Acellular matrix, preparation method thereof and application of acellular matrix in promoting hair growth

Abstract

The invention relates to an acellular matrix, a preparation method thereof and medical application of promoting hair growth, and belongs to the field of hair growth and hair care medical beauty such as scalp care. Source tissue of the acellular matrix leaving the living body is firstly soaked in a protective solution containing a lysosome stabilizer and an isotonic agent at low temperature, so that cell membranes and lysosomes can be prevented from being broken, and active ingredients in the extracellular matrix are prevented from being degraded and damaged; then, a double-low-concentration method is adopted for mild decellularization treatment, the finally prepared decellularized matrix is complete in ECM structure, the content of active factors (such as bFGF and Wnt2b) is higher, and the tissue regeneration inducing capacity is higher; the acellular matrix can be micronized and mixed with a suspending aid to prepare an injectable suspension, or microneedled, and can be applied to the head in various ways to promote hair regeneration.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a decellularized matrix preparation and a preparation method thereof, and uses thereof in promoting hair care and hair growth. Background Art

[0002] Hair loss can be roughly divided into anagen effluvium and telogen effluvium. The causes of anagen effluvium are varied. Hair loss affects about half of men and more than a quarter of women worldwide. Young and middle-aged people have become the main force of the "hair loss army". Common types of hair loss include androgenetic alopecia (AGA), alopecia areata, trichotillomania, telogen effluvium, and senile alopecia. As we all know, androgenetic alopecia, also known as male pattern baldness, is the complete or partial loss of all or most of the hair on the patient's head. Among them, AGA patients will have a reduction in hair follicles and a decrease in hair volume in the affected scalp area. The incidence of AGA increases with age. Despite the high incidence of this disease, there are relatively few effective treatments for patients. At present, some of the specific mechanisms of hair growth disorders are not fully understood, but there is scientific evidence that hair loss is caused by a disorder or disorder in the relationship or interaction between hair follicle development and the hair cycle.

[0003] As we all know, the hair follicle is a complex micro-organ in the skin that is responsible for the growth, periodic replacement of hair, and interaction with surrounding tissues; the main structure, components, and functions of the hair follicle are as follows. The hair follicle structure includes the following 7 parts: Bulge: The upper part of the hair follicle, which contains hair follicle stem cells (HFSC) and melanocyte stem cells, which are essential for hair color and regeneration.

[0004] Matrix: The area beneath the bulge of the hair follicle where hair growth occurs.

[0005] Outer Root Sheath (ORS): The outer structure of the hair follicle, composed of multiple layers of cells, provides protection for the hair.

[0006] Inner Root Sheath (IRS): A multi-layered structure surrounding the hair that affects its shape and direction.

[0007] Dermal Papilla (DP): A group of cells located at the base of the hair follicle that regulates hair growth.

[0008] Sebaceous glands: connected to hair follicles, secrete oil to lubricate and protect the skin and hair.

[0009] Arrector Pili Muscle: A tiny muscle attached to the hair follicles that, when contracted, causes the hair to stand upright.

[0010] The main components of hair follicles include collagen (the main structural protein of hair follicles, providing support and strength); elastin (makes hair follicles elastic, allowing a certain degree of stretching and rebound); proteoglycans (help maintain moisture and structure of tissues); fibronectin (promotes cell adhesion and migration, which is important for hair growth and wound healing); it also contains its own cytokines and growth factors (such as FGF, IGF, Wnt signaling molecules, etc.), which regulate the proliferation, differentiation and hair growth cycle of hair follicle cells.

[0011] Functions of hair follicles: including the periodic growth of hair through the proliferation and differentiation of their matrix cells; the periodic replacement of hair follicles: hair follicles undergo cyclical changes in the growth phase (anagen), regression phase (catagen) and rest phase (telogen); hair follicle stem cells provide new cells to maintain hair growth during the regeneration phase of the hair cycle. As well as sensory functions (hair follicles are connected to nerve endings and participate in sensory conduction); temperature regulation (the contraction and relaxation of the arrector pili muscles help regulate body temperature); protective effects (hair provides a physical barrier for the skin to protect it from external damage); and cosmetic functions. Hair (hair) has important aesthetic value and significant social functions.

[0012] The growth of hair follicles shows a cyclical replacement. The state and condition of hair follicles have a decisive influence on hair growth. Hair does not grow continuously, but grows and falls off through cyclic replacement. The three main stages of normal hair follicle cyclic replacement and how they affect hair growth: Growth phase: accounts for 85%, and can last for 2-8 years. This is the early stage of hair growth, the stage of active hair growth, which can last for several years. During the growth phase, the hair follicle matrix cells proliferate rapidly, differentiate very actively, and push the hair fibers upward, causing the hair length to increase. The duration of the growth phase determines the maximum length that the hair can reach.

[0013] Catagen: accounts for 1-2%, lasts for 2-4 weeks. This is the anagen phase, a transitional phase that lasts for several weeks. The hair follicles begin to atrophy and hair growth stops. During the catagen phase, the structure of the hair follicles changes, and the hair papilla gradually separates from the follicle matrix. At the end of the catagen phase, the hair matrix cells stop producing hair, and the hair becomes a specific shape, namely a rod. The end of this phase marks the beginning of the next phase of the hair growth cycle.

[0014] Telogen: accounts for 10-15%, lasting 2-3 months. The telogen is the final stage of the hair growth cycle, during which the hair follicles completely stop moving and shrink, which lasts for several months. In this stage, the hair follicles are almost inactive, and the old hair fibers gradually separate from the follicles and eventually fall off. After the telogen, the hair follicles will re-enter the growth phase, and new hair will begin to grow to replace the fallen hair.

[0015] The effects of hair follicle cycle replacement on hair growth include: Hair density: The shedding of hair in the resting phase and the growth of new hair in the growth phase together determine the density of hair.

[0016] Hair length: The duration of the growth phase, which affects the maximum length the hair can grow to.

[0017] Hair color: As we age, melanocyte activity in hair follicles may decrease, causing hair color to lighten.

[0018] Hair quality: During the periodic replacement process, the health of hair follicles affects the thickness and elasticity of hair.

[0019] Hair growth pattern: The cyclic replacement pattern of hair follicles in different individuals may result in different hair growth patterns, such as sparse or dense hair.

[0020] At present, stem cell transplantation is one of the optional strategies to achieve hair follicle functional regeneration. For example, stem cell therapy has received much attention as a potential new therapy. The principle of preventing and treating AGA is the reactivation of hair follicle stem cells, thereby improving hair follicle growth and hair regeneration. There are two main types of sources of transplanted stem cells, autologous and allogeneic. Some studies have shown that injection of a mixture containing skin epithelial stem cells and mesenchymal stem cells can induce the production of new hair follicles. Importantly, hair follicle stem cells (HFSC) periodically switch between active and inactive phases, thereby maintaining a stable stem cell population in the hair follicle; however, this ability weakens with age.

[0021] Hair follicles contain a variety of cell resources, such as melanocytes, epithelial cells, and stem cells of different developmental origins, which can continuously renew and regulate hair growth and hair homeostasis. There are two main types of stem cells in hair follicles; the first is HFSC, which is located in the outer root sheath (ORS) in the arrector pili muscle attachment area and the proximal area of ​​the isthmus, both of which are called the "bulge". The second is the dermal papilla cell (DPC), which is responsible for controlling the induction and growth of hair and participates in the formation of new hair follicles. AGA is a non-scarring alopecia, and HFSCs are not damaged, only the progenitor cells are damaged. This fact makes androgenic alopecia potentially reversible. The replication potential of DPCs in the scalp affected by androgenic alopecia is reduced, and it is also found that their shape, size and characteristic markers are prone to loss. In recent years, some clinics have carried out simple stem cell culture and transplantation. Due to the regenerative potential of stem cells, stem cell therapy has attracted much attention among doctors and patients with androgenic alopecia; although studies on the efficacy of stem cell therapy in AGA have been published one after another; the efficacy of this therapy in treating AGA still needs further observation.

[0022] Studies and investigations have shown that hair follicle epithelial stem cells exist among hair follicle stem cells, and these epithelial stem cells have the function of regulating the hair cycle, see JID Symposium Proceedings, 8: 28-38 (2003); the report said that the relationship between epithelium and mesenchyme is a key factor in hair follicle formation.

[0023] Hair transplantation is one of the effective methods to solve baldness, which is to remove hair roots from donor areas that still have normal function (such as along the back and sides of the head) and then transplant them to the scalp areas where the hair roots have died; surgical procedures, such as scalp plastic surgery, including, for example, scalp reduction surgery, scalp flap surgery and tissue extension or tissue expansion.

[0024] Drug therapy, including the use of minoxidil and finasteride. Finasteride and minoxidil are the only two drugs approved by the FDA for hair regrowth; the main disadvantage of the above drug therapy is that it has little effect on the treatment of hair loss after stopping the medication, so the above treatment methods are only temporary means of treating hair loss, not permanent means.

[0025] Currently available alternative methods include, for example, hair transplantation and scalp plastic surgery, as well as various pharmacological (or drug) therapies; despite a lot of research and development on hair growth methods, unfortunately, these methods are still relatively cumbersome, inconvenient to use, and have unstable effects. There is currently no effective method that can fundamentally prevent or treat hair loss.

[0026] On the other hand, various decellularization technologies, including physical, chemical, and biological methods, are used at home and abroad. For the reagents and methods used for decellularization, please refer to Table 1 in CN201910804397.2; the patent name is: A tissue repair material and its preparation method. For homologous or heterologous tissues, the extracellular matrix (ECM) material after removing immunogenic substances is also called a decellularized product preparation. Some technicians also call it a decellularized patch or tissue regeneration scaffold; currently, this type of product (ECM) has been widely used in the fields of tissue engineering and clinical regenerative medicine, such as for the repair and reconstruction of heart valves, blood vessels, nerves, tendons, bones, cartilage, skin, esophagus, trachea, abdominal wall, and many other tissues. Sufficient research has shown that by using appropriate decellularization technology, less immunogenic substances can be prepared, while retaining various components in the original ECM, and retaining a well-structured ECM, which can achieve endogenous induction of in situ tissue regeneration including stem cell regeneration and tissue repair. Existing studies have shown that the tissue repair and reconstruction effects of ECM products are closely related to the material tissue source and decellularization process. Currently, porcine small intestinal submucosa (SIS) is a hot topic and focus of research. It has the advantages of being easy to obtain, having multiple sources, no ethical issues, and low risk of virus transmission. It also has the characteristics of scalable production and is favored by both technical leaders in the industry and experts in academia. For many years, our company's technical team has used porcine small intestinal submucosa (SIS) as the main raw material, repeatedly studied and optimized the decellularization process, and carried out further deep processing. For this purpose, we have carried out the research and development, production and application of multiple series of Class III medical devices.

[0027] Although acellular matrix, or sometimes called extracellular matrix (ECM) preparations, have been used to prevent and treat various types of hair loss in adults, they have also shown certain potential and interest. ECM is composed of a variety of macromolecules, including collagen, elastin, proteoglycans, glycoproteins and fibronectin; ECM not only provides structural support for cells, but also participates in regulating cell behavior, including cell adhesion, migration, proliferation and differentiation. In hair growth, the role of ECM is mainly reflected in the following aspects: 1. Structural support: ECM provides a stable support structure for hair follicles, maintains their shape and integrity, and contributes to the normal growth of hair.

[0028] 2. Cell signaling: ECM interacts with cell surface receptors, triggering intracellular signaling, affecting the behavior of hair follicle stem cells and the hair growth cycle.

[0029] 3. Cell adhesion: The active molecules on the ECM promote the adhesion and anchoring of hair follicle cells by binding to the integrin receptors on the cell surface.

[0030] 4. Nutrient delivery: ECM helps in the delivery of nutrients and oxygen, providing the necessary nutrients and environment for hair growth.

[0031] 5. Cell migration and differentiation: Changes in the composition and structure of ECM can guide the migration and differentiation of hair follicle stem cells and other cells, promoting the formation of new hair.

[0032] 6. Immunomodulation: ECM can regulate local immune responses, affect the inflammatory state of hair follicles, and thus affect hair growth.

[0033] 7. Regulation of hair growth cycle: ECM is closely related to each stage of the hair growth cycle, including the growth phase, regression phase and resting phase.

[0034] 8. Wound healing and regeneration: After skin injury, the repair and regeneration of ECM are essential for the recovery of hair follicles and hair growth.

[0035] The effects of ECM in promoting hair care and hair growth are as follows: first, it improves the hair growth cycle, prolongs the growth period, and shortens the regression period or resting period so that the hair can enter the growth period in time; second, it promotes the increase of hair density and thickness; third, it enhances the elasticity and resistance to breakage of hair.

[0036] ECM plays a multifaceted role in promoting hair growth, however, the current treatment effect is not satisfactory. One of the important reasons may be that during the whole process of preparation of the decellularized matrix, including the timely and proper pretreatment of the target tissue after ex vivo (referred to as S1), different decellularization processes (sometimes too harsh decellularization conditions, high concentration of reagents, long time, and repeated treatments), the natural three-dimensional structure of the ECM is damaged to varying degrees, and different treatment processes may have different effects on the bioactive components and / or growth factors naturally contained in the ECM, resulting in more loss or more destruction of the content, reduced activity in promoting tissue regeneration, or even complete loss; which directly affects the efficacy of ECM preparations in promoting hair growth; in specific use, the induction ability of various hair stem cells or related hair regeneration cells is poor, including the lack of sufficient corresponding cell growth factors and cell channel signal support, so that the regeneration of injured or damaged hair tissue cannot be effectively promoted; ultimately, it is difficult to show a significant effect in promoting hair regeneration.

[0037] The main references are as follows Non-patent articles "New progress in research on the role of Wnt signaling pathway in hair follicle papilla cells in inducing hair follicle formation and growth" Chinese Journal of Cell Biology 2021, 43(7): 1550–1560 Author and unit: Yuan Mu Department of Stem Cell and Regenerative Medicine, Daping Hospital, Army Medical University "Research progress on stem cell therapy for androgenic alopecia" Journal of China Pharmaceutical University 2023, 54(3): 372 - 379 Author and institution: Yan Wenjing School of Life Sciences and Technology, China Pharmaceutical University "Study on the mechanism of action of basic fibroblast growth factor in treating alopecia induced by chemotherapy drugs" 2021 China Medical University PhD Dissertation, 82 pages Author and unit: Qin Jie, First Affiliated Hospital of China Medical University Wnt Ligands Secreted by Subepithelial Mesenchymal Cells Are Essentialfor the Survival of Intestinal Stem Cells and Gut Homeostasis. Valenta et al., 2016, Cell Reports 15, 911–918 “MAP3K2-regulated intestinal stromal cells define a distinct stemcell niche” 《Nature》volume 592, pages606–610 (2021) Author and affiliation: Su Bing, Shanghai Jiao Tong University School of Medicine and Shanghai Institute of Immunology This article discovered that at the bottom of intestinal stem cells, there is a new type of intestinal mesenchymal cell called MRISC; it revealed the role and mechanism of MRISC cells in the repair of intestinal epithelial tissue damage by specifically regulating intestinal stem cell microenvironment signals during intestinal inflammation and damage, providing new ideas for intestinal repair and regeneration.

[0038] Existing patent technology literature: US20140086867A1; US20210299036A1; CN201610511579.7; Regarding the decellularization protective solution, CN201610511580.X (abbreviated as D4), the invention name is: an optimized antioxidant decellularization protective solution; Biodisel (Beijing) Biotechnology Co., Ltd. (inventors are Shi Zhen and Shi Weiyun) has published an invention patent; the protective solution has a specific pH value, specific crystal and colloidal osmotic pressure, and appears as a light red liquid. It is obvious that the main function of the protective solution of the invention is at least anti-oxidation. In addition to using DMEM cell culture medium powder, the formula also contains nine other ingredients, which are as follows: (2) L-histidine hydrochloride 2.87-3.83 g / L; (3) chondroitin sulfate 25-30 g / L; (4) low molecular weight dextran 20-35 g / L; (5) hydroxypropyl methylcellulose 2-8 g / L; (6) allopurinol 0.5-0.8 g / L; (7) HEPES buffer 20-25 ml / L; (8) dexamethasone hydrochloride 1-2 mg / L; (9) reduced glutathione 1.5-3 g / L; (10) levofloxacin 0.1-0.2 g / L.

[0039] First, DMEM cell culture medium powder is used in the protective solution. DMEM is a widely used cell culture medium modified from Eagle's Minimum Essential Medium (MEM) by Dulbecco. The ingredients of DMEM powder include: amino acids (all essential amino acids required for cell growth); vitamins (including vitamin B group, vitamin C, vitamin D, etc.), minerals (such as trace elements and macroelements such as calcium, phosphorus, potassium, magnesium, iron), glucose (as a source of energy for cells), sodium chloride (to maintain the osmotic pressure of the culture medium), sodium bicarbonate (to help maintain the pH balance in the culture environment), L-glutamine (which is unstable in the solution), sodium pyruvate (as an auxiliary energy source for cells); phenol red (pH indicator, to help monitor the pH of the culture medium).

[0040] DMEM mainly provides cells with necessary nutrients to support cell growth in vitro; it is actually unnecessary in the protective solution of D4 invention and seems redundant. It has actually deviated from the original purpose and meaning of D4 invention; it is also far away from the theme of the invention "antioxidant decellularized protective solution".

[0041] Secondly, because DMEM is a cell culture medium, its composition itself is relatively complex, for the reasons mentioned above; which results in the decellularization protective solution of the invention being actually a formula on top of a formula, with obvious double complexity; it is to promote cell growth at the beginning and then to perform decellularization; which makes the ultimate purpose of using the protective solution somewhat contradictory; at least inconsistent, or even completely contrary.

[0042] Thirdly, the D4 protective solution has, on the one hand, a crystal osmotic pressure between 330-380 mOsm / kgH2O, derived from histidine hydrochloride; in Example 1, each liter of protective solution contains 2.87 g of histidine hydrochloride; on the other hand, a colloid osmotic pressure between 310-350 mOsm / kgH2O, derived from chondroitin sulfate, hydroxypropyl methylcellulose and low molecular weight dextran, and the molecular structures and molecular weights of these three components themselves fluctuate greatly; Chondroitin sulfate sodium (CS) is an acidic mucopolysaccharide composed of D-glucuronic acid and 2-acetylamino-2-deoxy-sulfate-D-galactose, with a molecular structure of (C14H21NO14S)n. Depending on the position of the sulfate group, if it is at the 4th position of galactose, it is called chondroitin sulfate A; if it is at the 6th position, it is called chondroitin sulfate C; the key is that the molecular weight of chondroitin sulfate fluctuates widely, ranging from 10,000 to 50,000 Daltons; the properties of chondroitin sulfate also vary greatly due to different sources and different extraction methods.

[0043] Hydroxypropyl methyl cellulose (HPMC) is listed in Part II of the 2005 edition of the Chinese Pharmacopoeia. Its methyl substitution degree is 1.0-2.0, its hydroxypropyl substitution degree is 0.1-0.34, and its relative molecular mass is generally 10,000 to 150,000. Therefore, the specific molecular weight of HPMC will vary significantly due to the different degrees of hydroxypropyl substitution and methyl substitution.

[0044] The molecular weight of low molecular weight dextran ranges from 25,000 to 45,000, resulting in large fluctuations in molecular weight.

[0045] And these three components that can produce colloid osmotic pressure Dosage , also varies within a large range, such as the lower and upper limits of the dosage of HPMC in the D4 protective solution, which differ by 4 times (the lowest is 2 g / L, the highest is 8 g / L); that is, in fact, for these three components, on the one hand, the molecular weight of each component itself varies greatly; on the other hand, the dosage of these three components also varies greatly; therefore, in actual operation, there are only many factors of variation for these three components that can produce colloid osmotic pressure, which will directly affect the changes in the colloid osmotic pressure parameters in the D4 protective solution, that is, there are many uncertain factors; or in order to achieve the preset target colloid osmotic pressure value, it is necessary to add unnecessary debugging troubles and multiple verifications.

[0046] Fourthly, it is particularly noteworthy that the osmotic pressure in the D4 protective solution is actually composed of the crystal osmotic pressure and the colloid osmotic pressure, which can reach at least 600mOsm / kgH2O; this is actually not in line with the protective purpose that the protective solution is supposed to achieve; it may even produce opposite or obviously adverse side effects.

[0047] Fifthly, the composition of the protective solution invented by D4 is actually very complex, in fact there are nearly twenty kinds of ingredients; because of the large number of ingredients, the actual operation difficulty and complexity of preparing the ingredients and corresponding dosages required for the decellularized protective solution are increased, adding more uncertainty, which in turn easily leads to more fluctuations and changes in the effects.

[0048] Sixthly, the decellularization protective solution invented by D4 uses at least two reagent-grade products for cell culture; one is the aforementioned DMEM cell culture medium powder, and the other is HEPES buffer (a non-ionic amphoteric buffer, whose component is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); these two components alone are not cheap. For example, the market price of 20 ml of Thermo Fisher HEPES is more than 400 yuan, which directly leads to the high cost of using the antioxidant decellularization protective solution.

[0049] Seventh, D4 uses an antioxidant decellularization protective solution throughout the entire process; but in fact, during the decellularization process, the cell membrane has been completely ruptured, resulting in the fact that the D4 protective solution is essentially unable to play the protective role it claims. Summary of the invention

[0050] The invention was developed by the company's senior American R&D personnel, who reviewed numerous domestic and foreign literature on stem cells, hair development, and hair follicle stem cells, carefully read authoritative articles, carefully studied the principles of hair growth and hair loss at all levels, and conducted in-depth discussions to understand the mechanism of action between hair loss-related factors / factors, and found the key molecules or pathways.

[0051] Studies have found that in a variety of tissues, stem cells can proliferate and drive tissue regeneration, which mainly depends on signal transduction on the cells, but the premise is that good tissue regeneration factors and external microenvironment need to be provided, but this problem has not been truly solved.

[0052] Hair follicle stem cells are adult stem cells that are sometimes dormant in the body. Only under certain conditions can they show strong proliferation ability and then differentiate into epidermis, hair follicles, and sebaceous glands to participate in the process of hair regeneration. Hair follicle stem cells live in each hair follicle and are rapidly activated and divided in a new hair growth cycle. There are currently literature reports that the use of acellular matrix can activate hair follicle stem cells in vitro or in vivo, thereby promoting hair growth and preventing hair loss in the beauty field.

[0053] The technical problems solved by the present invention are as follows: The first technical problem: Currently, in the decellularization process, no tissue protection solution is used or only antioxidant protection is used, or the composition is too complex, there is no effective protection solution for ECM before decellularization, or the protection system for ECM is not comprehensive, especially to prevent protease degradation.

[0054] The second technical problem is that the decellularization process is harsh, such as high concentration or long decellularization process time, which leads to a large loss of active ingredients and serious damage.

[0055] The third technical problem is that similar products have low levels of active ingredients or cell growth factors that promote hair growth, making it difficult to stimulate the regenerative vitality / activity of hair stem cells DPC and / or HFSC, and the effect of promoting hair growth is not obvious and lasting.

[0056] In order to solve the above technical problems and overcome the shortcomings of the prior art, the present invention discloses the following technical solutions.

[0057] The first purpose of the present invention is to invent a medical protective solution for mammalian living tissue, which is used for soaking under low temperature conditions before decellularization treatment, that is, a tissue structure or extracellular matrix (also known as ECM) protective solution, specifically, a medical xenogeneic animal tissue protective solution, which is used for soaking under low temperature conditions before decellularization treatment of the target tissue.

[0058] The protective solution comprises at least two components, an isotonic solution and a lysosome stabilizer, and may be referred to as F2 solution; In practice, F2 solution is essentially an isotonic solution containing at least a small amount of lysosomal stabilizer.

[0059] The protective solution is used to prevent the active components and / or various cell growth factors in the extracellular matrix (ECM) from being degraded or destroyed; the low temperature condition is 2-8°C.

[0060] Furthermore, the aforementioned isotonic solution is an isotonic whole intestinal solution with a pH value of 7.2-8.2; The isotonic whole intestinal solution contains 60 grams of polyethylene glycol, 5.68 grams of anhydrous sodium sulfate, 1.46 grams of sodium chloride, 0.74 grams of potassium chloride, 1.68 grams of sodium bicarbonate, and the balance is deionized water or double distilled water per liter of protective solution.

[0061] The polyethylene glycol is pharmaceutical grade PEG4000 of the Pharmacopoeia 2020 edition.

[0062] Furthermore, the isotonic whole intestinal lavage solution is a compound polyethylene glycol electrolyte powder (II), the detailed composition of which is ： Na ion 125mmol / L, K ion 10mmol / L, HCO3 ion 20mmol / L, SO4 ion 40mmol / L, Cl ion 35mmol / L, the osmotic pressure can also be adjusted to 280-320mOsmol / L.

[0063] Isotonic whole bowel lavage solution is originally a relatively mild laxative, mainly used for intestinal cleansing before intestinal endoscopy or intestinal surgery; it can also be used to treat functional constipation.

[0064] In the present invention, the applicant uses it (isotonic whole intestinal lavage solution) as a new use invention; that is, it is used in different technical fields, solves different technical problems, and achieves different technical purposes and technical effects).

[0065] Furthermore, the aforementioned lysosome stabilizer may be one or a combination of hydrocortisone, prednisone, prednisolone, methylprednisolone, and triamcinolone acetonide; Furthermore, the aforementioned lysosome stabilizer is hydrocortisone.

[0066] Furthermore, the concentration of hydrocortisone in each liter of the protective solution is 1 to 10 mg.

[0067] Furthermore, the aforementioned lysosomal stabilizer may be more preferably a nonsteroidal anti-inflammatory drug (NSAID), including one or a combination of sodium and potassium salts of salicylates, arylpropionic acids, anilines, indoles, arylacetic acids and their derivatives, wherein the NSAID is selected from aspirin, diclofenac, loxoprofen, ibuprofen, acetaminophen, celecoxib, etodolac, pranoprofen, flurbiprofen axetil, lornoxicam, thiazide, tramadol and zaltoprofen, and one or a combination of pharmacologically acceptable sodium or potassium salts of these ingredients.

[0068] Furthermore, it is preferred that the lysosome stabilizer is salicylic acid; A more preferred lysosome stabilizer is aspirin; Furthermore, the concentration of aspirin in each liter of the protective solution is 0.1 to 1 gram.

[0069] Specifically, in the embodiment of this patent, preferably, the lysosome stabilizer is aspirin with a concentration of 0.2 g / L (protective solution).

[0070] Further, the target tissue is immersed in F2 protective solution under low temperature (2-8°C) for at least one hour.

[0071] The second object of the present invention is to invent a medical decellularized matrix (ECM), wherein the ECM source tissue is immersed in the aforementioned F2 protective solution after the ECM source tissue leaves the living body, that is, after warm ischemia and before decellularization treatment; The purpose of soaking ECM source tissue with protective liquid is mainly to reduce the degradation or destruction of effective components and natural structures in ECM after leaving living tissue due to adverse external factors; thereby significantly increasing the content of various active ingredients that can activate stem cells in the final ECM preparation, so as to further enhance the regenerative performance and vitality of ECM in promoting tissue and cell regeneration.

[0072] Further, the ECM source tissue is one or more combinations of dermis, pericardium, organ membrane, peritoneum, bladder membrane, small intestinal submucosa, gastric submucosa or other membrane tissues or important organs; Furthermore, the ECM source tissue may be porcine small intestinal submucosa; Furthermore, the pig is a commercialized Duroc-Changda three-way cross commercial meat pig.

[0073] Furthermore, the pig is a Duroc-Changda three-way cross commercial pig; weighing 30-100 kg; preferably a feeder pig, 30-60 kg; and 80-120 days old.

[0074] Furthermore, the decellularized matrix contains one or a combination of Wnt2b and bFGF.

[0075] After rapid removal of impurities and rinsing, immediately place in the aforementioned F2 protective solution and soak for at least one hour until decellularization is performed. The third object of the present invention is to invent a method for preparing an injectable acellular matrix particle preparation.

[0076] The method for preparing the injectable acellular matrix microparticle preparation comprises the following three steps: Step 1 Preparation of decellularized matrix microparticles Immersion in protective solution and pretreatment: The target tissue that has left the living body is immersed in the aforementioned protective solution (F2 solution) at low temperature (2-8 degrees Celsius) to protect the active components and / or various cell growth factors in the extracellular matrix from being degraded and destroyed; pretreatment, such as removing impurities and removing lipids.

[0077] Inactivation, decellularization, DNA removal, and freeze-drying; Preparation of microparticles: The freeze-dried ECM is frozen with liquid nitrogen and then crushed and sieved to obtain microparticles with a diameter of 50 μm-400 μm.

[0078] Aliquot and sterilize.

[0079] Step 2 Preparation of suspending agent Prepare a liquid containing glycerol, sodium hyaluronate and phosphate buffer to obtain a suspending agent; the phosphate buffer is used to adjust the pH value and osmotic pressure of the suspending agent; The suspension is dispensed into containers that match its sterilization method, sealed, and sterilized by moist heat.

[0080] Step 3: The decellularized matrix microparticles and the suspending agent are packaged independently, sterilized separately, and fully mixed with a three-way pipe before use; the mixture is prepared into an injectable decellularized matrix microparticle preparation having good tissue repair and / or regeneration function.

[0081] Further, the specific details include the following three steps: Step 1 Preparation of decellularized matrix microparticles Pretreatment: Soak the fresh pig small intestine or the submucosa of the pig small intestine with the mucosal layer / muscular layer removed in F2 protective solution at low temperature (2-8℃) for at least one hour to protect the active ingredients in the extracellular matrix and various cell growth factors from degradation and destruction.

[0082] Inactivation: Take the submucosa of pig small intestine and treat it with a mixed solution of peracetic acid and ethanol under ultrasonic conditions at room temperature for inactivation.

[0083] Decellularization: The inactivated SIS was first washed with water under ultrasonic conditions, divided into small packages, quickly frozen overnight, thawed the next day, and the "washing-dividing-freezing-thawing" steps were repeated. A mixed solution containing 0.05% trypsin and 0.05% EDTA was used for treatment at 36±2℃, and then ultrasonically washed with PBS; treated in a 1.5% sodium chloride hypertonic solution at 36±2℃, and then washed with water; treated in a 20mM NaOH aqueous solution at 36±2℃; washed with water until neutral; treated with DNase to remove residual DNA.

[0084] Drying: Fix the decellularized SIS sheet on a mold and freeze-dry it.

[0085] Preparation of microparticles: Cut the freeze-dried SIS sheets into pieces, freeze them with liquid nitrogen, and crush them, and sieve them using stainless steel sieves of different mesh sizes to obtain decellularized matrix microparticles. The diameter of the obtained decellularized matrix microparticles is in the range of 50 μm-400 μm.

[0086] Packaging: Pack the decellularized matrix microparticles into syringes and seal them with stoppers.

[0087] Sterilization: sterilize the decellularized matrix particles pre-sealed in the syringe, and select a sterilization method suitable for the decellularized matrix and its packaging, such as cobalt 60 or electron beam radiation sterilization, or ethylene oxide sterilization.

[0088] Step 2: Preparation of suspending agent: prepare a suspending agent containing at least glycerol, sodium hyaluronate, phosphate buffer and other ingredients, wherein the concentration of glycerol component is 0.5%; the concentration of sodium hyaluronate component is 0.2%, and the molecular weight is in the range of 1-1.6 million Daltons.

[0089] Step 3: The decellularized matrix microparticles and the suspending agent are packaged independently, sterilized separately, and fully mixed with a three-way pipe before use; the mixture is prepared into an injectable decellularized matrix microparticle preparation with good tissue repair and regeneration functions.

[0090] The fourth purpose of the present invention is the use of a decellularized matrix of the present invention in the preparation of medical repair materials or medical devices for repairing tissue and / or organ defects; the tissue and organ are head appendages, hair, otology, face, periosteum, nerves, dura mater, spinal dura mater, tendons, ligaments, urinary tract, bladder, ureter, inguinal hernia, hiatal hernia, abdominal wall hernia, breast or uterine mucosa, oral soft tissue, joints, meniscus and other parts of the tissue and / or organ defects.

[0091] Furthermore, the use of the acellular matrix in the preparation of medical repair materials or medical devices is applied to the head to prevent various types of hair loss in adults; used to activate the growth of hair follicle stem cells; the hair follicle stem cells refer to one or a combination of DPC and HFSC of the hair follicles on the head. The medical use of the acellular matrix is ​​applied to the head to prevent hair loss, and to help promote hair follicle growth and hair regeneration.

[0092] Furthermore, the head application method includes external application on the scalp at the hair loss site, assisted introduction using microneedles, nanochips, ultrasonic ions, etc., subcutaneous injection of the scalp, microneedle patches on the scalp at the hair loss site, or as an in vitro culture agent for hair follicle-related tissues and related cells.

[0093] The head application method includes applying to the hair loss area, external scalp application, assisted introduction to the hair loss area using microneedles, nanochips, ultrasonic ions, etc., scalp injection to the hair loss area, microneedle patches to the hair loss area, or as an in vitro culture agent for hair follicle-related tissues and related cells.

[0094] Furthermore, the administration method is scalp injection at the hair loss site, and the injection is a mixed preparation composed of a mixture of decellularized matrix particles and a suspending agent; the mixing ratio is particles: suspending agent = 50mg-200mg: 1 ml; the particle size is in the range of 50μm-400μm.

[0095] The hair growth promoting ECM preparation provided by the present invention can be composed of ECM particles and a suspension, which are independently packaged, sterilized separately, and mixed before use. After mixing, the particles do not aggregate or settle, are evenly and stably dispersed, have low fluidity, and can be smoothly passed through a small diameter needle (27G and 30G) for minimally invasive injection.

[0096] The ECM microparticles are made from ECM of pig small intestinal submucosa or other sources as raw materials, and are prepared through the steps of inactivation, decellularization, freeze drying, granulation, packaging, sterilization and the like.

[0097] An ideal tissue regeneration agent should have good biocompatibility (safety), natural and long-lasting effects, and be easy to use (e.g., easy to inject, and can be flexibly applied to the head via non-invasive or minimally invasive methods such as microneedles).

[0098] ECM has unique advantages, but there are still some difficulties in preparing it into ECM, or further making it into a hair growth promoting preparation product, as follows: What kind of decellularization process should be adopted so that ECM can not only effectively remove immunogenic substances, but also ensure that the ECM components and structures are relatively intact, so that the ECM retains the original active ingredients and various cell growth factors of the ECM, which is conducive to the safe and effective endogenous induction of regeneration after implantation in the body.

[0099] During the decellularization process, how can we minimize or reduce the loss or degradation of various active components in the extracellular matrix? How can ECM materials be used conveniently, effectively and efficiently to promote tissue regeneration, including hair growth? How to prepare efficient, highly active and stable ECM preparations? How can we use ECM scientifically and rationally to promote tissue regeneration, as well as hair care and hair growth, such as in the form of microparticles or microneedles?

[0100] How to inject ECM microparticles (powder)? Before use, they should be mixed with the suspending solution before entering the scalp? Professional terms and related functions or principles are mainly applicable to the technical field and technical effects of the present invention.

[0101] Note: Although Chinese and English nouns or terms are different on the surface, their purposes and uses are basically the same; unless otherwise specified, the connotations of the above nouns are essentially equivalent. The terms / nouns should be understood and used according to the following text descriptions, unless otherwise specified in detail; other terms should be understood according to the level of ordinary technicians in the field; for example, in this patent, isotonic agents and isotonic solutions can usually be regarded as the same or equivalent meanings, unless there are special detailed descriptions or sufficient evidence, they cannot be regarded as the same or conditionally equivalent.

[0102] Warm ischemia time: the period from when blood supply to tissues or organs stops to when cold storage begins. During this period, the damage to tissues or organs is the most serious. This is because, during warm ischemia (after tissues or organs are separated from the body), although blood flow is interrupted, tissues and organs continue to metabolize. At this time, due to the lack of oxygen and various metabolic substrates, the metabolic level of tissues or organs is still high, so ischemic damage occurs faster and is more severe. After oxygen is consumed, anaerobic metabolism can still be carried out, but the metabolic products cannot be removed, which can cause acidosis, and the nutrients and enzyme systems necessary for metabolism are still consumed.

[0103] Low temperature: refers to 2-8°C; similar to the refrigeration temperature of a household refrigerator; the protective solution and the preservation solution, the fresh-keeping solution, can be substantially equivalent; the decellularized matrix preparation, the extracellular matrix preparation and the cell-free extracellular preparation or their combination, unless otherwise specified, can generally be used interchangeably in the present invention and are regarded as equivalent or similar, and can be regarded as substantially the same.

[0104] Extracellular Matrix (ECM) is a non-cellular component present in all tissues and organs. It not only provides the necessary physical support for cell tissues and provides a suitable place and microenvironment for the normal physiological activities of various cells, but also plays an important lever regulating role in tissue morphogenesis, cell chemotaxis and differentiation, as well as important physiological, biochemical and biomechanical aspects, thereby affecting or regulating the functions of tissues and organs. 50% of the cell's function is determined by the external microenvironment created by the extracellular matrix. The material composition of ECM is: structural proteins, such as collagen, elastin, fibrillin and other macromolecular components as the main framework structure, and some functional proteins such as fibronectin FN (fibronectin), laminin LN (laminin), etc. are attached; at the same time, it also carries various cell growth factors (such as fibroblast factor FGF, especially bFGF, as well as transforming growth factor TGF, vascular endothelial growth factor VEGF; it may also contain very small amounts of very important epidermal growth factor EGF and insulin-like growth factor-1 (IGF-1). In addition, the extracellular matrix also contains glycosaminoglycans (GAGs), proteoglycans and other active ingredients.

[0105] Exosomes: tiny vesicles secreted by cells, with a diameter of about 30-200nm, cup-shaped morphology, double-layer membrane structure, naturally present in biological fluids such as blood, urine, saliva, breast milk and cell culture medium. Almost all types of cells (immune cells, nerve cells, stem cells) can produce and release exosomes; exosomes contain protein rRNA and microRNA related to the cell source. Exosomes can directly activate receptor cells through cell membrane receptors, and can also transport proteins, mRNA, miRNA, lncRNA, circRNA, and even organelles into receptor cells to participate in intercellular communication. Exosomes play a key role in physiological processes such as immune response, inflammatory response, angiogenesis, apoptosis, and coagulation. The RNA and protein components contained in exosomes from different cell sources are not the same. Prior art literature indicates that exosomes in the ECM can also enhance angiogenesis and promote better blood supply to nourish hair follicles, but ECM exosome-based treatments have shown promise in stimulating hair growth, improving hair follicle function, and reducing inflammation; although the use of ECM exosomes to treat hair loss is still in the early stages of research and development, preclinical and early clinical studies have shown promising results. These studies have demonstrated that ECM exosomes have the ability to stimulate hair growth, improve hair follicle function, and reduce inflammation associated with certain types of hair loss. The dermal papilla cells (DPCs) in the hair bulb at the base of the hair follicle play an important role in the development of the hair follicle. It can be said that without the division of DPCs, hair follicles cannot be formed, and the three cycles of hair cannot be formed. Studies have shown that exosomes from the ECM can activate hair follicle stem cells, thereby promoting the development of new hair follicles; this is also the reason for the use of exosomes in the present invention. F2 The important reason for using protective solution and double low concentration gentle method to remove cells is that one of its purposes is to preserve more and better exosomes in ECM.

[0106] Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are a type of heteropolysaccharide, mainly found in animal connective tissues. They are important raw materials involved in normal physiological activities of tissues and repair and regeneration of tissue damage; they are important components of ECM; according to the monosaccharide residues, the type of linkages between residues, and the number and position of sulfate groups, glycosaminoglycans can be divided into five main categories, namely hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate and heparin (HP).

[0107] Hyaluronic acid (HA), also known as hyaluronic acid or hyaluronic acid salt, is a glycosaminoglycan and one of the main components of the extracellular matrix (ECM). It participates in tissue reconstruction, expansion of intercellular spaces, inflammatory response and many other cell physiological and tissue repair processes. Studies have shown that HA has obvious cellular effects and can significantly contribute to cell chemotaxis and migration, as well as cell proliferation. Its mechanism occurs after the cell surface HA binding protein binds to HA. The adhesion molecule CD44 is the cell surface receptor of HA and is a widely distributed cell surface glycoprotein that participates in specific adhesion between cells and between cells and matrix. Ru-Ming Liu, et al. published an article in Experimental Cell Research Volume 345, Issue 2, 15 July 2016, Pages 218-229, indicating that hyaluronic acid can also promote the proliferation of mesenchymal stem cells by activating the Wnt / β--Catenin signaling pathway.

[0108] Acellular Tissue Matrix (ACTM) or acellular tissue matrix: refers to the use of specific reagents and treatment methods to fully remove or inactivate cells, viruses, DNA and other components in animal organs or tissues that may produce immune rejection reactions, thereby maximally retaining the integrity of the original natural three-dimensional structure, and retaining the cell growth factors and active functional components in the original matrix as much as possible; Acellular matrix is ​​widely used in clinical practice for tissue repair and regeneration (congenital defects and acquired trauma) due to its natural three-dimensional (3D) structure, containing bioactive factors, being degraded by the host, and being easy to induce the translocation and differentiation of receptor stem cells. Acellular matrix is ​​a new type of tissue regeneration and repair material with good biological scaffold properties.

[0109] Xenogeneic animal decellularized regenerative repair biomaterials, including but not limited to, biogenic materials used for various types of superficial or deep injuries (whether internal or external, acute or chronic). In this patent, it mainly refers to the ECM three-dimensional structure remaining after processing such as decellularization from animal-derived tissues, containing various cytokines and biologically active components; the animal-derived tissue raw materials described in the patent of this invention can be derived from dermis, small intestinal submucosa, bladder matrix, pericardium or other tissues; the physical appearance of the decellularized matrix preparation can be one of powder, granules, colloid, paste, membrane or a combination thereof.

[0110] Dermal papilla cells (DPCs) are a group of dermal-derived cells located at the base of hair follicles. They play a leading role in the growth and development of hair follicles, cycle regulation, and maintenance of hair growth. Abnormalities in the structure or function of dermal papilla cells are the main initiating factors leading to imbalanced hair follicle growth. The hidden spatial location of the dermal papilla brings many difficulties to the isolation and culture of the dermal papilla, but DPCs have very important physiological functions. The research on dermal papilla cells has never been interrupted, and has gradually become a hot spot in the research of hair follicle growth and cycle regulation. In terms of cytology, dermal papilla cells can be divided into mesenchymal stem cells, which belong to the category of multifunctional stem cells. The so-called multifunctionality means that they can differentiate into stem cells of various tissue structures. The dermal papilla cells cultured in vitro are morphologically similar to fibroblasts, but they are essentially different from fibroblasts. In terms of growth mode, the most obvious feature of cultured dermal papilla cells is the multilayered cell mass formed before cell fusion, that is, the characteristic of agglutinative growth. However, the agglutinative growth characteristics of hair papilla cells will gradually weaken with the increase in the number of cell passages. Generally, hair papilla cells after the 7th generation will lose the ability to grow in an agglutinative manner. Agglutinative growth is one of the important biological characteristics of hair papilla cells and is closely related to the biological functions of hair papilla cells. Studies have shown that exosomes from ECM can activate hair follicle stem cells and promote the development of new hair follicles; this is also an important reason for using F2 protective solution in the present invention and adopting a dual low concentration and gentle method to remove cells. One of its purposes is to preserve more and better exosomes in ECM.

[0111] Hair follicle stem cells (HFSC) are the original cells in the hair follicles. Hair and body hair grow from hair follicles. However, the hair follicle stem cells responsible for hair growth are usually dormant, but they will quickly activate and divide in a new round of hair growth cycle. When the adjacent parts are damaged, the hair follicle stem cells can migrate from the original bulge and participate in the repair of the damaged parts. The hair follicle is the basic unit of hair, and hair grows and differentiates from the cells in the hair follicle. There is a group of hair follicle stem cells in each hair follicle, providing new cells to the growing hair follicles. Human hair follicle stem cells are a type of adult stem cells and have the common characteristics of adult stem cells. Many tissue functions of the human body depend on the healthy activity of stem cells. Damage or reduction in the number of stem cells will lead to human aging.

[0112] Wnt signaling is closely related to tissue regeneration; Wnt ligand is a secreted glycoprotein that binds to Frizzled receptors and then forms a larger complex on the cell surface; the conserved Wnt / β-Catenin pathway regulates the pluripotency of stem cells and determines the differentiation fate of cells during development. This developmental cascade integrates signals from other pathways, including basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP), and is present in a variety of cell types and tissues; Wnt polypeptides form a family of highly conserved secreted signaling molecules that regulate cell-cell interactions during tissue regeneration; Wnt family factors are signaling molecules that play a role in a variety of cell pathways and cell-to-cell interactions; Wnt signaling and tissue regeneration, the term "Wnt" or "Wnt gene product" or "Wnt polypeptide" when used herein includes a native sequence Wnt polypeptide, a Wnt polypeptide fragment, a chimeric Wnt polypeptide, or a functionally active variant of the aforementioned species. The terms "WNT" and "Wnt" are used interchangeably herein. As used herein, "a bioactivating substance that can activate the Wnt pathway" can be, for example, a bioactivating substance that can activate the canonical Wnt pathway (also known as the β-catenin pathway) or a bioactivating substance that can activate a non-canonical Wnt pathway (planar cell polarity pathway; PCP pathway, also known as the Ca2+ pathway). Examples of typical bioactivating substances that can activate the Wnt pathway may also include some non-canonical bioactive substances that activate the Wnt pathway.

[0113] Basic fibroblast growth factor (bFGF, or type II (fibroblast growth factor-2, FGF-2), has been reported in the literature to have an effect on hair follicle proliferation and cycle conversion in hair follicle organ culture models, animal models, and human experiments. Studies have shown that in the current work, a variety of growth factors have been found to regulate hair growth and hair follicle cycle in the process of hair loss, including bFGF, EGF, adipose stem cell culture medium, etc. It was found that in the hair loss mouse model, some growth factors alone or synergistically promote hair growth, especially bFGF has the potential to treat hair loss. Studies have confirmed that FGF can induce β-Catenin and Shh pathways in mice, accelerating the transition of hair follicles from the resting phase to the growth phase; bFGF plays an important role in the proliferation and cycle conversion of hair follicle cells after hair loss, and helps to enhance the Shh signaling pathway.

[0114] The Sonic hedgehog (Shh) pathway plays a very important role in the growth and development of hair follicles and the cycle transition. Shh is a key gene in the pathway and is upregulated in the early growth phase. It is one of the factors that regulate the growth and cycle transition of hair follicles. Although the direct link between the induction of hair follicle cell apoptosis and Shh has not yet been found, the disruption of Shh signaling has been found to be a key event in the mechanism of hair loss.

[0115] Supplementary explanation: The acellular matrix preparation of the present invention, when used as a hair growth promoter, can promote the production of growth factors of hair papilla cells; for example, stimulating endogenous vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), etc. Therefore, the hair growth promoter of the present invention can also be called a growth factor production promoter of hair papilla cells, a vascular endothelial growth factor (VEGF) production promoter of hair papilla cells, a fibroblast growth factor (FGF) production promoter of hair papilla cells, and an insulin-like growth factor-1 (IGF-1) production promoter of hair papilla cells.

[0116] In short, during the entire preparation process of the decellularized matrix of xenogeneic animals, including the first moment after leaving the living body, is the obtained target tissue subjected to targeted pretreatment, and is a professional, targeted or specific (tissue or membrane) protective solution used? As soon as the target tissue leaves the living body, that is, in the early stage (after leaving the living body's constant temperature) or early period, can the tissue be preserved or pretreated in a non-destructive or minimally destructive manner, that is, can it be immersed in a low-temperature and high-quality protective solution? Is the entire decellularization process gentle, minimally damaging, fast and efficient, and suitable and matched with the corresponding target tissue? And are the subsequent deep processing (freeze drying and granulation) and sterilization processes of ECM appropriately matched? These key or important technical details will directly affect whether the natural three-dimensional structure and various effective active ingredients (such as Wnt and bFGF and other biologically active factors) in the final ECM product can be sufficiently well protected and preserved, that is, whether the natural biological structure, effective ingredients and biologically active factors of ECM can be effectively protected in three dimensions without dead ends; to achieve high-quality protection of quality, quantity and integrity (equivalent to four guarantees, preservation / good preservation / preservation / integrity).

[0117] Therefore, this technical field needs to develop an ECM preparation with good biocompatibility, natural biological components and structure, endogenous induction of regeneration, including promotion of hair regeneration, controllable maintenance time, minimally invasive injection (appropriate particle size), convenient clinical use, wide application range, and suitable for large-scale production.

[0118] To further describe the purpose of the present invention in detail, an extracellular matrix or tissue protection solution is invented; Of course, in this technical field, technicians with IPC main classification number A61L27 can also reasonably and logically deduce that the protective solution of the present invention is also applicable to other equivalent or similar tissues to be decellularized, such as immersing target tissues such as dermis, bladder membrane, pericardium, peritoneum, amnion, etc. in the protective solution at low temperature (2-8 degrees Celsius). The protective solution can prevent the rupture of cell membranes and lysosomes, and prevent the degradation and destruction of various active ingredients and growth factors in the extracellular matrix (ECM).

[0119] Further, the target tissue disclosed in this embodiment is the small intestinal submucosa tissue or ECM from other sources, that is, the present invention discloses a protective solution of the extracellular matrix of the small intestinal submucosa tissue (SIS) or other sources; Furthermore, the ECM protective solution of the small intestinal submucosal layer or other sources protects the membrane of the ECM in the small intestinal tissue or other sources. Before pretreatment or decellularization, the protective solution can protect the cell membrane and the lysosomal membrane (inner and outer double membranes) from rupture; so as to further protect various cell growth factors (such as bFGF and / or Wnt) and other functional proteins in the extracellular matrix from degradation and destruction by lysosomal proteases, etc.

[0120] Furthermore, the protective solution of the present invention is not only a protective solution for cell membranes and lysosomal membranes, but also essentially an isotonic protective solution for the extracellular matrix with high sodium and low potassium. It is completely different from the antioxidant protective solution in the prior art and does not belong to the same inventive concept.

[0121] Furthermore, the extracellular matrix protective solution disclosed in the present invention comprises at least the following two components: an isotonic solution and a lysosome stabilizer.

[0122] The first type of ingredients: isotonic solution, which may also include buffers such as lactic acid-sodium lactate, citric acid-sodium citrate, succinic acid-sodium succinate, etc.; Furthermore, in the F2 solution, the sodium ion concentration is 145mmol / L+ / -10%, and the potassium ion concentration is 4mmol / L+ / -10%; the pH value is adjusted to 7.2--8.2, which is weakly alkaline, which is basically consistent with the pH value of small intestinal fluid 7.6, both of which are weakly alkaline and stored at low temperature.

[0123] The second category of ingredients: lysosome stabilizers, including glucocorticoids (hydrocortisone), prostaglandins (PGI, PGE and tissue protease inhibitors, scopolamine can inhibit Ca2+ influx. (Note: Promoting the stability of lysosomal membranes in tissue cells has been considered to be the mechanism of anti-inflammatory effects of corticosteroids; cortisone is a 21-carbon steroid hormone, which is one of the main hormones released by the adrenal glands in stress response; it can control and regulate the release of lysosomal enzymes into the cell fluid).

[0124] Furthermore, it may specifically include cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone and the like.

[0125] Furthermore, the aforementioned lysosomal stabilizer may also preferably be a nonsteroidal anti-inflammatory drug (NSAID), including one or a combination of sodium or potassium salts of salicylates, arylpropionic acids, anilines, indoles, arylacetic acids and their derivatives, wherein the NSAID is selected from aspirin, diclofenac, loxoprofen, ibuprofen, acetaminophen, celecoxib, etodolac, pranoprofen, flurbiprofen axetil, lornoxicam, thiamide, tramadol and zaltoprofen, and one or a combination of pharmacologically acceptable sodium or potassium salts of these ingredients.

[0126] Furthermore, it is preferred that the lysosome stabilizer is salicylic acid; A more preferred lysosome stabilizer is aspirin; Furthermore, the concentration of aspirin in each liter of the protective solution is 0.1 to 1 gram.

[0127] In addition, in the present invention F2 The following ingredients may be further added to the liquid as needed, for example: preservatives (ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, chlorphenesin, phenoxyethanol, etc.); anti-inflammatory agents (for example, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, cypress phenol, zinc oxide, allantoin, etc.); Of course, on the basis of the present invention, an antioxidant may also be added to the F2 protective solution.

[0128] Furthermore, it may also specifically include plant-derived antioxidants, animal-derived antioxidants and / or antioxidant free radicals such as allopurinol.

[0129] Furthermore, it can also specifically include N-acetylcysteine ​​(NAC). NAC is an N-acetylated derivative of cysteine. NAC has good antioxidant and anti-inflammatory effects and can generate glutathione (GSH) in the body. The presence of SH can remove H20OH and HOCl. And NAC is easily deacetylated to cysteine ​​to form glutathione; in addition, NAC can also reduce pro-inflammatory mediators; according to the existing medical uses of NAC, its powerful expectorant effect has three mechanisms: one is to destroy the disulfide bonds in sputum mucin and reduce the viscosity of sputum; the second is to enhance the cleaning effect of respiratory cilia on the airway; the third is to increase alveolar surfactant. NAC pharmacological benefits, traditionally NAC is a commonly used expectorant, suitable for respiratory diseases such as bronchitis and bronchiectasis accompanied by high secretion of sputum. At present, there is a new medical understanding of it, including its efficacy and pharmacological mechanism, expanded pharmacological effects and new dosage forms, such as oral preparations (tablets, granules) and nebulized liquid medicine. More importantly for the present invention, NAC also has antioxidant and anti-inflammatory effects, can generate glutathione (GSH); NAC can remove oxygen free radicals. In addition, some scholars have found that NAC has a synergistic antibacterial effect and can enhance the antibacterial activity of piperacillin and ciprofloxacin against pathogens. NAC has a synergistic antibacterial mechanism, including: 1. NAC can reduce the number of glycolipids in epithelial cells and inhibit the adhesion of pathogens; 2. NAC can destroy the biofilm of bacteria, enhance the bactericidal effect of antibacterial drugs, and reduce bacterial resistance. It can be seen that in addition to the expectorant effect, NAC also has antioxidant, anti-inflammatory, and synergistic antibacterial pharmacological effects; the use is further broadened. This is an additional invention point added on the basis of the F2 liquid of the present invention, and it is also the main reason for selecting NAC as an important antioxidant, which can be attributed to the second invention point outside the present invention.

[0130] It is further preferred that the NAC concentration of N-acetylcysteine ​​is 0.2-1 g / L, and more preferably 0.6 g / L.

[0131] On the other hand, antioxidant adjuvants may be added on or in addition to the original basis of the present invention, such as phosphoric acid, citric acid, ascorbic acid, succinic acid, fumaric acid, cephalin, hexametaphosphate (ester), etc.; as well as antibiotics (such as ciprofloxacin and / or streptomycin and / or chlortetracycline) and anti-inflammatory drugs (such as tranexamic acid, thiotaurine, hypotaurine, etc.), etc.

[0132] The inventor would like to particularly or emphatically explain that the original technical concept of the present invention is indeed partially based on or borrowed from the ideas of organ transplant preservation fluid (such as University of Wisconsin UW fluid, HTK fluid, etc.), and there are certain similarities or similarities; but in terms of specific technical details or deep working principles, the two are essentially different and significantly different.

[0133] Common organ transplant preservation fluids are as follows: UW fluid is characterized by high potassium (125mmol / l) and low sodium (29mmol / l), which is similar to the composition of intracellular fluid in tissues. High concentrations of K+ ions will inhibit the outflow of K+ from the cell along the concentration gradient; it also contains hydroxyethyl starch (HES), a colloidal carrier that can retain fluid in the intravascular space to prevent extracellular swelling; lactobionic acid and kapok, both of which are membrane-impermeable substances, can reduce tissue edema; glutathione, as a reducing agent, can remove oxygen free radicals and maintain the integrity of cell membranes; adenosine is a substrate for the synthesis of high-energy phosphate (adenosine triphosphate, ATP), providing energy support for organ metabolism; allopurinol is a xanthine oxidase inhibitor that has a protective effect in ischemia; UW fluid is mainly used for organ preservation before liver, pancreas and kidney transplantation; it is used to preserve the vitality and / or activity of tissue cells.

[0134] HTK solution is mainly composed of histidine, with added tryptophan and ketoglutaric acid to promote ATP production. Low potassium reduces calcium overload, and added glycine, alanine, and LK614 (iron chelator) can reduce damage from oxygen free radicals.

[0135] Celsior solution, with lactobionic acid as the osmotic pressure component and histidine as the buffer system, is a high-sodium, low-potassium extracellular fluid-type protective solution. Low potassium limits the opening of voltage-dependent calcium channels, restricts the entry of calcium ions into cells, and avoids smooth muscle cell membrane depolarization. It is suitable for myocardial smooth muscle and is used as a preservation solution during heart transplantation.

[0136] HCA solution is a hypertonic citrate purine solution with high potassium and low sodium (intracellular fluid type). It uses histidine as a buffer system and dextran to maintain osmotic pressure. It is mainly used as a preservation solution during kidney transplantation.

[0137] First, UW fluids are mainly used for preserving organs before transplantation; however, the F2 fluid of the present invention does not need to preserve the vitality and / or activity of tissue cells; the F2 fluid is mainly used to maintain the osmotic pressure of the extracellular fluid and prevent various types of rupture of the cell membrane.

[0138] Second aspect: F2 The fluid does not need to provide energy levels similar to those in the organ preservation fluid, that is, it does not need to supplement sugars (such as glucose, lactose, sucrose, etc.) to maintain the vitality and survival of cells in tissues / organs; in other words, F2 The main purpose of the fluid is to maintain the stability of the cell membrane and prevent it from rupturing. Further, it is to prevent the rupture of the cell lysosomal membrane and the release of proteases from the cells, which will cause rapid degradation and serious damage to the extracellular matrix.

[0139] The third aspect: the present invention will F2The osmotic pressure molar concentration of the protective solution is set at 280-300mOsmol / kg, which is a low-temperature osmotic pressure and slightly lower than that of the organ transplant preservation solution; this is mainly because F2 The temperature of the protective solution is always maintained at 2-8°C; at the same time, the pH is adjusted to be slightly alkaline, about 7.2-8.2, preferably 7.6; this is mainly due to the fact that the original tissue, i.e. the small intestinal tissue, especially the digestive fluid in the small intestinal mucosa is weakly alkaline and this pH can also effectively inhibit the protease activity of lysosomes (when a small number of cells or lysosomal membranes are occasionally ruptured).

[0140] Fourth aspect: UW solution (University of Wisconsin solution) refers to the preservation solution used to preserve donor organs in organ transplantation; it mainly contains lactobionic acid, which is the main non-permeable anion with a relatively large molecular weight, which can reduce the swelling of cells during refrigeration, and contains cotton sugar, hydroxyethyl starch and adenosine.

[0141] It mainly uses hydroxyethyl starch (50g / L) to maintain (colloid) osmotic pressure. Hydroxyethyl starch (Heta starch) is a synthetic colloid solution widely used in clinical practice, and it is also a natural polysaccharide. Hydroxyethyl starch (HES) is a high molecular weight complex formed by hydroxyethylation of the glucose ring of branched starch in corn or potatoes. Natural starch cannot be used as a plasma substitute because natural starch is unstable and easily hydrolyzed by endogenous amylase. After starch is hydroxyethylated, its decomposition and elimination in the blood can be delayed, significantly prolonging its residence time in the blood vessels. HES has the effect of expanding blood volume; however, the present invention does not use hydroxyethyl starch products, that is, it does not contain colloid solutions.

[0142] In the present invention, the target tissue is immersed in a protective solution (F2 solution) before decellularization, and then double low concentration decellularization is used to prepare ECM.

[0143] Furthermore, the preparation method of the hair growth promoting ECM microparticles is to use ECM from the submucosa of porcine small intestine or other sources as raw materials, and soak them in low-temperature F2 liquid to protect the active ingredients in the extracellular matrix from being degraded and destroyed.

[0144] The remaining process steps, such as inactivation, decellularization, freeze drying, granulation, packaging, sterilization and other processing steps, are basically the same as the conventional method. The difference that needs to be pointed out is that the decellularization reagent and concentration are different. The present invention uses a mixed solution of 0.05% trypsin and 0.05% EDTA to decellularize (i.e., a dual low concentration and gentle method) to prepare a decellularized matrix.

[0145] The mechanism of action of the present invention is as follows: the use of F2 protective solution can, on the one hand, prevent the pathological or physiological lysis of the cell membrane in the target tissue caused by various external non-physiological environmental factors after leaving the living tissue, and strive to maintain the integrity of the cell membrane structure.

[0146] Furthermore, F2 solution can also prevent the lysosomes in the cell from being free and independent after the cell membrane is lysed, causing the lysosomes to rupture easily and then releasing various proteases in the lysosomes.

[0147] F2 protective solution can prevent lysosome rupture, thereby preventing / reducing the release of various proteases and preventing them from degrading ECM, including WNT pathway active factors (WNT ligands and / or agonists, including wnt2b); thereby maintaining the activity of various active ingredients and cell growth factors in ECM powder to the greatest extent; This type of ECM can better promote the activation or regeneration of various stem cells; for example, it can promote the activation and regeneration of hair follicle stem cells, thereby achieving the medical purpose of hair care and hair regeneration.

[0148] Compared with the prior art, the present invention has the following beneficial effects: The protective solution provided by the present invention has a simple formula, can protect both cell membranes and lysosome membranes, and can inhibit the activity of lysosomal proteases, thereby achieving the effects of double membrane multiple protection and increasing the content of Wnt2b.

[0149] The raw materials of the protective liquid provided by the present invention are easy to obtain, are all commercial products, and are cheap.

[0150] The decellularized matrix preparation prepared by the present invention is just a low-temperature immersion protection treatment added before the decellularization treatment of the original in vitro tissue; it is simple and convenient, easy to master, easy to operate, and highly feasible.

[0151] The ECM product provided by the present invention, especially the prepared ECM microparticles, has the advantages of being able to fully remove the immunogenic components in SIS and having good biocompatibility, while being able to retain the natural three-dimensional structure of SIS and the active ingredients, including collagen and various growth factors, to the greatest extent.

[0152] The ECM preparation prepared by the present invention can be applied to the head or scalp to prevent hair loss and or induce hair regeneration.

[0153] The hair growth preparation of the present invention is richer in various growth factors; on the one hand, it promotes scalp blood circulation and increases blood supply to the hair papilla; on the other hand, it can improve hair follicle nutrition, slow down the process of hair follicle miniaturization, increase the number of hair follicles, and promote hair growth, thereby achieving the dual effects of preventing hair loss and maintaining hair growth.

[0154] Compared with several types of hair growth agents in clinical practice (minoxidil, finasteride, including some plant essences and plant extract hair growth factors, which have been used for many years, but still have some side effects, the hair growth effect of their use is short-lived and will gradually disappear with the passage of time; compared with single-ingredient products that cannot or are difficult to induce the regeneration of hair HFSC / secretion of hair functional components such as collagen, which will completely degrade and disappear after a period of use; the ECM preparation of the present invention has medium- and long-term efficacy. Consumers only need to inject a small amount several times, and the hair is grown in the original part (no complicated operations such as scalp transplantation are involved); thereby reducing unnecessary troubles and economic burdens on consumers; it can effectively and permanently induce the regeneration of autologous hair HFSC, promote DPC activation and growth and development; the effect is stable and lasting, and only a limited number of re-injections are required.

[0155] The term "comprising" (and related terms such as "including" or "having" or "including") in this patent includes those embodiments, such as any composition of matter, method or process "consists of the described features" or "consists essentially of the described features". Isotonic agents and isotonic solutions have the same functions in essence or in specific practical applications; isotonic agents are prepared into solids (such as powders, etc.) just for the convenience of transportation or packaging; although such isotonic agents are solids (such as powders), deionized water or double distilled water can usually be added to them during actual use or before use to quickly turn them into liquid isotonic solutions. Based on this principle, isotonic agents and isotonic solutions are essentially the same, or at least equivalent.

[0156] The applicant needs to explain that in this patent, when referring to numbers or numerical ranges, the use of data terms means that the numbers or numerical ranges referred to may have experimental changes or variability ranges or statistical experimental errors, and the point values ​​of the data may also be regarded as approximate values, that is, the numbers or numerical ranges may vary, and the variation is usually -10% to +10% of the numbers or numerical ranges.

[0157] In addition, the numerical values ​​in the test results, unless otherwise specified, are usually the average values ​​of the test results of at least three samples or examples.

[0158] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar to or equivalent to the methods, equipment, and materials in the embodiments of the present invention can also be used to realize the present invention.

[0159] The following is an explanation of the embodiments of the present invention by specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0160] Before describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing the specific embodiments rather than for limiting the protection scope of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0161] Specific embodiments are used to describe the principles and schemes of the present invention; it should be understood that they are only for illustration and convenience of understanding the ideas of the present invention, but are not limited to this; the embodiments do not limit the scope of the present invention in any way. In the following embodiments, various processes and methods that are not described in detail are common knowledge methods used in the field; they may also include omissions of conventional techniques and methods used in the field of medical biomaterials. DETAILED DESCRIPTION

[0162] Example 1 Preparation of F2 protective solution and suspending solution (one of the main points of the present invention) 1) F2 protective solution, including two types of components, namely isotonic agent and lysosome stabilizer.

[0163] The first step: isotonic solution, in the present invention, a commercial isotonic whole intestinal lavage solution (from Shenzhen Wanhe Pharmaceutical Co., Ltd., trade name, Heshuang; common name: compound polyethylene glycol electrolyte powder (II), that is, polyethylene glycol 4000, 60 grams, anhydrous sodium sulfate 5.68 grams, sodium chloride 1.46 grams, potassium chloride 0.75 grams, sodium bicarbonate 1.68 grams; add water to make liters; that is, a whole intestinal isotonic solution with 125mmol / L sodium ion, 10mmol / L potassium ion, 20mmol / L bicarbonate ion, 40mmol / L sulfate ion, and 35mmol / L chloride ion is prepared; after preparation, fully mix; and adjust the pH value to 7.4.

[0164] Step 2: Add a small amount of lysosome stabilizer, i.e. NSAID drug. Specifically, choose aspirin, with a dosage of 0.2 g (mass concentration ratio) of aspirin per liter of protective solution, to the solution (isotonic whole bowel lavage solution) prepared in the first step, stir well to mix, and store at 4 degrees Celsius for use.

[0165] 2) Prepare the suspension solution with a glycerol concentration of 0.5% (W / V) and a sodium hyaluronate concentration of 0.2% (W / V). The molecular weight of hyaluronic acid is 1.2-1.5 million Daltons. Use phosphate buffer to adjust the pH of the suspension solution to 7.10 and the osmotic pressure to 330 mOsmol / L. Store at room temperature until use.

[0166] Example 2 Preparation of conventional ECM microparticles (ie, control group ECM, not soaked in protective solution).

[0167] (1) Inactivation: Take the submucosa of the small intestine of pigs and use a mixed aqueous solution of 1% peracetic acid and 20% ethanol to soak at room temperature for 60 minutes under ultrasonic conditions for inactivation.

[0168] (2) Decellularization: The inactivated SIS was first washed with water under ultrasonic conditions, packaged into small packages, quickly frozen overnight, thawed the next day, and the "washing-packaging-freezing-thawing" freeze-thaw steps were repeated; a mixed aqueous solution containing 0.05% trypsin and 0.05% EDTA was used for ultrasonic treatment at 36±2°C, and then ultrasonic washing was performed with PBS; treatment was performed in a 15% sodium chloride hypertonic solution at 36±2°C, and then washed with water; treatment was performed in a 25 mM NaOH aqueous solution at 36±2°C under ultrasonic conditions; then ultrasonic washing was performed with water until neutral; DNase treatment was added to remove residual DNA.

[0169] (3) Freeze-drying: Stack the SIS sheets, fix them on a mold, and freeze-dry them.

[0170] (4) Granulation: The freeze-dried SIS was cut into small pieces, frozen with liquid nitrogen and then crushed, and sieved using stainless steel sieves of different mesh sizes to obtain ECM particles with a particle size ranging from 50 μm to 400 μm.

[0171] (5) Packaging: Weigh the ECM microparticles and dispense them into COP syringes in amounts of, for example, 100 mg, 200 mg or 300 mg, and seal them with stoppers.

[0172] (6) Sterilization: Select cobalt 60 sterilization method with a radiation dose of 25kGy-30kGy.

[0173] Example 3 Preparation of ECM Microparticles (i.e. Preparation of Highly Active ECM by Soaking in Low-Temperature F2 Protective Solution) The process steps are basically the same as those of Example 2, except that before or during pretreatment, that is, after the target tissue leaves the living body (animal living tissue is usually between 35-39°C, more commonly 36-38°C), the F2 protective solution prepared in Example 1 is immediately used for the target tissue (may also include the target organ), the porcine small intestinal submucosa (SIS), (Note: Of course, in this technical field, technicians with IPC main classification number A61L27 can also reasonably and logically deduce that the protective solution of the present invention is also applicable to other equivalent or similar tissues or organs to be decellularized); the protective solution of the present invention is used to soak the decellularized tissue at low temperature (2-8°C) for at least one hour to protect the active ingredients and / or various cell growth factors in the extracellular matrix from being easily degraded and destroyed.

[0174] The remaining steps (such as inactivation, decellularization, lyophilization, granulation, packaging and sterilization) are exactly the same as those in Example 2.

[0175] Example 4 Detection of active ingredients in ECM (bFGF, Wnt2b) The relevant components in the ECM prepared in Example 2 and Example 3 were detected using an ELISA kit.

[0176] It should be noted that the ECM samples prepared in the two examples were pre-treated before testing using urea heparin solution (non-enzymatic degradation method) and operated according to the literature method. For the detection of relevant active ingredients (growth factors) in the ECM prepared in Examples 2-3, in the present invention, the detection of ECM active ingredients mainly targets typical regenerative growth factors, which are also cytokines closely related to stem cell regeneration, such as hair growth, namely basic fibroblast growth factor (bFGF), using ELISA method; the test results are: The bFGF content in the ECM of Example 2 was 12.76+ / -0.71 pg / mg, and the Wnt2b content was 48.79+ / -0.18 pg / mg; The content of bFGF in the ECM of Example 3 (immersed in the F2 protective solution of the present invention) was 20.18+ / -0.51 pg / mg; the content of Wnt2b was 76.11+ / -0.48 pg / mg.

[0177] Summary: After the target tissue leaves the living body (36-38°C), it is immersed in a cryoprotective solution and then subjected to subsequent treatments including decellularization. This type of immersion treatment with a protective solution can significantly increase the content of various functional and / or growth factors and proteins in the decellularized matrix (ECM).

[0178] According to the principles of the present invention and the actual test results, it can be logically and scientifically deduced that the ECM prepared by the present invention (immersed in preservation solution) can better retain the regeneration-related active ingredients in the ECM (such as Wnt protein, bFGF, etc.). This can explain that the ECM prepared by the present invention has a better natural ECM structure and can better combine with the integrins on the surface of target cells or stem cells, promote the activation of stem cell signals or tissue regeneration in the recipient site, stimulate the activity of major stem cells, and start the repair and regeneration of various target tissues; in this patent, in particular, the stimulation includes hair regeneration-related tissues, such as promoting the activation of DPC and HFSC stem cells; and provides a rich and efficient material basis and a strong microenvironment (Niche) guarantee for hair regeneration and regrowth.

[0179] Example 5 Experiment on the hair growth promoting effect of two types of ECM mixed preparations The ECM microparticles prepared in Example 2 and Example 3 were fully mixed with the suspension prepared in Example 1 to prepare injectable suspensions, named A3 and A2. Animal experiments were then conducted, and the preliminary results of the animal hair growth promotion test results showed that the hair growth promotion effect of the animal experiment of the A3 group was significantly better than that of the A2 group.

Claims

1. A medical protective solution for mammalian living tissue, which is used for soaking under low temperature conditions before decellularization, characterized in that: The protective solution includes an isotonic solution and a lysosome stabilizer; the protective solution is used to prevent the active ingredients and / or various cell growth factors in the decellularized matrix (ECM) from being degraded or destroyed; the low temperature condition is 2--8°C; the isotonic solution is an isotonic whole intestinal solution with a pH value of 7.2-8.2; and the lysosome stabilizer is aspirin.

2. A medical protective solution according to claim 1, characterized in that: The isotonic whole enteral solution in the medical protective solution is a commercial isotonic whole enteral solution; per liter of solution, there are 60 grams of polyethylene glycol, 5.68 grams of anhydrous sodium sulfate, 1.46 grams of sodium chloride, 0.74 grams of potassium chloride, 1.68 grams of sodium bicarbonate, and the remainder is deionized water; the polyethylene glycol is pharmaceutical grade PEG4000 of the Pharmacopoeia 2020 edition.

3. A medical protective solution according to claim 1, characterized in that: The lysosome stabilizer in the medical protective solution is aspirin, and the final concentration in each liter of protective solution is 0.1-1 gram.

4. A medical decellularized matrix, characterized in that: The decellularized matrix, after its source tissue leaves the living tissue and before the decellularization treatment, is immersed in the medical protective solution of claim 1 to significantly increase the content of active ingredients in the ECM.

5. The decellularized matrix according to claim 4, characterized in that The decellularized matrix is ​​the submucosal layer of the pig small intestine, and contains one or a combination of Wnt2b and bFGF components.

6. An injectable medical acellular matrix preparation, characterized in that: The preparation consists of a decellularized matrix and a suspending agent, which are packaged independently, sterilized separately, and mixed before use; the decellularized matrix is ​​microparticles with a diameter of 50 μm-400 μm; the suspending agent contains glycerol, sodium hyaluronate, and phosphate buffer components; the source tissue of the decellularized matrix is ​​first soaked in the medical protective solution of claim 1 at low temperature after leaving the living body, and then a mixed solution of 0.05% trypsin and 0.05% EDTA is used to gently remove cells.

7. A method for preparing an injectable acellular matrix preparation according to claim 6, characterized in that: It includes the following three steps: Step 1: Preparation of decellularized matrix microparticles 1). Soaking and pretreatment in protective solution: Soak the target tissue away from the living body in the protective solution of claim 1 under low temperature conditions to protect the active ingredients and / or various cell growth factors in the extracellular matrix from degradation and destruction; Pretreatment refers to the removal of impurities and fat; 2). Inactivation, decellularization, DNA removal, and freeze-drying; 3) Preparation of microparticles: freeze the lyophilized ECM with liquid nitrogen, crush it, and sieve it to obtain microparticles with a diameter of 50 μm-400 μm; package and sterilize; Step 2: Preparation of suspending agent a). Prepare a liquid containing glycerol, sodium hyaluronate and phosphate buffer, wherein the concentration of glycerol is 0.5%; The concentration of sodium hyaluronate is 0.2%, and the molecular weight is between 1 million and 1.6 million Daltons; phosphate buffer is used to adjust the pH value and osmotic pressure of the suspending agent; b). Pack the suspending agent into containers that match its sterilization method, seal them, and sterilize them with moist heat; Step 3: The decellularized matrix microparticles and the suspending agent are packaged and sterilized separately, and are fully mixed using a three-way pipe before use; the mixture is prepared into an injectable decellularized matrix microparticle preparation having good tissue repair and / or regeneration promoting functions.

8. Use of a decellularized matrix according to any one of claims 4 to 6 in preparing medical repair materials or medical devices, characterized in that: It is used to repair tissue and / or organ defects; the tissue and organ are defects in head appendages, hair, ear, face, periosteum, nerves, dura mater, spinal dura mater, tendons, ligaments, urinary tract, bladder, ureter, inguinal hernia, hiatal hernia, abdominal wall hernia, oral soft tissue, joints and meniscus.

9. Use of a decellularized matrix according to any one of claims 4 to 6 in preparing medical repair materials or medical devices, characterized in that: The invention is applied to the head to prevent various types of hair loss in adults and help promote the growth of hair follicles. The head application method is to apply it externally to the scalp at the hair loss site, use microneedles, nanochips and ultrasonic ion methods to assist in the introduction, subcutaneous injection of the scalp, microneedle patches on the scalp at the hair loss site, or use it as an in vitro culture medium for hair follicle-related tissues and related cells to activate the growth of hair follicle stem cells. The hair follicle stem cells refer to one or a combination of DPCs and HFSCs of the hair follicles on the head.