. Hair follicle inactivation technology, with the help of a dermatoscope and a hair follicle inactivation needle, the hair follicles before transplantation are inactivated, and no hair grows after the operation.

Embodiment 1:

As shown in FIG3 , the present invention provides a technical solution: a technical method for treating chloasma by hair follicle stem cell transplantation, the technical method comprising the following steps:
Step S1, extraction of hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extract a hair follicle unit, and preferably extract single or double follicles, preferably white hair follicles.

Step S5, transplantation of hair follicle stem cells, using hair follicles as support to transplant the outer hair root sheath containing hair follicle stem cells, under the positioning of the implantation positioning needle of the hair follicle transplantation, the above outer hair root sheath is implanted into a specific layer of the chloasma area using the hair follicle implantation needle, thereby solving the inflammation of chloasma melanocytes and reducing the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, rebuild the skin barrier of chloasma, and achieve the purpose of curing chloasma.

In this embodiment, the specific method of step S1 is to extract hair follicles, use FUE technology to extract and separate the outer root sheath containing hair follicle stem cells, extract a hair follicle unit, and preferably extract single or double follicles, preferably white hair follicles.

In this embodiment, in step S2, the hair follicles are separated by removing the epidermal tissue from the hair follicle unit under a 2-15 times magnifying glass or electron microscope to obtain a single hair follicle outer root sheath containing hair follicle stem cells.

In this embodiment, in step S3, the inactivation of hair follicles is selected according to the severity of chloasma. For patients with small area, white skin and mild symptoms, inactivation treatment is adopted to inactivate the hair follicles before transplantation. The method is: using a hair follicle inactivation needle under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle; for patients with large area, darker pigment and severe disease, the hair follicles before transplantation may not be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required.

In this embodiment, in step S4, hair follicle stem cells are cultured in vitro by placing the extracted and separated hair follicles of the outer hair root sheath containing hair follicle stem cells into a culture medium for culture, so as to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells.

In the present embodiment, in step S5, the transplantation of hair follicle stem cells is performed by transplanting the outer hair root sheath containing the hair follicle stem cells based on the hair follicles. Under the positioning of the hair follicle implantation positioning needle, the hair follicle implantation needle is used to implant the outer hair root sheath into a specific layer of the chloasma area, thereby solving the inflammation of chloasma melanocytes and reducing the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, rebuild the skin barrier of chloasma, and achieve the purpose of curing chloasma.

Embodiment 2:

As shown in FIG3 , the technical method of treating vitiligo with hair follicle stem cell transplantation provided by the present invention uses the technical method in Example 1 to transplant hair follicle stem cells to a specific level of chloasma, promote the proliferation and differentiation of hair follicle stem cells and the formation of tissues, repair the epidermal barrier, and enhance the ability to defend against ultraviolet rays.

In this embodiment, the cure rate of vitiligo treated by hair follicle stem cell transplantation reaches over 80%.

Technical difficulties and surgical steps of the present invention:
Technical difficulties: "Survival rate" refers to whether the hair follicle stem cells can survive directly after transplantation, which is related to the later treatment effect. The autologous hair follicle stem cells obtained through extraction, after being cultured and activated, are more active when transplanted into the melasma lesion area, thus ensuring the postoperative efficacy.

Surgical steps: 1. Use FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells and culture them to promote the differentiation and proliferation of hair follicle stem cells. By activating and improving the activity of hair follicle stem cells through cultivation, the hair follicle stem cells are promoted to quickly transform and proliferate, and almost no hair grows after the operation; 2. "Implantation" is another key point related to the survival of hair follicle stem cells. If the implantation is too shallow, the survival rate is low and the effect is not good. If the implantation is too deep, the repair effect on the skin barrier will be lost and the efficacy will be reduced. Only by accurately implanting at a specific level of white spots can the inflammation of chloasma melanocytes be solved and the secretion of melanin particles be reduced; at the same time, it can promote epidermal thickening, enhance the ability to defend against ultraviolet rays, and rebuild the skin barrier of chloasma to solve the problems in the above-mentioned background technology.

Principle of the invention: The invention uses skin B-ultrasound and dermoscopy to accurately report data to evaluate the condition of chloasma, uses FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells and culture them, promotes the differentiation and proliferation of hair follicle stem cells, activates and improves the activity of hair follicle stem cells through cultivation, and promotes the rapid transformation and proliferation of hair follicle stem cells; then the hair follicle stem cells and the outer hair root sheath are accurately transplanted to a specific layer of the epidermis of chloasma to solve the inflammation of chloasma melanocytes and reduce the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, and rebuild the skin barrier of chloasma.

The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:
1. The present invention uses a unique in vitro separation and culture technology of the outer hair root sheath to culture hair follicle stem cells in vitro, and transplants the outer hair root sheath to treat chloasma.

2. The present invention implants hair follicle stem cells into a specific layer of the epidermis through B-ultrasound positioning and hair follicle transplantation positioning needles, thereby resolving the inflammation of melanocytes in chloasma and reducing the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the ability to defend against ultraviolet rays, and rebuild the skin barrier of chloasma.

3. The hair follicle stem cells extracted by the present invention have the characteristics of not being affected by the body's endocrine and hormones, which is equivalent to implanting an anti-inflammatory agent of melanocytes in the chloasma area, playing a role in permanently inhibiting the excessive secretion of melanin particles.

4. The present invention changes the traditional method of treating chloasma through drugs and photoelectric beauty treatment, subverts the treatment concept of chloasma, and treats chloasma through minimally invasive surgery of hair follicle stem cell transplantation. The efficacy is significantly better than the current treatment methods of chloasma.

5. The present invention has been approved by the patient's informed consent and the hospital ethics committee. It does not violate medical ethics and is harmless to patients. Because part of the outer root sheath of the hair follicle is transplanted, it complies with national laws and regulations.

The clinical research program involved in the present invention was discussed and approved by the hospital ethics committee, and all patients gave informed consent to the trial. The test data and clinical experience obtained neither violate medical ethics nor are harmful to patients. Because only part of the outer root sheath of the hair follicle is transplanted, it complies with national laws and regulations.

Embodiments of the present invention

1. The new technical method for stem cell transplantation for treating chloasma is characterized by an in vitro preparation method of hair follicle stem cells, which comprises the following steps:
Step S1, extracting hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extracting a hair follicle unit, preferably single or double hair follicles, preferably white hair follicles;
Step S2, separation of hair follicles, separation of the hair follicle unit to remove the epidermal tissue of the single outer hair root sheath containing hair follicle stem cells under a 2-15 times magnifying glass or electron microscope, with an accuracy rate of up to 100%, avoiding damage to the hair follicle stem cells and the outer hair root sheath during the separation process;
Step S3, inactivation of hair follicles. Whether to inactivate hair follicles is determined according to the severity of chloasma. Inactivation treatment is performed on patients with small area, white skin and mild symptoms. The hair follicles before transplantation are inactivated: a hair follicle inactivation needle is used under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle. For patients with large area, darker pigment and severe disease, the hair follicles before transplantation may not be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required.
Step S4, in vitro culture of hair follicle stem cells, placing the extracted and separated hair follicles of the outer hair root sheath containing hair follicle stem cells into a special culture medium and culturing them at 22-26° C. for 1-4 hours to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells, wherein the main components of the special culture medium are tranexamic acid injection, compound glycyrrhizin injection, vitamin C injection and normal saline;
Step S5, in vitro positioning of the transplantation layer of hair follicle stem cells for the treatment of melasma, through the combined use of dermatoscope and/or skin CT and/or skin B-ultrasound, the insertion layer of the hair follicle implantation needle located in vitro is the specific layer of the hair follicle outer root sheath transplantation, and the in vitro positioning can provide preoperative technical support for the precise transplantation of the outer root sheath containing hair follicle stem cells.

2. The new technical method for treating chloasma with stem cell transplantation surgery according to claim 1 is characterized by the step S1 of the in vitro preparation method of hair follicle stem cells: extraction of hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extracting a follicle unit, preferably single or double follicles, preferably white hair follicles, and colored hair follicles can also be selected; hair follicle stem cell transplantation to treat chloasma, especially transplanting white hair follicles for disease treatment, is one of the creative ideas of the present invention.

3. According to claim 2, the new technical method for treating chloasma with stem cell transplantation surgery is characterized by the step S2 of the in vitro preparation method of hair follicle stem cells: separation of hair follicles, separating the hair follicle unit under a 2-15 times magnifying glass or electron microscope to remove the epidermal tissue from the single outer hair root sheath containing hair follicle stem cells, with an accuracy rate of up to 100%, avoiding damage to the hair follicle stem cells and the outer hair root sheath during the separation process. The in vitro separation of the outer hair root sheath without the epidermal part is the second creativity of the present invention.

4. According to claim 3, the new technical method for treating chloasma with stem cell transplantation surgery lies in the step S3 of the in vitro preparation method of hair follicle stem cells: inactivation of hair follicles. Whether to inactivate hair follicles is selected according to the severity of chloasma. For patients with small area, white skin and mild symptoms, inactivation treatment is adopted. The hair follicles before transplantation are inactivated. The method is: using a hair follicle inactivation needle under a microscope or a magnifying glass to remove the hair follicle papilla to prevent it from entering the next hair follicle cycle; for patients with large area, darker pigmentation and severe disease, the hair follicles before transplantation do not need to be inactivated; in addition, if the hair follicles are white, no inactivation treatment is required. The selective hair follicle inactivation method adapted to local conditions is the third creativity of the present invention.

5. The new technical method for treating chloasma with stem cell transplantation surgery according to claim 4 is characterized by the step S4 of the in vitro preparation method of hair follicle stem cells: in vitro culture of hair follicle stem cells, placing the hair follicles treated in steps 1, 2 and 3 into a special culture medium, and culturing at 22-26°C for 1-4 hours to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells. The main components of the special culture medium are tranexamic acid injection, compound glycyrrhizin injection, vitamin C injection and normal saline; the combined use of non-toxic and harmless tranexamic acid injection, compound glycyrrhizin injection and vitamin C injection for in vitro culture of hair follicle stem cells is the fourth creativity of the present invention.

6. The new technical method for treating chloasma with stem cell transplantation surgery according to claim 5 is characterized by the step S5 of the in vitro preparation method of hair follicle stem cells: in vitro positioning of the transplantation layer of hair follicle stem cells for treating chloasma, using a dermatoscope and/or skin CT and/or skin B-ultrasound alone or in combination, the layer of the hair follicle implantation needle obtained by in vitro positioning is the specific layer of the hair follicle outer root sheath transplantation, and this layer of transplantation needle will produce special dermatoscope, skin CT, and skin B-ultrasound images, providing reliable preoperative technical support for the precise transplantation of the outer root sheath containing hair follicle stem cells to treat chloasma; the in vitro use of a dermatoscope, skin CT, and skin B-ultrasound to locate the special image of the hair follicle transplantation needle or the implantation needle at a specific layer of chloasma is the fifth creativity of the present invention.

Industrial Applicability

The present invention provides a new treatment method for chloasma, proposes a subversive treatment theory for chloasma, and successfully treats chloasma under the guidance of this theory. The present invention uses skin B-ultrasound and dermoscopy imaging to accurately report data to evaluate the condition of chloasma, uses FUE technology to extract and separate the outer root sheath containing hair follicle stem cells and culture them, promotes the differentiation and proliferation of hair follicle stem cells, and activates and improves the activity of hair follicle stem cells through cultivation, promoting the rapid transformation and proliferation of hair follicle stem cells; then the hair follicle stem cells and the outer root sheath are accurately transplanted to the specific layer of the epidermis of chloasma to treat pathological chloasma melanocytes and reduce the secretion of melanin particles; at the same time, it can promote epidermal thickening, enhance the defense against ultraviolet rays, and rebuild the skin barrier of chloasma. This invention fundamentally solves the problem that stubborn chloasma cannot be cured.

Sequence Listing Free Content

Based on the pathogenesis of chloasma and basic research on chloasma, the present invention proposes for the first time in the world a technical method for repairing the skin barrier of chloasma and a treatment method for pathological chloasma melanocytes, thereby providing an innovative technical solution for completely curing chloasma. Through this invention, clinical observations were conducted on 3 patients with chloasma, and the therapeutic effect was significant.

Claims

The new technical method of stem cell transplantation for treating chloasma is characterized by an in vitro preparation method of hair follicle stem cells, which comprises the following steps:

Step S1, extraction of hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extracting a hair follicle unit, preferably single or double hair follicles, preferably white hair follicles;

Step S2, separation of hair follicles, separation of the hair follicle unit to remove the epidermal tissue of the single outer hair root sheath containing hair follicle stem cells under a 2-15 times magnifying glass or electron microscope, with an accuracy rate of up to 100%, avoiding damage to the hair follicle stem cells and the outer hair root sheath during the separation process;

Step S3, inactivation of hair follicles. Whether to inactivate hair follicles is determined according to the severity of chloasma. Inactivation treatment is performed on patients with small area, white skin and mild symptoms. The hair follicles before transplantation are inactivated: a hair follicle inactivation needle is used under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle. For patients with large area, darker pigment and severe disease, the hair follicles before transplantation may not be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required.

Step S4, in vitro culture of hair follicle stem cells, placing the extracted and separated hair follicles of the outer hair root sheath containing hair follicle stem cells into a special culture medium and culturing them at 22-26° C. for 1-4 hours to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells, wherein the main components of the special culture medium are tranexamic acid injection, compound glycyrrhizin injection, vitamin C injection and normal saline;

Step S5, in vitro positioning of the transplantation layer of hair follicle stem cells for the treatment of melasma, through the combined use of dermatoscope and/or skin CT and/or skin B-ultrasound, the insertion layer of the hair follicle implantation needle located in vitro is the specific layer of the hair follicle outer root sheath transplantation, and the in vitro positioning can provide preoperative technical support for the precise transplantation of the outer root sheath containing hair follicle stem cells.
According to claim 1, the new technical method for treating chloasma with stem cell transplantation surgery is characterized by the step S1 of the in vitro preparation method of hair follicle stem cells: extraction of hair follicles, using FUE technology to extract and separate the outer hair root sheath containing hair follicle stem cells, extracting a follicle unit, preferably single or double follicles, preferably white hair follicles, and colored hair follicles can also be selected; hair follicle stem cell transplantation to treat chloasma, especially transplanting white hair follicles for disease treatment, is one of the creative ideas of the present invention.
According to claim 2, the new technical method for treating chloasma with stem cell transplantation surgery is characterized by the step S2 of the in vitro preparation method of hair follicle stem cells: separation of hair follicles, separating the hair follicle unit under a 2-15 times magnifying glass or electron microscope to remove the epidermal tissue from the single outer hair root sheath containing hair follicle stem cells, with an accuracy rate of up to 100%, avoiding damage to the hair follicle stem cells and the outer hair root sheath during the separation process. The in vitro separation of the outer hair root sheath without the epidermal part is the second creativity of the present invention.
According to claim 3, the new technical method of stem cell transplantation surgery for treating chloasma lies in the step S3 of the in vitro preparation method of hair follicle stem cells: inactivation of hair follicles. Whether to inactivate hair follicles is selected according to the severity of chloasma. For patients with small area, white skin and mild symptoms, inactivation treatment is adopted. The hair follicles before transplantation are inactivated by using a hair follicle inactivation needle under a microscope or a magnifying glass to remove the hair follicle papilla so that it cannot enter the next hair follicle cycle. For patients with larger area, darker pigmentation and more serious disease, the hair follicles before transplantation do not need to be inactivated. In addition, if the hair follicles are white, no inactivation treatment is required. The selective hair follicle inactivation method adapted to local conditions is the third creativity of the present invention.
According to claim 4, the new technical method for treating chloasma by stem cell transplantation surgery is characterized by the step S4 of the in vitro preparation method of hair follicle stem cells: in vitro culture of hair follicle stem cells, placing the hair follicles treated in steps 1, 2 and 3 into a special culture medium, and culturing at 22-26°C for 1-4 hours to enhance the activity of hair follicle stem cells, inhibit the formation of melanocytes, and promote the differentiation and proliferation of hair follicle stem cells. The main components of the special culture medium are tranexamic acid injection, compound glycyrrhizin injection, vitamin C injection and normal saline; the combined use of non-toxic and harmless tranexamic acid injection, compound glycyrrhizin injection and vitamin C injection for in vitro culture of hair follicle stem cells is the fourth creativity of the present invention.
According to claim 5, the new technical method for treating chloasma with stem cell transplantation surgery is characterized by the step S5 of the in vitro preparation method of hair follicle stem cells: in vitro positioning of the transplantation layer of hair follicle stem cells for treating chloasma, using a dermatoscope and/or skin CT and/or skin B-ultrasound alone or in combination, the layer of the hair follicle implantation needle obtained by in vitro positioning is the specific layer of the hair follicle outer root sheath transplantation, and this layer of transplantation needle will produce special dermatoscope, skin CT, and skin B-ultrasound images, providing reliable preoperative technical support for the precise transplantation of the outer root sheath containing hair follicle stem cells to treat chloasma; the in vitro use of a dermatoscope, skin CT, and skin B-ultrasound to locate the special image of the hair follicle transplantation needle or the implantation needle at a specific layer of chloasma is the fifth creativity of the present invention.