Homologous hair follicle stem cell spray for burnt hair regeneration as well as preparation method and application of homologous hair follicle stem cell spray

Abstract

The invention relates to a biomedical material, in particular to homologous hair follicle stem cell spray for burnt hair regeneration and a preparation method and application thereof, and the preparation method comprises the following steps: washing after enzymolysis of a skin sample, scraping cells from the treated skin sample, collecting the cells through a suspension, and filtering to obtain a cell suspension; mixing the cell suspension with a cell nutrient solution to obtain homologous hair follicle stem cell spray; the skin sample is homologous skin of burnt hair. Compared with the prior art, the problems that in the prior art, operation is complex, time is long, recovery is slow, a patient needs to recur for a long time, and life and work are affected are solved, and alopecia treatment which is easy to operate and fast in recovery is achieved.

Description

Technical Field

[0001] The invention relates to a biomedical material, in particular to a homologous hair follicle stem cell spray for regeneration of burned hair and a preparation method and application thereof. Background Art

[0002] Burns, whether caused by chemical (acid / alkali), flame, scald (including steam, hot water, and cooking oils), or rarely electrical injury, may be isolated to the head and neck area or part of an injury that affects a larger total body surface area. Functional issues are common and include intrinsic and extrinsic contractures leading to microscopic excisions, ectropion of the upper and lower eyelids, lip eversion, and nares retraction. After acute treatment of the life-threatening problem is concluded, wound healing is complete, and functional deficits are resolved, attention can be turned to improving the patient's aesthetic issues, such as partial or complete loss of the nose and external ears. The complete destruction of hair follicles is often associated with deep burns, and their nature also results in severe scarring, which makes hair transplantation more challenging. Although hair loss in previously hairy areas of the head and neck can be difficult to conceal with clothing, especially in the scalp, eyebrows, eyelashes, and beard areas, hair loss can have an equal or greater impact on the patient's self-esteem and quality of life than the scars themselves, and serve as a constant reminder of the cause of the trauma.

[0003] Treating patients with burn-related alopecia or hair loss is often a challenge for both surgeons and patients. Like other reconstructive procedures required in the post-burn phase, this is usually a multi-stage process that often requires several years of surgery. This is because graft uptake is not as reliable as healthy scar-free skin and may require repetition to achieve adequate density. Surgical hair transplantation techniques include follicular unit scalp strip excision technology and follicular unit extraction technology. This method is complex to operate, time-consuming, and slow to recover. Patients need to rest for a long time, which affects their lives and work. In addition, there is a 30-50% probability that the surviving hair will no longer grow, and the effect cannot satisfy the patient.

[0004] It can be seen that the above existing methods for treating hair loss obviously still have inconveniences and defects, and are in urgent need of further improvement. Stem cell therapy technology is a major leap forward in tissue engineering and a major advancement in the synergistic effect of biomaterials and cells to repair defective tissues. Using autologous homologous cells to promote autologous hair growth can fundamentally solve the problem of hair loss in patients, and it is simple to operate and has a fast recovery, which has become a goal that the industry urgently needs to improve. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the above problems and to provide a homologous hair follicle stem cell spray for burn hair regeneration and its preparation method and application, so as to solve the problems in the prior art of complex operation, long time, slow recovery, and patients needing long time to recuperate, which affects their life and work. This solution realizes a hair loss treatment with simple operation and fast recovery.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention discloses a method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair, comprising the following steps:

[0008] The skin sample is enzymatically hydrolyzed and then rinsed, cells are scraped from the treated skin sample to obtain a cell suspension, and then filtered to obtain a cell suspension;

[0009] Mixing the cell suspension with a cell nutrient solution to obtain a homologous hair follicle stem cell spray;

[0010] The skin sample is the same as the burned hair.

[0011] Preferably, the selected skin sample is homologous hair follicle tissue from the occipital region or the back of the head of the patient.

[0012] Preferably, the enzymatic hydrolysis is performed by using 0.1-0.2 g / mL type I collagenase at 30-40° C. for 15-20 min.

[0013] Preferably, the filtering is: using a filter to remove particles with a particle size > 100 μm.

[0014] Preferably, the cell nutrient solution comprises a combination of two or more of a lactate solution, a hair follicle stem cell complete culture medium and a growth factor, and the volume ratio of the cell suspension to the cell nutrient solution is 1-3:2.

[0015] Preferably, the lactate solution is composed of sodium chloride, sodium lactate and calcium chloride dissolved in water for injection, the concentration of sodium chloride is 0.05-0.15 mol / L, the concentration of sodium lactate is 0.02-0.03 mol / L, the concentration of calcium chloride is 0.001-0.002 mol / L, and the pH value of the lactate solution is 6.0-7.5.

[0016] Preferably, the hair follicle stem cell complete culture medium is a combination of one or more of KSFM cell culture medium, DMEM / F12 cell culture medium and 1640 cell culture medium, and the volume ratio of the lactate solution to the hair follicle stem cell complete culture medium is 4-3:7.

[0017] Preferably, the growth factor is a combination of one or more of basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor and insulin growth factor, and the concentration of the growth factor is 20-30 ng / mL.

[0018] Preferably, the cell nutrient solution also includes serum protein and a penicillin / streptomycin mixed solution.

[0019] The second aspect of the present invention discloses a homologous hair follicle stem cell spray for regeneration of burned hair, which is prepared by any of the above-mentioned preparation methods.

[0020] The third aspect of the present invention discloses the use of the homologous hair follicle stem cell spray for regeneration of burned hair as described above in the preparation of a spray for regeneration of burned hair.

[0021] The working principle of the present invention is:

[0022] The prepared cell suspension is an autologous cell suspension derived from the full-thickness skin tissue with hair follicles on the occipital side of the patient's head. Human hair follicle stem cells are mainly concentrated in the hair follicles of the scalp, especially in the back of the head and the areas on both sides of the head (i.e., the temporal areas), where the number of hair follicle stem cells is relatively large. The hair follicle stem cells in these areas have strong self-renewal and regeneration capabilities. They can divide and produce new hair follicle cells, thereby maintaining the growth cycle of hair. In addition, the scalp skin in the back of the head and the areas on both sides is thicker and has a rich blood supply, which provides a good nutrition and oxygen environment for hair follicle stem cells, promoting their growth and reproduction.

[0023] The cell suspension prepared by this invention is derived from the skin tissue of the occipital part of the patient's head, and is used for the patient's hair loss area, which belongs to homologous cell transplantation technology. This process uses enzymatic hydrolysis and filtration to obtain hair follicle stem cells with excellent proliferation characteristics and a high survival rate, which is conducive to the subsequent preparation of high-purity, high-survival cell suspension preparations. It has the following advantages: (1) The cell survival rate is high and the survival time is long, and it has good injectability. By spraying, the hair follicle stem cells are sprayed on the hair damage site, which is simple to operate; (2) When the cell spray liquid is injected into the area with sparse hair, it can wake up the sleeping hair and promote its natural growth. The effect is significant and the problem of hair loss is completely solved. At the same time, because the entire cell suspension preparation process is simple to operate, the damage area to the patient is small, and it does not affect the patient's normal work and life. The patient compliance is good, the separation method is simple, and the cost is low.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a method for preparing a hair follicle stem cell spray, which generally includes: preparing a cell nutrient solution and mixing a cell suspension. The hair follicle stem cell spray of the present invention is prepared by the above-mentioned preparation method, and the hair follicle stem cell spray is sprayed according to a certain ratio of the spray expansion area to the burn injury area, and has the characteristics of high cell survival rate and long survival time, in-situ spraying, and simple operation. In addition, the autologous cell suspension for promoting hair regeneration is derived from the full-thickness skin tissue with hair follicles at the occipital part of the patient's head, and the hair follicle tissue is decomposed by an enzymatic decomposition method to obtain an autologous hair follicle stem cell spray preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation process of homologous hair follicle stem cell spray.

[0027] Figure 2 This is the cell morphology of the cell suspension extracted in Example 1 in the cell culture medium (10x objective lens).

[0028] Figure 3 This is the cell morphology of the cell suspension extracted in Example 2 in the cell culture medium (10x objective lens). DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] In the following description, unless otherwise specified, conventional commercially available products are used and the methods used are well known in the art.

[0031] The present invention designs a spray derived from autologous hair follicle stem cells, which effectively repairs damaged skin, promotes skin regeneration and improvement, and repairs hair follicles. Spraying on skin tissue will not cause immune rejection.

[0032] A method for preparing a spray of autologous hair follicle stem cells that can be used for hair regeneration after burns, such as Figure 1 As shown, it includes the following steps:

[0033] (I) Selecting the appropriate donor skin area

[0034] The hair follicle tissue is obtained from the occipital or back of the patient's head. A preferred instrument such as a sterile scalpel is used to obtain a donor skin area with a thickness of 0.006-0.008 mm. The donor site must be clean, of appropriate depth, and without surrounding inflammation or infection. The hair follicles are extracted from the dermis of the donor tissue to obtain the required hair follicle tissue (skin sample).

[0035] (II) Enzyme decomposition

[0036] Place the treated hair follicle tissue in a heated decomposition chamber. Place several skin samples in 0.1-0.2g / mL type I collagenase for enzymatic hydrolysis at 30-40℃ for 15-20min. After decomposing the tissue, remove the hair follicle sample from the heated enzyme with sterile tweezers and place the sample dermis side up on a sterile tray.

[0037] (III) Flushing hair follicle tissue samples

[0038] Rinse the hair follicle tissue sample in a solution containing decomposing enzymes to wash away the residual enzymes. Rinse for 3-5 minutes.

[0039] (IV) Scraping cells from hair follicle tissue samples

[0040] Place the sample on a sterile tray with the dermis facing down. Use forceps to anchor the skin sample and gently scrape the epidermal surface with the blade of a scalpel. Completely expose the epidermal cells to obtain a cell suspension.

[0041] (V) Collecting cell suspension

[0042] Collect and prepare the cell suspension. Using sterile water, rinse the tray. Pipette and rinse several times to maximize cell collection. Finally, draw all of the cell suspension into the syringe.

[0043] (VI) Filtering the cell suspension

[0044] Spray the cell suspension into the cell filter in the cell suspension apparatus. The filter removes particles >100 μm and can effectively prevent nozzle clogging when spraying the cell suspension.

[0045] (VII) Preparation of Hair Follicle Stem Cell Spray Preparation

[0046] Prepare cell nutrient solution: the nutrient solution includes lactate solution, hair follicle stem cell culture medium and growth factors. Preferably, the lactate solution is composed of sodium chloride, sodium lactate, calcium chloride, and water for injection; preferably, the concentration of sodium chloride is 0.05-0.15 mol / L, the concentration of sodium lactate is 0.02-0.03 mol / L, the concentration of calcium chloride is 0.001-0.002 mol / L, and the pH value of the lactate solution is 6.0-7.5. The volume ratio of lactate solution to hair follicle stem cell culture medium in the nutrient solution is 4:7-3:7. The hair follicle stem cell culture medium base solution is selected from one or several combinations of KSFM cell culture medium, DMEM / F12 cell culture medium, 1640 cell culture medium, and then serum protein and penicillin / streptomycin mixed solution and one or several combinations of growth factors are added. The preferred growth factor is composed of one or more of basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor, and insulin growth factor, and the concentration of the growth factor is 20-30 ng / mL. The cell suspension filtered in the above step and the cell nutrient solution are mixed in a ratio of 1:3-1:2 to prepare a hair follicle stem cell spray preparation.

[0047] (VIII) Applying the cell suspension to the wound

[0048] The spray preparation is drawn up using a syringe and can be directly applied to partial thickness burn wounds. The spray preparation can be sprayed or dripped into the wound bed. The specific operation (i.e., spraying or dripping) depends on the amount of cell suspension used and the size of the wound bed.

[0049] Example 1

[0050] A method for preparing an autologous hair follicle stem cell spray comprises the following steps:

[0051] 1) Select the appropriate donor skin area

[0052] The hair follicle tissue is obtained from the back of the patient's head. The donor area with a thickness of 0.006 mm is obtained using a preferred instrument such as a sterile scalpel. The donor site must be clean, of appropriate depth, and without showing surrounding inflammation or infection. The hair follicles are extracted from the dermis of the donor tissue to obtain the final hair follicle tissue.

[0053] 2) Enzyme decomposition

[0054] Place the hair follicle specimen in a heated decomposition chamber. Heat one hair follicle sample in 0.2 g / mL type I collagenase at 40°C for 20 minutes. After decomposing the tissue, remove the hair follicle sample from the heated enzyme with sterile tweezers and place the sample dermis side up on a sterile tray.

[0055] 3) Rinse the hair follicle tissue sample

[0056] Rinse the hair follicle tissue sample containing the decomposition enzyme to rinse off the residual enzyme. Rinse for 3 minutes until all the decomposition enzyme is rinsed off.

[0057] 4) Scrape cells from hair follicle tissue samples

[0058] Place the sample on a sterile tray with the dermis facing down. Use forceps to anchor the skin sample and gently scrape the epidermal surface with the blade of a scalpel. Completely expose the epidermal cells to obtain a cell suspension.

[0059] 5) Collect cell suspension

[0060] Collect and prepare the cell suspension. Using sterile water, rinse the tray. Pipette and rinse several times to maximize cell collection. Finally, draw all of the cell suspension into the syringe.

[0061] 6) Filter the cell suspension

[0062] Spray the cell suspension into the cell filter in the cell suspension apparatus. The filter removes particles >100 μm and can effectively prevent nozzle clogging when spraying the cell suspension.

[0063] 7) Preparation of hair follicle stem cell spray preparation

[0064] Prepare cell nutrient solution: the nutrient solution includes lactate solution, preferably, the lactate solution is composed of sodium chloride, sodium lactate, calcium chloride, and water for injection, the concentration of sodium chloride is 0.08 mol / L, the concentration of sodium lactate is 0.03 mol / L, the concentration of calcium chloride is 0.002 mol / L, and the pH value of the lactate solution is 7. The hair follicle stem cell culture medium base solution is selected from KSFM complete cell culture medium, and the preferred growth factor is composed of basic fibroblast growth factor and epidermal growth factor, and the concentration of the growth factor is 20 ng / mL. The cell morphology of the hair follicle mixed cells in the cell culture medium is as follows Figure 2 As shown, Figure 2 The cells in the hair follicle stem cell spray preparation are polygonal or elliptical, with clear cell boundaries and evenly distributed in the field of view, and are arranged closely. The volume ratio of the lactate solution to the KSFM complete cell culture medium in the nutrient solution is 4:7. In this embodiment, the cell suspension filtered in the above step and the cell nutrient solution are prepared in a ratio of 1:3 to prepare the hair follicle stem cell spray preparation.

[0065] 8) Apply the cell suspension to the wound

[0066] The spray is drawn up with a syringe and can be applied directly to partial-thickness burn wounds. The spray can be sprayed or dripped into the wound bed, depending on the amount of cell suspension used and the size of the wound bed. Surgical glue, sutures, or staples can be used to secure the dressing to the wound when necessary. Auxiliary dressings with moderate water absorption and minimal adsorption can be used for wound dressing.

[0067] Example 2

[0068] A method for preparing an autologous hair follicle stem cell spray comprises the following steps:

[0069] 1) Select the appropriate donor skin area

[0070] The hair follicle tissue is obtained from the occipital part of the patient's head. The donor area with a thickness of 0.008 mm is obtained using a preferred instrument such as a sterile scalpel. The donor site must be clean, of appropriate depth, and without showing surrounding inflammation or infection. The hair follicles are extracted from the dermis of the donor tissue to obtain the final hair follicle tissue.

[0071] 2) Enzyme decomposition

[0072] Place the processed hair follicle tissue in a heated decomposition chamber. Place one hair follicle sample in 0.2g / mL type I collagenase and heat at 30°C for 15 minutes. After decomposing the tissue, use sterile tweezers to remove the skin sample from the heated enzyme and place the dermis side of the hair follicle tissue sample on a sterile tray.

[0073] 3) Rinse the skin sample

[0074] Rinse the skin sample in a solution containing degrading enzymes to wash away any residual enzymes. Rinse for 5 minutes.

[0075] 4) Scrape cells from hair follicle tissue samples

[0076] Place the sample on a sterile tray with the dermis facing down. Use forceps to anchor the skin sample and gently scrape the epidermal surface with the blade of a scalpel. Completely expose the epidermal cells to obtain a cell suspension.

[0077] 5) Collect cell suspension

[0078] Collect and prepare the cell suspension. Using sterile water, rinse the tray. Pipette and rinse several times to maximize cell collection. Finally, draw all of the cell suspension into the syringe.

[0079] 6) Filter the cell suspension

[0080] Spray the cell suspension into the cell filter in the cell suspension device. The filter removes particles >100 μm and can effectively prevent nozzle clogging when spraying the cell suspension. The filtered cell suspension is used together with the subsequent suspension of the remaining skin sample.

[0081] 7) Preparation of hair follicle stem cell spray preparation

[0082] Prepare cell nutrient solution

[0083] The nutrient solution includes a lactate solution. Preferably, the lactate solution is composed of sodium chloride, sodium lactate, calcium chloride, and water for injection. The concentration of sodium chloride is 0.15 mol / L, the concentration of sodium lactate is 0.03 mol / L, the concentration of calcium chloride is 0.0015 mol / L, and the pH value of the lactate solution is 7.5. The hair follicle stem cell culture medium solution is selected from 1640 cell complete culture medium. The preferred growth factors are basic fibroblast growth factor and epidermal growth factor. The concentration of the growth factor is 30 ng / mL. The cell morphology of the hair follicle mixed cells in the cell culture medium is as follows: Figure 3 As shown, Figure 3 The cells in the experiment are polygonal or elliptical, with clear cell boundaries and evenly distributed in the field of vision, and are arranged relatively tightly. The cell nucleus is obvious, located in the center of the cell or biased to one side, the chromatin in the nucleus is evenly distributed, and clear nucleoli can be seen in some cell nuclei, indicating that these cells may have higher activity. Compared with Example 1, the number of cells is larger and the cell viability is higher. The method separates hair follicle stem cells in a short time by enzymatic hydrolysis and filtration, and maintains high activity and growth in the culture medium. The volume ratio of lactate solution to KSFM complete cell culture medium in the nutrient solution is 3:7. In this embodiment, the cell suspension filtered in the above step and the cell nutrient solution are prepared in a ratio of 1:2 to prepare a hair follicle stem cell spray preparation.

[0084] 8) Apply the cell suspension to the wound

[0085] The spray is drawn up with a syringe and can be applied directly to partial-thickness burn wounds. The spray can be sprayed or dripped into the wound bed. The specific operation (i.e. spraying or dripping) depends on the amount of cell suspension used and the size of the wound bed. Surgical glue, sutures or staples are used for fixation.

[0086] In summary, the present invention adopts enzymatic hydrolysis and filtration methods to prepare stem cell suspension, and simultaneously prepares cell culture solution containing growth factors and polymer permeate. The two are mixed in a certain proportion to prepare a cell spray solution. The results show that the cells extracted from the hair follicle tissue on both sides of the patient's occipital region have better cell viability than the cells extracted from the back of the head region, and are in the logarithmic growth phase. The prepared cell spray can be sprayed in situ, which is easy to operate.

[0087] The above description of the embodiments is to facilitate the understanding and use of the invention by those of ordinary skill in the art. It is obvious that those familiar with the cell field technology can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to go through creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair, characterized in that: The steps include: The skin sample is enzymatically hydrolyzed and then rinsed, cells are scraped from the treated skin sample to obtain a cell suspension, and then filtered to obtain a cell suspension; Mixing the cell suspension with a cell nutrient solution to obtain a homologous hair follicle stem cell spray; The skin sample is the same as the burned hair.

2. The method for preparing a homologous hair follicle stem cell spray for burn hair regeneration according to claim 1, characterized in that: The skin sample is homologous hair follicle tissue of the occipital part or the back of the head; the enzymatic hydrolysis is: using 0.1-0.2g / mL type I collagenase to perform enzymatic hydrolysis at 30-40°C for 15-20min.

3. The method for preparing a homologous hair follicle stem cell spray for burn hair regeneration according to claim 1, characterized in that: The filtering is: using a filter to remove particles with a particle size greater than 100 μm.

4. The method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair according to claim 1, characterized in that: The cell nutrient solution comprises a combination of two or more of a lactate solution, a hair follicle stem cell complete culture medium and a growth factor, and the volume ratio of the cell suspension to the cell nutrient solution is 1-3:

2.

5. The method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair according to claim 4, characterized in that: The lactate solution is composed of sodium chloride, sodium lactate and calcium chloride dissolved in water for injection, the concentration of sodium chloride is 0.05-0.15 mol / L, the concentration of sodium lactate is 0.02-0.03 mol / L, the concentration of calcium chloride is 0.001-0.002 mol / L, and the pH value of the lactate solution is 6.0-7.

5.

6. The method for preparing a homologous hair follicle stem cell spray for burn hair regeneration according to claim 4, characterized in that: The hair follicle stem cell complete culture medium is a combination of one or more of KSFM cell culture medium, DMEM / F12 cell culture medium and 1640 cell culture medium, and the volume ratio of the lactate solution to the hair follicle stem cell complete culture medium is 4-3:

7.

7. The method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair according to claim 4, characterized in that: The growth factor is a combination of one or more of basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor and insulin growth factor, and the concentration of the growth factor is 20-30 ng / mL.

8. The method for preparing a homologous hair follicle stem cell spray for regeneration of burned hair according to claim 4, characterized in that: The cell nutrient solution also includes serum protein and a penicillin / streptomycin mixed solution.

9. A homologous hair follicle stem cell spray for regeneration of burned hair, characterized in that: The method is described in any one of claims 1 to 8.

10. Use of the homologous hair follicle stem cell spray for regeneration of burned hair as claimed in claim 9 in the preparation of a spray for regeneration of burned hair.

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