A decellularized matrix, preparation method thereof, and use thereof in promoting hair growth

Abstract

The present invention relates to a decellularized matrix, its preparation method, and its medical use for promoting hair growth, belonging to the field of hair growth and beauty treatments, such as scalp care. The source tissue of the decellularized matrix, after leaving the living body, is first immersed in a protective solution containing a lysosome stabilizer and an isotonic agent at low temperature to prevent the rupture of cell membranes and lysosomes, and to inhibit the degradation and destruction of active components in the extracellular matrix. A dual-low concentration method is then used for gentle decellularization. The resulting decellularized matrix has an intact ECM structure, a higher content of active factors (such as bFGF and Wnt2b), and a stronger ability to induce tissue regeneration. The decellularized matrix can be micronized and mixed with a suspending fluid to form an injectable suspension, or microneedled, and applied to the head in various ways to promote hair regeneration.

Description

Technical Field

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

[0002] Hair loss can be broadly categorized as anagen effluvium and telogen effluvium. The causes of anagen effluvium vary widely; it affects approximately half of men and over a quarter of women worldwide. Young and middle-aged individuals have become the primary victims of hair loss. Common types of hair loss include androgenetic alopecia (AGA), alopecia areata, trichotillomania, telogen effluvium, and senile alopecia. Androgenetic alopecia, also known as male pattern baldness, is the complete or partial loss of all or most of the hair on the head. AGA patients experience smaller hair follicles and a decrease in hair volume in the affected scalp area. The incidence of AGA increases with age. Despite its high prevalence, effective treatments are relatively rare. While the specific mechanisms of hair growth disorders remain somewhat unclear, scientific evidence suggests that hair loss is caused by a disruption or disruption 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 consists of the following 7 parts:

[0004] Bulge: The upper part of the hair follicle, which contains hair follicle stem cells (HFSC) and melanocyte stem cells, which are crucial for hair color and regeneration.

[0005] Matrix: The area beneath the bulge of the hair follicle where hair growth occurs.

[0006] Outer Root Sheath (ORS): The outer structure of the hair follicle, composed of multiple layers of cells, that provides protection for the hair.

[0007] Inner Root Sheath (IRS): A multi-layered structure surrounding the hair follicle that influences its shape and direction.

[0008] Dermal Papilla (DP): A cluster of cells located at the base of the hair follicle that regulates hair growth.

[0009] Sebaceous glands: connected to hair follicles, secrete oil to lubricate and protect the skin and hair.

[0010] Arrector Pili Muscle: A tiny muscle attached to the hair follicle that, when contracted, causes the hair to stand upright.

[0011] The main components of hair follicles include collagen (the main structural protein of hair follicles, providing support and strength); elastin (which makes hair follicles elastic, allowing a certain degree of stretching and rebound); proteoglycans (which help maintain the moisture and structure of the tissue); and fibronectin (which promotes cell adhesion and migration, and is important for hair growth and wound healing). They also contain their 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.

[0012] The functions of hair follicles include the cyclical growth of hair through the proliferation and differentiation of their matrix cells; the cyclical replacement of hair follicles, which undergo a cycle of growth (anagen), regression (catagen), and rest (telogen); and the provision of new cells by hair follicle stem cells during the regeneration phase of the hair cycle, maintaining hair growth. Hair follicles also have sensory functions (hair follicles are connected to nerve endings and participate in sensory transmission); temperature regulation (the contraction and relaxation of the arrector pili muscles help regulate body temperature); protective functions (hair provides a physical barrier to the skin, protecting it from external damage); and cosmetic functions. Hair (hair) has important aesthetic value and significant social functions.

[0013] 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. There are three main stages of normal hair follicle replacement and how they affect hair growth:

[0014] 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 the hair can reach.

[0015] Catagen: accounts for 1-2% and lasts for 2-4 weeks. This is the late anagen phase of hair growth, a transitional stage that lasts for several weeks. Hair follicles begin to shrink and hair growth stops. During the catagen phase, the structure of the hair follicle changes, and the hair papilla gradually separates from the hair follicle matrix. At the end of the catagen phase, hair matrix cells stop producing hair, and the hair becomes a specific shape, namely rod-shaped. The end of this phase marks the beginning of the next phase of the hair growth cycle.

[0016] Telogen phase: accounts for 10-15%, lasting 2-3 months. The telogen phase 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 phase ends, the hair follicles will re-enter the growth phase, and new hair will begin to grow to replace the fallen hair.

[0017] The effects of hair follicle cyclical replacement on hair growth include:

[0018] Hair density: The shedding of hair during the resting phase and the growth of new hair during the growth phase together determine the density of hair.

[0019] Hair Length: The duration of the growth phase, which affects the maximum length the hair can grow to.

[0020] Hair color: As we age, the activity of melanocytes in hair follicles may decrease, causing hair color to lighten.

[0021] Hair quality: During the periodic replacement process, the health of hair follicles affects the thickness and elasticity of hair.

[0022] Hair growth pattern: The cyclic replacement pattern of hair follicles in different individuals may lead to different hair growth patterns, such as sparse or thick hair.

[0023] Currently, 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 treatment. The principle of its prevention and treatment of AGA is the reactivation of hair follicle stem cells, thereby improving hair follicle growth and hair regeneration. There are two main types of sources for 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 generation of new hair follicles. Importantly, hair follicle stem cells (HFSCs) periodically switch between active and inactive phases, thereby maintaining a stable stem cell population in the hair follicle; however, this ability weakens with age.

[0024] Hair follicles contain a diverse array of cellular resources, including melanocytes, epithelial cells, and stem cells of diverse developmental origins, which continuously renew and regulate hair growth and homeostasis. There are two main types of stem cells in the hair follicle: HFSCs, located within the outer root sheath (ORS) at the arrector pili muscle attachment area and the proximal region of the isthmus, both areas known as the "bulge." The second type, dermal papilla cells (DPCs), control hair induction and growth and participate in the formation of new hair follicles. AGA is a non-scarring form of hair loss, in which HFSCs are intact, but only progenitor cells are damaged. This fact suggests that androgenic alopecia may be reversible. DPCs in the scalp affected by androgenic alopecia have reduced replicative potential and have been found to be prone to loss of shape, size, and characteristic markers. In recent years, some clinics have implemented simple stem cell culture and transplantation. Due to the regenerative potential of stem cells, stem cell therapy has attracted considerable attention among physicians and patients with androgenic alopecia. While studies on the efficacy of stem cell therapy in AGA have been published, further investigation is needed to determine its efficacy.

[0025] 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); it is reported that the relationship between the epithelium and mesenchyme is a key factor in hair follicle formation.

[0026] Hair transplantation is one of the effective methods to address baldness, in which hair roots are removed from donor areas that still have normal function (such as along the back and sides of the head) and then transplanted to areas of the scalp where the hair roots have died; surgical procedures such as scalp plastic surgery, including scalp reduction surgery, scalp flap surgery and tissue extension or tissue expansion.

[0027] Drug therapies, including minoxidil and finasteride. Finasteride and minoxidil are the only two FDA-approved medications for hair regrowth. The main drawback of these medications is that they are largely ineffective after discontinuation of treatment, making them temporary, not permanent, solutions.

[0028] Currently available alternative methods include, for example, hair transplantation and scalp plastic surgery, as well as various pharmacological (or drug) therapies; although there has been a lot of research and development on hair growth methods, unfortunately, these methods still have defects and shortcomings such as being more cumbersome, inconvenient to use, and having unstable effects. At present, there is no effective method that can fundamentally prevent or treat hair loss.

[0029] On the other hand, various decellularization techniques, including physical, chemical, and biological methods, are being developed both domestically and internationally. For details on the reagents and methods used for decellularization, please refer to Table 1 in CN201910804397.2. The patent title is: "A Tissue Repair Material and Its Preparation Method." The extracellular matrix (ECM) material, after the removal of immunogenic substances from homologous or heterologous tissues, is also known as a decellularized product preparation. Some technicians also refer to it as a decellularized patch or tissue regeneration scaffold. Currently, this type of product (ECM) is widely used in tissue engineering and clinical regenerative medicine, such as for the repair and reconstruction of heart valves, blood vessels, nerves, tendons, bone, cartilage, skin, esophagus, trachea, abdominal wall, and many other tissues. Extensive research has demonstrated that using appropriate decellularization techniques can produce materials with minimal immunogenicity while preserving the various components of the original ECM and maintaining a well-structured ECM, enabling endogenous induction of in situ tissue regeneration, including stem cell regeneration and tissue repair. Existing research shows 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 research hotspot and focus. SIS has the advantages of easy access, a wide range of sources, no ethical issues, and a low risk of viral transmission. Its scalable production characteristics have earned it the favor of both technical leaders in the industry and experts in academia. For many years, our company's technical team has repeatedly researched and optimized the decellularization process using porcine small intestinal submucosa (SIS) as the primary raw material, and has also performed further deep processing. To this end, we have carried out the research and development, production, and application of multiple series of Class III medical devices.

[0030] Although acellular matrix, or sometimes also called extracellular matrix (ECM) preparations, have been used to prevent and treat various types of hair loss in adults, they also show 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:

[0031] 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.

[0032] 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.

[0033] 3. Cell adhesion: Active molecules on the ECM promote the adhesion and anchoring of hair follicle cells by binding to integrin receptors on the cell surface.

[0034] 4. Nutrient delivery: ECM helps in the delivery of nutrients and oxygen, providing the necessary nutrients and environment for hair growth.

[0035] 5. Cell migration and differentiation: Changes in the composition and structure of the ECM can guide the migration and differentiation of hair follicle stem cells and other cells, promoting the formation of new hair.

[0036] 6. Immune regulation: ECM can regulate local immune responses, affect the inflammatory state of hair follicles, and thus affect hair growth.

[0037] 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.

[0038] 8. Wound healing and regeneration: After skin injury, the repair and regeneration of ECM are crucial for the recovery of hair follicles and hair growth.

[0039] 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 enters 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.

[0040] The ECM plays a multifaceted role in promoting hair growth, yet current treatments are unsatisfactory. One key reason for this may be that the entire acellular matrix preparation process, including timely and proper pretreatment of the target tissue after ex vivo removal (referred to as S1), and the varying degrees of damage to the ECM's natural three-dimensional structure during different decellularization processes (sometimes involving harsh decellularization conditions, high reagent concentrations, prolonged periods, and repeated treatments), can also have varying effects on the bioactive components and growth factors naturally present in the ECM, leading to significant loss or destruction of their content, reduced tissue regeneration activity, or even complete loss. This, in turn, directly impacts the ECM's efficacy in promoting hair growth. Furthermore, during specific use, the ECM preparations may have poor induction capacity for various hair stem cells or related hair regeneration cells, including a lack of sufficient support from the corresponding cell growth factors and cell pathway signals, resulting in an inability to effectively promote the regeneration of damaged or injured hair tissue. Ultimately, this results in a lack of significant hair regeneration benefits.

[0041] The main references are as follows

[0042] Non-patent articles

[0043] "New progress in research on the role of the Wnt signaling pathway in the induction of hair follicle formation and growth by hair follicle papilla cells"

[0044] Chinese Journal of Cell Biology 2021, 43(7): 1550–1560

[0045] Author and unit: Yuan Mu, Department of Stem Cell and Regenerative Medicine, Daping Hospital, Army Medical University

[0046] "Research Progress on Stem Cell Therapy for Androgenic Alopecia"

[0047] Journal of China Pharmaceutical University 2023, 54(3): 372-379

[0048] Author and institution: Yan Wenjing, School of Life Sciences and Technology, China Pharmaceutical University

[0049] "Study on the mechanism of action of basic fibroblast growth factor in the treatment of chemotherapy-induced hair loss"

[0050] 2021 China Medical University Doctoral Dissertation, 82 pages

[0051] Author and institution: Qin Jie, First Affiliated Hospital of China Medical University

[0052] Wnt Ligands Secreted by Subepithelial Mesenchymal Cells Are Essentialfor the Survival of Intestinal Stem Cells and Gut Homeostasis.

[0053] Valenta et al., 2016, Cell Reports 15, 911–918

[0054] “MAP3K2-regulated intestinal stromal cells define a distinct stemcell niche”

[0055] 《Nature》volume 592, pages606–610 (2021)

[0056] Author and Affiliation: Su Bing, Shanghai Jiao Tong University School of Medicine and Shanghai Institute of Immunology

[0057] This article discovered that there is a new type of intestinal mesenchymal cell called MRISC at the bottom of intestinal stem cells; 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.

[0058] Existing patent technology literature:

[0059] US20140086867A1;

[0060] US20210299036A1;

[0061] CN201610511579.7;

[0062] Regarding the decellularization protective solution, patent CN201610511580.X (abbreviated as D4), titled "An Optimized Antioxidant Decellularization Protective Solution," a publicly available invention patent from Biodisel (Beijing) Biotechnology Co., Ltd. (inventors Shi Zhen and Shi Weiyun). This solution has a specific pH value, specific crystallinity and colloidal osmotic pressure, and appears as a light red liquid.

[0063] It is obvious that the main function of the protective solution of the invention is at least antioxidant. In addition to using DMEM cell culture medium powder, the formula also contains nine other ingredients; specifically, they 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.

[0064] First, DMEM cell culture medium powder is used in the protective medium. 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 B vitamins, vitamin C, vitamin D, etc.), minerals (such as trace elements and macroelements such as calcium, phosphorus, potassium, magnesium, and 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 solution), sodium pyruvate (as an auxiliary energy source for cells); and phenol red (a pH indicator to help monitor the pH of the culture medium).

[0065] DMEM mainly provides cells with necessary nutrients to support cell growth in vitro; it is actually unnecessary in the protective solution invented by D4 and seems to be redundant. It has actually deviated from the original purpose and meaning of the D4 invention; it is also far away from the theme of the invention, "antioxidant decellularized protective solution."

[0066] Secondly, because DMEM is a cell culture medium, its ingredients are inherently complex for the reasons stated above; this results in the invention's decellularized protective fluid being, in fact, a formula on top of a formula, with obvious dual complexity; it promotes cell growth in the first place, and then performs decellularization; this makes the ultimate purpose of applying the protective fluid somewhat contradictory; at least inconsistent, or even completely contrary.

[0067] Thirdly, the D4 protective solution has, on the one hand, a crystalloid osmotic pressure between 330-380 mOsm / kg H2O, derived from histidine hydrochloride; in Example 1, each liter of protective solution contains 2.87 grams of histidine hydrochloride; on the other hand, a colloidal osmotic pressure between 310-350 mOsm / kg H2O, derived from chondroitin sulfate, hydroxypropyl methylcellulose, and low-molecular-weight dextran. These three components themselves have significant fluctuations in their molecular structures and molecular weights.

[0068] Chondroitin sulfate sodium (CS) is an acidic mucopolysaccharide composed of D-glucuronic acid and 2-acetylamino-2-deoxy-sulfated D-galactose, with a molecular formula 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. Crucially, the molecular weight of chondroitin sulfate fluctuates widely, ranging from 10,000 to 50,000 Daltons. The properties of chondroitin sulfate also vary significantly depending on its source and extraction method.

[0069] Hydroxypropyl methylcellulose (HPMC), as listed in Part II of the 2005 edition of the Chinese Pharmacopoeia, has a methyl degree of substitution of 1.0 to 2.0, a hydroxypropyl degree of substitution of 0.1 to 0.34, and a relative molecular mass of generally 10,000 to 150,000. Therefore, the specific molecular weight of HPMC will vary significantly due to the presence of different degrees of hydroxypropyl and methyl substitution.

[0070] The molecular weight of low molecular weight dextran ranges from 25,000 to 45,000, resulting in large fluctuations in molecular weight.

[0071] And these three components that can produce colloid osmotic pressure Dosage, and also vary within a large range. For example, the lower and upper limits of HPMC dosage in D4 protective solution differ by 4 times (the lowest is 2 g / L, the highest is 8 g / L). 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 many multiple factors of variation for only these three components that can generate colloid osmotic pressure, which will directly affect the changes in the colloid osmotic pressure parameters in 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.

[0072] 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 not effectively consistent with the protective purpose that the protective solution is actually intended to achieve; it may even produce opposite or obviously adverse side effects.

[0073] Fifthly, the protective solution invented by D4 is actually very complex, with nearly twenty ingredients in fact. Due to 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 results.

[0074] Sixthly, the decellularization protective fluid 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, composed of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); these two ingredients alone are not cheap. For example, the market price of 20 ml of Thermo Fisher Scientific HEPES is more than 400 yuan, which directly leads to the high cost of using this antioxidant decellularization protective fluid.

[0075] Seventh, D4 uses an antioxidant decellularization protective solution throughout the entire process; however, in reality, the cell membrane is completely ruptured during the decellularization process, resulting in the D4 protective solution essentially being completely unable to play the protective role it claims. Summary of the Invention

[0076] This invention was developed by the company's senior American R&D personnel, who extensively reviewed numerous domestic and international literature on stem cells, hair development, and hair follicle stem cells, carefully read authoritative articles, and carefully studied the various levels of principles of hair growth and hair loss. They conducted in-depth research and understood the mechanism of action between hair loss-related factors / factors, and found the key molecules or pathways involved.

[0077] 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 prerequisite is to provide good tissue regeneration factors and external microenvironment, but this problem has not been truly solved.

[0078] Hair follicle stem cells are adult stem cells that sometimes remain dormant in the body. Only under specific conditions can they display a strong proliferation capacity, subsequently differentiating into the epidermis, hair follicles, and sebaceous glands, participating in the process of hair regeneration. Hair follicle stem cells reside within each hair follicle and rapidly activate and divide during a new hair growth cycle. Currently, there are reports that the use of acellular matrices can activate hair follicle stem cells in vitro or in vivo, thereby promoting hair growth and preventing hair loss in the cosmetic field.

[0079] 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 provided, or the composition is too complex, and there is no effective protection solution for ECM before decellularization, or the protection system for ECM is not comprehensive, especially to prevent protease degradation.

[0080] 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.

[0081] 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 long-lasting.

[0082] In order to solve the above technical problems and overcome the shortcomings of the prior art, the present invention discloses the following technical solutions.

[0083] 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 is used for soaking under low temperature conditions before decellularization treatment of the target tissue.

[0084] The protective solution, comprising at least two components of an isotonic solution and a lysosomal stabilizer, may be referred to as F2 solution;

[0085] In practice, F2 solution is essentially an isotonic solution containing at least a small amount of lysosomal stabilizer.

[0086] 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.

[0087] Furthermore, the aforementioned isotonic solution is an isotonic whole intestinal solution with a pH value of 7.2-8.2;

[0088] 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, and 1.68 grams of sodium bicarbonate per liter of protective solution, and the balance is deionized water or double-distilled water.

[0089] The polyethylene glycol is pharmaceutical grade PEG4000 specified in the 2020 edition of the Pharmacopoeia.

[0090] 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 root ion 20mmol / L, SO4 root ion 40mmol / L, Cl ion 35mmol / L, the osmotic pressure can also be adjusted to 280-320mOsmol / L.

[0091] 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.

[0092] In the present invention, the applicant has invented its (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).

[0093] Furthermore, the aforementioned lysosome stabilizer may be one or a combination of hydrocortisone, prednisone, prednisolone, methylprednisolone, and triamcinolone acetonide;

[0094] Furthermore, the aforementioned lysosome stabilizer is hydrocortisone.

[0095] Furthermore, the concentration of hydrocortisone in each liter of the protective solution is 1 to 10 mg.

[0096] Furthermore, the aforementioned lysosomal stabilizer may be more preferably 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, as well as one or a combination of pharmacologically acceptable sodium or potassium salts of these ingredients.

[0097] Furthermore, it is preferred that the lysosomal stabilizer is a salicylic acid;

[0098] More preferably, the lysosome stabilizer is aspirin;

[0099] Furthermore, the concentration of aspirin in each liter of the protective solution is 0.1 to 1 gram.

[0100] Specifically, in the embodiment of this patent, preferably, the lysosome stabilizer is aspirin with a concentration of 0.2 g / L (protective solution).

[0101] Furthermore, the target tissue is immersed in F2 protective solution at low temperature (2-8°C) for at least one hour.

[0102] 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.

[0103] The purpose of soaking ECM source tissue in protective liquid is mainly to reduce the degradation or destruction of effective components and natural structures in ECM after leaving living tissue by adverse external factors; thereby significantly increasing the content of various active ingredients that can activate stem cells in the final ECM preparation, thereby further enhancing the regenerative performance and vitality of ECM in promoting tissue and cell regeneration.

[0104] Furthermore, 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;

[0105] Furthermore, the ECM source tissue may be porcine small intestinal submucosa;

[0106] Furthermore, the pig is a commercial Duchang three-way hybrid commercial meat pig.

[0107] Furthermore, the pig is a commercial meat pig of Duchang-Changda three-way cross; weighing 30-100 kg; preferably a feeder pig, weighing 30-60 kg; and aged 80-120 days.

[0108] Furthermore, the acellular matrix contains one or a combination of Wnt2b and bFGF.

[0109] 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.

[0110] The third object of the present invention is to invent a method for preparing an injectable acellular matrix microparticle preparation.

[0111] The method for preparing the injectable acellular matrix microparticle preparation comprises the following three steps:

[0112] Step 1 Preparation of acellular matrix microparticles

[0113] Immersion in protective solution and pretreatment: The target tissue removed from the living body is immersed in the aforementioned protective solution (F2 solution) at low temperature (2-8 degrees Celsius) to protect the active components of the extracellular matrix and various cell growth factors from degradation and destruction; pretreatment, such as removing impurities and delipidation.

[0114] Inactivation, decellularization, DNA removal, and freeze-drying;

[0115] Preparation of microparticles: The freeze-dried ECM is frozen in liquid nitrogen and then crushed and sieved to obtain microparticles with a diameter of 50 μm-400 μm.

[0116] Aliquot and sterilize.

[0117] Step 2 Preparation of suspending agent

[0118] 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;

[0119] The suspension agent is divided into containers that match its sterilization method, sealed, and sterilized by moist heat.

[0120] 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 / or regeneration function.

[0121] Furthermore, the specific details include the following three steps:

[0122] Step 1 Preparation of acellular matrix microparticles

[0123] Pretreatment: Soak the fresh porcine small intestine or the submucosa of the porcine small intestine with the mucosal layer / muscular layer removed in F2 protective solution at low temperature (2-8°C) for at least one hour to protect the active ingredients in the extracellular matrix and various cell growth factors from degradation and destruction.

[0124] 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.

[0125] 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" freeze-thaw steps were repeated. The cells were treated with a mixed solution containing 0.05% trypsin and 0.05% EDTA at 36±2°C, and then ultrasonically washed with PBS; treated in a 1.5% sodium chloride hypertonic solution at 36±2°C, and then washed with water; treated in a 20mM NaOH aqueous solution at 36±2°C; washed with water until neutral; and treated with DNase to remove residual DNA.

[0126] Drying: Fix the decellularized SIS sheet on a mold and freeze-dry.

[0127] Microparticle preparation: The freeze-dried SIS sheets were cut into pieces, frozen in liquid nitrogen, and then pulverized. The particles were sieved using stainless steel sieves of varying mesh sizes to obtain decellularized matrix microparticles. The resulting decellularized matrix microparticles had a diameter ranging from 50 μm to 400 μm.

[0128] Packaging: Pack the acellular matrix microparticles into syringes and seal them with stoppers.

[0129] Sterilization: Sterilize the decellularized matrix particles pre-sealed in the syringe by selecting a sterilization method suitable for the decellularized matrix and its packaging, such as cobalt 60 or electron beam radiation sterilization, or ethylene oxide sterilization.

[0130] 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 million to 1.6 million Daltons.

[0131] 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.

[0132] The fourth object 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 tissues and organs 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.

[0133] Furthermore, the acellular matrix is used in the preparation of medical repair materials or medical devices for application to the head to prevent various types of hair loss in adults and to activate the growth of hair follicle stem cells; the hair follicle stem cells are either DPCs or HFSCs, or a combination thereof, derived from hair follicles on the head. The medical use of the acellular matrix is application to the head to prevent hair loss and promote hair follicle growth and hair regeneration.

[0134] Furthermore, the head application method includes external application on the scalp in the hair loss area, assisted introduction using microneedles, nanochips, ultrasonic ions, etc., subcutaneous injection of the scalp, microneedle patches on the scalp in the hair loss area, or as an in vitro culture agent for hair follicle-related tissues and related cells.

[0135] The head application method includes applying to the hair loss area, applying to the scalp externally, introducing into the hair loss area with the aid of microneedles, nanochips, ultrasonic ions, etc., injecting into the hair loss area, applying microneedle patches to the hair loss area, or using it as an in vitro culture medium for hair follicle-related tissues and related cells.

[0136] Furthermore, the administration method is to inject the mixture into the scalp of the hair loss area, and the mixture is composed of a mixture of acellular 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.

[0137] The hair growth-promoting ECM preparation provided by the present invention can be composed of ECM particles and a suspending liquid, 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 small-diameter needles (27G and 30G) for minimally invasive injection.

[0138] The ECM microparticles are prepared from ECM of porcine small intestinal submucosa or other sources through the steps of inactivation, decellularization, freeze drying, granulation, packaging, sterilization and the like.

[0139] Ideal tissue regeneration preparations should have good biocompatibility (safety), natural and long-lasting effects, and be easy to use (e.g., easy to inject, or flexible to apply to the head via non-invasive or minimally invasive methods such as microneedles).

[0140] 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:

[0141] What kind of decellularization process should be adopted to enable ECM to effectively remove immunogenic substances while ensuring that the ECM components and structure 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 into the body.

[0142] During the decellularization process, how can we minimize or reduce the loss or degradation of various active components in the extracellular matrix?

[0143] How can ECM materials be used conveniently, effectively and efficiently to promote tissue regeneration, including hair growth?

[0144] How to prepare efficient, highly active and stable ECM preparations?

[0145] How can ECM be used scientifically and rationally to promote tissue regeneration, as well as hair care and growth, such as in the form of microparticles or microneedles?

[0146] How to inject ECM microparticles (powder)? Before use, they should be mixed with the suspending solution before entering the scalp?

[0147] Professional terms and related functions or principles are mainly applicable to the technical field and technical effects of the present invention.

[0148] Note: Although Chinese and English nouns or terms may superficially differ, their purposes and uses are essentially the same. Unless otherwise specified, the aforementioned nouns are essentially equivalent. Terms / nouns should be understood and used primarily according to the following text, unless otherwise specified. Other terms should be understood according to the level of understanding of a person of ordinary skill in the art. For example, in this patent, "isotonic agent" and "isotonic solution" are generally considered to have the same or equivalent meanings. They are not considered to be the same or conditionally equivalent unless otherwise specified or with sufficient evidence.

[0149] Warm ischemia time refers to the period from the cessation of blood supply to the start of cold storage. During this period, tissue or organ damage is most severe. This is because, during warm ischemia (after the tissue or organ is removed from the body), although blood flow is interrupted, the tissue or organ continues to metabolize. During this period, due to a lack of oxygen and various metabolic substrates, the metabolic level of the tissue or organ remains high, resulting in faster onset and more severe ischemic damage. Furthermore, even after oxygen is depleted, anaerobic metabolism can still occur, but metabolic products cannot be eliminated, which can cause acidosis and continue to deplete essential metabolic nutrients and enzyme systems.

[0150] Low temperature: refers to 2-8°C; similar to the refrigeration temperature of a household refrigerator; protective solution and preservation solution, fresh-keeping solution, can be substantially equivalent; acellular matrix preparations, extracellular matrix preparations and cell-free extracellular preparations or their combinations, 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.

[0151] Extracellular Matrix (ECM): It 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; it 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 function 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 based on structural proteins such as collagen, elastin, fibrillin and other macromolecular components as the main framework structure, and some functional proteins such as fibronectin FN and laminin LN 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 but very important epidermal growth factor EGF and insulin-like growth factor-1 (IGF-1). In addition, the extracellular matrix also contains active ingredients such as glycosaminoglycans (GAGs) and proteoglycans.

[0152] Exosomes are tiny vesicles secreted by cells, approximately 30-200 nm in diameter, with a cup-shaped morphology and a double-layer membrane structure. They are naturally present in biological fluids such as blood, urine, saliva, breast milk, and cell culture media. Almost all cell types (immune cells, neural cells, and stem cells) can produce and release exosomes. Exosomes contain proteins, rRNA, and microRNAs related to their cell origin. Exosomes can directly activate recipient cells through cell membrane receptors and can also transport proteins, mRNA, miRNA, lncRNA, circRNA, and even organelles into recipient cells to participate in intercellular communication. Exosomes play a key role in physiological processes such as immune response, inflammation, angiogenesis, apoptosis, and coagulation. Exosomes from different cell sources contain different RNA and protein components. Existing technical literature shows 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 bottom of the hair follicle play an important role in the development of the hair follicle. It can be said that without DPC division, 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 use of exosomes in the present invention. F2 The important reason for using protective solution and dual low-concentration gentle method to remove cells is, among other things, to preserve more and better exosomes in the ECM.

[0153] Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are a type of heteropolysaccharide that mainly exists in animal connective tissue. They are important raw materials involved in normal tissue physiological activities and tissue damage repair and regeneration; they are important components of ECM; based on 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: hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate, and heparin (HP).

[0154] Hyaluronic acid (HA), also known as hyaluronic acid or hyaluronate, is a glycosaminoglycan and a major component of the extracellular matrix (ECM). It participates in many cellular physiological and tissue repair processes, including tissue reconstruction, expansion of intercellular spaces, inflammatory responses, and more. Studies have shown that HA has significant cellular effects, significantly promoting cell chemotaxis, migration, and proliferation. This occurs through the binding of HA to cell-surface HA-binding proteins. The adhesion molecule CD44 is a cell-surface receptor for HA and is a widely distributed cell-surface glycoprotein involved in specific cell-to-cell and cell-matrix adhesion. Ru-Ming Liu et al., publishing an article in Experimental Cell Research, Volume 345, Issue 2, 15 July 2016, Pages 218-229, demonstrated that HA can also promote the proliferation of mesenchymal stem cells by activating the Wnt / β-Catenin signaling pathway.

[0155] Acellular Tissue Matrix (ACTM), or acellular tissue matrix, is a matrix made by using specific reagents and treatment methods to fully remove or inactivate cells, viruses, DNA, and other components in animal organs or tissues that may cause immune rejection, thereby maximizing the integrity of the original natural three-dimensional structure and preserving the cell growth factors and active functional components of the original matrix. Due to its natural three-dimensional (3D) structure, the presence of bioactive factors, host degradation, and the ability to induce the migration and differentiation of recipient stem cells, acellular matrix is widely used in clinical practice for tissue repair and regeneration (both congenital defects and acquired trauma). Acellular matrix is a new type of tissue regeneration and repair material with excellent biological scaffold properties.

[0156] Xenogeneic animal decellularized regenerative repair biomaterials, including but not limited to biogenic materials used for various types of superficial or deep injuries (whether caused by internal or external factors, or acute or chronic), in this patent, mainly refer to the ECM three-dimensional structure remaining after animal-derived tissues are processed by decellularization processes, etc., containing various cytokines and bioactive components; the animal-derived tissue raw materials described in the patent of this invention can be derived from the 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.

[0157] Dermal papilla cells (DPCs) are a group of dermal-derived cells located at the base of the hair follicle. They play a dominant role in hair follicle growth, development, cycle regulation, and the maintenance of hair growth. Abnormalities in the structure or function of DPCs are the primary initiating factors leading to imbalanced hair follicle growth. The secluded location of the DPCs presents significant challenges in their isolation and culture. However, DPCs play a crucial physiological role, leading to ongoing research on DPCs and their potential to become a hot topic in the study of hair follicle development and cycle regulation. Cytologically, DPCs can be classified as mesenchymal stem cells, belonging to the category of multipotent stem cells. Multipotency refers to the ability of DPCs to differentiate into a variety of tissue structures. While cultured in vitro, DPCs resemble fibroblasts in morphology, they are inherently different. Their most distinctive growth pattern is the formation of multilayered cell clumps prior to fusion, a characteristic known as aggregative growth. However, the agglutinative growth properties of dermal papilla cells gradually weaken with increasing cell passages. Generally, dermal papilla cells lose this ability to grow in agglutinatively after passage 7. Agglutinative growth is a key biological characteristic of dermal papilla cells and is closely related to their biological functions. Studies have shown that exosomes derived from the ECM can activate hair follicle stem cells, thereby promoting the development of new hair follicles. This is a key reason for using F2 protective solution and employing a dual-low-concentration, gentle decellularization method in this invention. One of its goals is to preserve more and better-quality exosomes within the ECM.

[0158] Hair follicle stem cells (HFSCs) are the primitive cells within the hair follicle, from which both head and body hair grow. However, these stem cells, responsible for hair growth, are typically dormant but rapidly activate and divide during a new hair growth cycle. When an adjacent area is damaged, these cells migrate from their original bulge and participate in repair. The hair follicle is the basic unit of hair, and hair grows from the cells within it. Within each hair follicle resides a population of HFSCs, which provide new cells to the growing follicle. Human HFSCs are a type of adult stem cell, sharing common characteristics. Many tissue functions in the human body rely on the healthy activity of stem cells, and damage or a decrease in stem cell numbers contributes to aging.

[0159] Wnt signaling is closely linked to tissue regeneration. Wnt ligands are secreted glycoproteins that bind to Frizzled receptors, forming a larger complex on the cell surface. The conserved Wnt / β-Catenin pathway regulates stem cell pluripotency and, during development, determines cell differentiation fate. This developmental cascade integrates signals from other pathways, including basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), and bone morphogenetic proteins (BMPs), and is present in various 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 cellular pathways and cell-cell interactions. Regarding Wnt signaling and tissue regeneration, the terms "Wnt," "Wnt gene product," or "Wnt polypeptide," as used herein, encompass native-sequence Wnt polypeptides, Wnt polypeptide fragments, chimeric Wnt polypeptides, or functionally active variants of any of the foregoing. 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 (the planar cell polarity pathway; the 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.

[0160] Basic fibroblast growth factor (bFGF), or fibroblast growth factor-2 (FGF-2), has been reported to regulate hair follicle proliferation and hair cycle transitions in hair follicle organ culture models, animal models, and human experiments. Studies have shown that various growth factors, including bFGF, EGF, and adipose-derived stem cell culture media, regulate hair growth and hair follicle cycle during hair loss. In a mouse model of hair loss, some growth factors, either individually or synergistically, have been found to promote hair growth, with bFGF in particular showing potential for treating hair loss. Studies have demonstrated that FGF can induce the β-Catenin and Shh pathways in mice, accelerating the transition of hair follicles from the telogen phase to the anagen phase. bFGF plays a crucial role in hair follicle cell proliferation and cycle transitions after hair loss and helps enhance the Shh signaling pathway.

[0161] The Sonic hedgehog (Shh) pathway plays a crucial role in hair follicle growth, development, and hair cycle transitions. Shh, a key gene in this pathway, is upregulated during the early growth phase and is one of the factors regulating hair follicle growth and cycle transitions. Although a direct link between Shh and the induction of hair follicle cell apoptosis has not yet been established, disruption of Shh signaling has been shown to be a key event in the mechanism of hair loss.

[0162] Supplementary explanation: The acellular matrix preparation of the present invention, when used as a hair growth promoter, can promote the production of growth factors in hair papilla cells; for example, it can stimulate 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 referred to as a hair papilla cell growth factor production promoter, a hair papilla cell vascular endothelial growth factor (VEGF) production promoter, a hair papilla cell fibroblast growth factor (FGF) production promoter, or a hair papilla cell insulin-like growth factor-1 (IGF-1) production promoter.

[0163] 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 professional, targeted or specific (tissue or membrane) protective fluid used?

[0164] Can the target tissue be preserved or pretreated in a non-destructive or minimally destructive manner immediately after leaving the living body, that is, in the early stage (after leaving the living body at constant temperature) or early period, that is, can it be immersed in a low-temperature and high-quality protective solution?

[0165] Is the entire decellularization process gentle, minimally damaging, fast and efficient, and suitable and compatible with the corresponding target tissue?

[0166] And are the subsequent deep processing (freeze drying and granulation) and sterilization processes of ECM appropriately matched?

[0167] 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 bioactive factors) in the final ECM product can be sufficiently well protected and preserved. In other words, whether the natural biological structure, effective ingredients and bioactive factors of the ECM can be effectively protected in three dimensions without blind spots; and whether high-quality protection of quality, quantity and integrity can be achieved (equivalent to the four guarantees: preservation / good preservation / preservation / integrity).

[0168] 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.

[0169] To further describe the purpose of the present invention in detail, an extracellular matrix or tissue protection solution is invented;

[0170] 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 dermal membrane, bladder membrane, pericardium, peritoneum, and amnion 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).

[0171] Furthermore, the target tissue disclosed in this embodiment is small intestinal submucosa tissue or ECM from other sources, that is, the present invention discloses a protective solution for small intestinal submucosa tissue (SIS) or extracellular matrix from other sources;

[0172] Furthermore, the ECM protective solution of the small intestinal submucosa 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; thereby further protecting 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.

[0173] Furthermore, the protective solution of the present invention is not only a protective solution for cell membranes and lysosomal membranes, but also essentially a high-sodium, low-potassium extracellular matrix isotonic protective solution; it is completely different from the antioxidant protective solution in the prior art and does not belong to the same inventive concept.

[0174] Furthermore, the extracellular matrix protection solution disclosed in the present invention comprises at least the following two components: an isotonic solution and a lysosome stabilizer.

[0175] 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.

[0176] 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 of 7.6, both of which are weakly alkaline and stored at low temperature.

[0177] The second category of ingredients: lysosomal stabilizers, specifically including glucocorticoids (hydrocortisone), prostaglandins (PGI, PGE and cathepsin inhibitors, and hyoscine can inhibit Ca2+ influx. (Note: Promoting the stability of lysosomal membranes in tissue cells has been considered 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 gland in stress response; it can control and regulate the release of lysosomal enzymes into the cell fluid).

[0178] Furthermore, specific examples include cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, and the like.

[0179] 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, as well as one or a combination of pharmacologically acceptable sodium or potassium salts of these ingredients.

[0180] Furthermore, it is preferred that the lysosomal stabilizer is a salicylic acid;

[0181] More preferably, the lysosome stabilizer is aspirin;

[0182] Furthermore, the concentration of aspirin in each liter of the protective solution is 0.1 to 1 gram.

[0183] In addition, in the present invention F2 The following ingredients may be further added to the liquid as needed, for example: preservatives (ethyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorphenesin, phenoxyethanol, etc.); anti-inflammatory agents (for example, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin, etc.);

[0184] Of course, on the basis of the present invention, an antioxidant may also be added to the F2 protective solution.

[0185] Furthermore, it may specifically include plant-derived antioxidants, animal-derived antioxidants and antioxidant free radicals such as allopurinol.

[0186] Further specific examples include N-acetylcysteine (NAC). NAC is an N-acetylated derivative of cysteine. It exhibits excellent antioxidant and anti-inflammatory properties and can generate glutathione (GSH) in the body. The presence of SH can scavenge H₂O₁₄ and HOCl. NAC easily deacetylates to cysteine, which then forms glutathione. Furthermore, NAC can reduce pro-inflammatory mediators. According to existing medical uses, NAC has a potent expectorant effect through three mechanisms: first, it disrupts disulfide bonds in sputum mucins, reducing sputum viscosity; second, it enhances the airway clearing function of respiratory cilia; and third, it increases alveolar surfactant. Regarding the pharmacological benefits of NAC, NAC has traditionally been a commonly used expectorant, suitable for respiratory diseases associated with hypersecretion of sputum, such as bronchitis and bronchiectasis. New medical insights are emerging, including its efficacy and pharmacological mechanisms, expanding its pharmacological effects, and exploring new dosage forms, such as oral preparations (tablets and granules) and nebulized solutions. More importantly for the present invention, NAC also has antioxidant and anti-inflammatory properties, generating glutathione (GSH) and scavenging oxygen free radicals. Furthermore, researchers have discovered that NAC exhibits synergistic antibacterial effects, enhancing the antibacterial activity of piperacillin and ciprofloxacin against pathogens. The mechanisms of NAC's synergistic antibacterial effect include: 1. NAC reduces the amount of glycolipids in epithelial cells, inhibiting pathogen adhesion; and 2. NAC disrupts bacterial biofilms, enhancing the bactericidal effect of antibacterial drugs and reducing bacterial resistance. This demonstrates that, in addition to its expectorant properties, NAC possesses antioxidant, anti-inflammatory, and synergistic antibacterial properties, further broadening its applications. This is a new inventive feature of the present invention's F2 solution and the primary reason for selecting NAC as a key antioxidant. It constitutes a second key feature of the present invention, independent of the present invention.

[0187] It is further preferred that the NAC concentration of N-acetylcysteine is 0.2-1 g / L, and more preferably 0.6 g / L.

[0188] On the other hand, in addition to or on the basis of the original invention, antioxidant adjuvants may be added, 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.

[0189] The inventors would like to emphasize that the original technical concept of the present invention is indeed partially based on or borrowed from the ideas of organ transplant preservation fluids (such as University of Wisconsin UW solution, HTK solution, etc.), and there are certain similarities or similarities; however, in terms of specific technical details or underlying working principles, the two are essentially different and significantly different.

[0190] Common organ transplant preservation fluids are as follows:

[0191] UW fluid is characterized by high potassium (125 mmol / l) and low sodium (29 mmol / l), similar to the composition of intracellular fluid. High K+ concentrations inhibit the efflux of K+ from cells along their concentration gradient. UW fluid also contains hydroxyethyl starch (HES), a colloidal carrier that retains fluid within the intravascular space and prevents extracellular swelling. Lactobionic acid and kapokose, both membrane-impermeable substances, reduce tissue edema. Glutathione, a reducing agent, scavenges oxygen free radicals and maintains cell membrane integrity. Adenosine, a substrate for the synthesis of high-energy phosphate (adenosine triphosphate, ATP), provides energy support for organ metabolism. Allopurinol, a xanthine oxidase inhibitor, has a protective effect during ischemia. UW fluid is primarily used for organ preservation prior to liver, pancreas, and kidney transplantation, and is used to preserve the viability and / or activity of tissue cells.

[0192] HTK solution is mainly composed of histidine, with added tryptophan and ketoglutaric acid to promote ATP production. Low potassium reduces calcium overload, and the addition of glycine, alanine, and LK614 (iron chelator) can reduce damage from oxygen free radicals.

[0193] Celsior solution, with lactobionic acid as its osmotic pressure component and histidine as its 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 depolarization of smooth muscle cell membranes. It is suitable for myocardial smooth muscle and is used as a preservation solution during heart transplantation.

[0194] 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.

[0195] First, UW fluids are mainly used for organ preservation before transplantation; however, the F2 fluid of the present invention does not need to preserve the vitality and activity of tissue cells; the F2 fluid mainly maintains the osmotic pressure of the extracellular fluid and prevents various types of cell membrane rupture.

[0196] Second aspect: F2 The fluid does not need to provide the energy level similar to that 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. It further prevents the rupture of the cell lysosomal membrane and the release of proteases in the cells, which causes rapid degradation and serious damage to the extracellular matrix.

[0197] The third aspect of the present invention isF2 The 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).

[0198] Fourth aspect: UW solution (University of Wisconsin solution) refers to the preservation solution used to preserve donor organs during organ transplantation; it mainly contains lactobionic acid, which is the main non-permeable anion with a relatively large molecular weight, which can reduce cell swelling during cold storage, and also contains cotton sugar, hydroxyethyl starch and adenosine.

[0199] It mainly uses hydroxyethyl starch (50g / L) to maintain (colloidal) osmotic pressure. Hydroxyethyl starch (Heta starch) is a synthetic colloidal solution widely used in clinical practice. It is also a natural polysaccharide. Hydroxyethyl starch (HES) is a high-molecular complex formed by hydroxyethylation of the glucose ring of amylopectin 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 colloidal solutions.

[0200] 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.

[0201] Furthermore, the preparation method of the hair growth-promoting ECM microparticles is to use ECM from porcine small intestinal submucosa or other sources as raw materials, and soak them in low-temperature F2 solution to protect the active ingredients in the extracellular matrix from degradation and destruction.

[0202] The remaining process steps, such as inactivation, decellularization, lyophilization, granulation, packaging, and sterilization, are essentially the same as those in conventional methods. The difference lies in the decellularization reagent and concentration. The present invention utilizes a mixed solution of 0.05% trypsin and 0.05% EDTA for decellularization (i.e., a dual low-concentration, gentle method) to prepare the decellularized matrix.

[0203] The mechanism of action of the present invention is as follows: the use of F2 protective solution can, on the one hand, prevent the cell membrane in the target tissue from being pathologically or physiologically lysed by various external non-physiological environmental factors after leaving the living tissue, and strive to maintain the integrity of the cell membrane structure.

[0204] 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.

[0205] F2 protective solution can prevent lysosome rupture, thereby preventing / reducing the release of various proteases and preventing their degradation of ECM, including WNT pathway active factors (WNT ligands and / or agonists, including wnt2b); thereby maximizing the activity of various active ingredients and cell growth factors in ECM powder;

[0206] 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.

[0207] Compared with the prior art, the present invention has the following beneficial effects:

[0208] The protective solution provided by the present invention has a simple formula, can protect both cell membranes and lysosomal membranes, and can inhibit the protease activity of lysosomes, thereby achieving the effects of double membrane multiple protection and increasing the Wnt2b content.

[0209] The raw materials of the protective liquid provided by the present invention are easily available, are all commercial products, and are inexpensive.

[0210] The acellular matrix preparation prepared by the present invention is simply a low-temperature immersion protection treatment added before the decellularization treatment of the original in vitro tissue; it is simple, convenient, easy to master, easy to operate and highly feasible.

[0211] The ECM product provided by the present invention, especially the prepared ECM microparticles, has the advantage that it can fully remove the immunogenic components in SIS and has good biocompatibility, while also retaining the natural three-dimensional structure of SIS and its active ingredients, including collagen and various growth factors, to the greatest extent.

[0212] The ECM preparation prepared by the present invention can be applied to the head or scalp to prevent hair loss and or induce hair regrowth.

[0213] The hair growth preparation of the present invention is richer in various growth factors; on the one hand, it promotes blood circulation in the scalp and increases blood supply to the hair papilla; on the other hand, it can improve the nutrition of hair follicles, slow down the miniaturization process of hair follicles, increase the number of hair follicles, and promote hair growth, thereby achieving the dual effects of preventing hair loss and maintaining hair growth.

[0214] Compared to several types of hair growth agents currently used in clinical practice (minoxidil, finasteride, and some plant essences and plant extract hair growth agents), which have been used for many years but still have some side effects, their hair growth effects are short-lived and gradually disappear over time; and compared to single-ingredient products that cannot or have difficulty in inducing the regeneration of autologous hair growth hormone (HFSC) and secretion of hair functional components such as collagen, and which completely degrade and disappear after a period of use; the ECM preparation of the present invention has medium- to long-term efficacy. Consumers only need a few small injections, and hair growth is achieved in the original location (no complex procedures such as scalp transplantation are involved), thereby reducing unnecessary trouble and financial burden on consumers; it can effectively and durably induce the regeneration of autologous hair growth hormone (HFSC), promote the activation and growth of hair growth hormone (DPC), and has a stable and long-lasting effect, requiring only a limited number of re-injections.

[0215] In this patent, the term "comprising" (and related terms such as "including" or "having" or "including") includes those embodiments, such as any composition of matter, method, or process "consists of" or "consists essentially of" the described features. Isotonic agents and isotonic solutions have the same functions in substance or in specific practical applications. Isotonic agents are prepared as solids (such as powders) simply 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 convert them into liquid isotonic solutions. Based on this principle, isotonic agents and isotonic solutions are substantially the same, or at least equivalent.

[0216] 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 generally -10% to +10% of the said numbers or numerical ranges.

[0217] 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.

[0218] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may 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 understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar to, or equivalent to, the methods, equipment, and materials described in the embodiments of the present invention may be used to implement the present invention.

[0219] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the spirit of the present invention.

[0220] Before describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection 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.

[0221] Specific embodiments are used to describe the principles and solutions of the present invention; it should be understood that they are only for illustration and to facilitate understanding of 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 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

[0222] Example 1 Preparation of F2 Protective Solution and Suspending Solution (One of the Key Points of the Invention)

[0223] 1) F2 protective solution, including two types of components, namely isotonic agent and lysosomal stabilizer.

[0224] The first step is to add water to an 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)) is used, i.e., 60 g of polyethylene glycol 4000, 5.68 g of anhydrous sodium sulfate, 1.46 g of sodium chloride, 0.75 g of potassium chloride, and 1.68 g of sodium bicarbonate; to make 1 liter; that is, to prepare a whole intestinal isotonic solution with 125 mmol / L of sodium ions, 10 mmol / L of potassium ions, 20 mmol / L of bicarbonate ions, 40 mmol / L of sulfate ions, and 35 mmol / L of chloride ions; after preparation, thoroughly mix; and adjust the pH value to 7.4.

[0225] 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 prepared in the first step (isotonic whole bowel lavage solution), stir thoroughly to mix, and store at 4 degrees Celsius for use.

[0226] 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 should be 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 ready to use.

[0227] Example 2 Preparation of conventional ECM microparticles (ie, control group ECM, not soaked in protective solution).

[0228] (1) Inactivation: The submucosa of the small intestine of pigs was inactivated by soaking in a mixed aqueous solution of 1% peracetic acid and 20% ethanol under ultrasonic conditions at room temperature for 60 minutes.

[0229] (2) 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 freeze-thaw steps of "washing-dividing-freezing-thawing" were repeated; a mixed aqueous solution containing 0.05% trypsin and 0.05% EDTA was used for ultrasonic treatment at 36±2℃, and then ultrasonic washing was performed with PBS; treatment was performed in a 15% sodium chloride hypertonic solution at 36±2℃, and then washed with water; treatment was performed in a 25mM NaOH aqueous solution at 36±2℃; ultrasonic washing was then performed with water until neutral; DNA enzyme treatment was added to remove residual DNA.

[0230] (3) Freeze-drying: Stack the SIS sheets, fix them on a mold, and freeze-dry them.

[0231] (4) Granulation: The freeze-dried SIS was cut into small pieces, frozen in liquid nitrogen, and then crushed. The pieces were sieved using stainless steel sieves of different mesh sizes to obtain ECM particles with a particle size ranging from 50 μm to 400 μm.

[0232] (5) Packaging: Weigh the ECM microparticles and dispense them into COP syringes in portions of 100 mg, 200 mg, or 300 mg, and seal them with stoppers.

[0233] (6) Sterilization: Select cobalt 60 sterilization method with a radiation dose of 25kGy-30kGy.

[0234] Example 3 Preparation of ECM Microparticles (i.e., Preparation of Highly Active ECM by Soaking in Low-Temperature F2 Protective Solution)

[0235] The process steps are basically the same as those in 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 (which may also include the target organ), the porcine small intestinal submucosa (SIS). (Note: Of course, in this technical field, technicians with the 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 components and / or various cell growth factors in the extracellular matrix from degradation and destruction.

[0236] The remaining steps (such as inactivation, decellularization, lyophilization, granulation, packaging and sterilization) are exactly the same as those in Example 2.

[0237] Example 4 Detection of active ingredients in ECM (bFGF, Wnt2b)

[0238] The relevant components in the ECM prepared in Example 2 and Example 3 were detected using an ELISA kit.

[0239] It should be noted that the ECM samples prepared in the two examples were pretreated prior to testing using a urea-heparin solution (non-enzymatic degradation method) and the procedures were performed according to the literature. Regarding the detection of relevant active components (growth factors) in the ECM prepared in Examples 2-3, the present invention detected the active components of the ECM, mainly targeting a typical regenerative growth factor, a cytokine closely related to stem cell regeneration, such as hair growth, namely basic fibroblast growth factor (bFGF), using ELISA. The test results were:

[0240] 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;

[0241] The bFGF content in the ECM of Example 3 (immersed in the F2 protective solution of the present invention) was 20.18+ / -0.51 pg / mg; and the Wnt2b content was 76.11+ / -0.48 pg / mg.

[0242] Summary: After the target tissue leaves the living body (36-38°C), it is immersed in a cryoprotective solution and then undergoes subsequent treatments including decellularization. This type of protective solution immersion treatment can significantly increase the content of various functional and / or growth factors and proteins in the decellularized matrix (ECM).

[0243] Based on the principles of the present invention and 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 indicate 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 cells or stem cells in the target area, promoting the activation of stem cell signals or tissue regeneration in the recipient area, stimulating the activity of major stem cells, and initiating 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.

[0244] Example 5: Experimental study on the hair growth promoting effect of two types of ECM mixed preparations

[0245] The ECM microparticles prepared in Examples 2 and 3 were thoroughly mixed with the suspension prepared in Example 1 to form injectable suspensions, designated A3 and A2. Animal experiments were then conducted. Preliminary results showed that the hair growth-promoting effect of group A3 was significantly superior to that of group A2.

Claims

1. A medical protective solution for leaving mammalian living tissue, characterized in that: Before decellularization, the protective solution is immersed in low temperature conditions for use, and includes an isotonic solution and a lysosome stabilizer. The isotonic solution in the medical protective solution is a commercially available isotonic whole intestinal solution with a pH value of 7.2-8.

2. 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, and 1.68 grams of sodium bicarbonate, and the remainder is deionized water. The polyethylene glycol is pharmaceutical grade PEG4000 in the 2020 edition of the Pharmacopoeia. The low temperature condition is 2-8°C. The lysosome stabilizer is aspirin.

2. 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.

3. A medical acellular matrix, characterized in that: The source tissue of the acellular matrix is immersed in the medical protective solution of claim 1 after leaving the living tissue and before decellularization, so as to significantly increase the content of active ingredients in the ECM.

4. The acellular matrix according to claim 3, characterized in that The acellular matrix is the submucosa of the pig small intestine and contains one or a combination of Wnt2b and bFGF components.

5. An injectable medical acellular matrix preparation, characterized in that: The preparation consists of a decellularized matrix and a suspending agent, which are independently packaged, 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; 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 gently decellularized using a mixed solution of 0.05% trypsin and 0.05% EDTA.

6. A method for preparing an injectable medical acellular matrix preparation according to claim 5, characterized in that: It includes the following three steps: Step 1: Preparation of acellular matrix microparticles 1) Soaking in protective solution and pretreatment: soaking the target tissue from the living body in the protective solution according to claim 1 under low temperature conditions; Pretreatment refers to removing impurities and fat; 2). Inactivation, decellularization, DNA removal, and freeze-drying; 3) Microparticle preparation: freeze the lyophilized ECM in liquid nitrogen, crush it, and sieve it to obtain microparticles with a diameter of 50 μm-400 μm; then package and sterilize; Step 2: Preparation of suspending agent a). Prepare a liquid containing glycerol, sodium hyaluronate, and phosphate buffer, wherein the glycerol concentration is 0.5%; The concentration of sodium hyaluronate is 0.2%, and the molecular weight is between 1 million and 1.6 million Daltons; the 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 acellular matrix microparticles and the suspending agent are packaged independently, sterilized separately, and fully mixed using a three-way pipe before use; the mixture is prepared into an injectable medical acellular matrix preparation with good tissue repair and regeneration functions.

7. Use of the acellular matrix according to any one of claims 3 to 4 in preparing medical repair materials or medical devices, characterized in that: The medical repair material or medical device is used to repair defects in tissues and organs; the tissues and organs are hair, 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.

8. Use of the acellular matrix according to any one of claims 3 to 4 in preparing medical repair materials or medical devices, characterized in that: The product is applied to the head to prevent various types of hair loss in adults and help promote hair follicle growth. The head application method includes external application to the scalp in the area of hair loss, assisted introduction using microneedles, nanochips and ultrasonic ions, subcutaneous injection of the scalp, microneedle patches on the scalp in the area of hair loss, or 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.