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

Abstract

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

Description

[0001] Technical Field This invention relates to the field of biomaterials technology, specifically to a decellularized matrix preparation, its preparation method, and its use in promoting hair care and hair growth. Background Technology

[0002] Hair loss can be broadly divided into anagen (growth phase) hair loss and telogen (resting phase) hair loss. The causes of anagen hair loss are diverse; hair loss affects approximately half of all men and over a quarter of all women worldwide. Young and middle-aged adults have become the main force of the "hair loss army." Common types of hair loss include androgenetic alopecia (AGA), alopecia areata, trichotillomania, telogen effluvium, and senile alopecia. As is well known, androgenetic alopecia, also known as male pattern baldness, is characterized by the complete or partial loss of all or most of the hair on the scalp; in AGA patients, the affected scalp areas show shrinkage of hair follicles and a decrease in hair volume; the incidence of AGA increases with age. Although this condition has a high incidence, there are relatively few effective treatments for patients. Currently, the specific mechanisms of hair growth disorders are not fully understood, but scientific evidence suggests that hair loss is caused by disorders or disturbances in the relationship or interaction between hair follicle development and the hair cycle. As is well known, hair follicles are complex micro-organs in the skin, responsible for hair growth, cyclical replacement, and interaction with surrounding tissues; the main structure, components, and functions of hair follicles are as follows. The hair follicle structure consists of the following 7 parts:

[0003] Bulge: The upper part of the hair follicle, containing hair follicle stem cells (HFSC) and melanocyte stem cells, which are crucial for hair color and regeneration.

[0004] Hair follicle matrix: The area below the bulge of the hair follicle where hair growth occurs.

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

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

[0007] Dermal papilla (DP): A cluster of cells located at the base of the hair follicle that plays a regulatory role in hair growth.

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

[0009] Arrector Pili Muscle: A tiny muscle connected to the hair follicle; when it contracts, it causes the hair to stand on end.

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

[0011] The functions of hair follicles include: the periodic growth of hair through the proliferation and differentiation of their stromal cells; the periodic replacement of hair follicles, which undergo anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase; the provision of new cells by hair follicle stem cells during the regeneration phase of the hair cycle to maintain hair growth; sensory functions (hair follicles are connected to nerve endings and participate in sensory transmission); thermoregulation (contraction and relaxation of the arrector pili muscles help regulate body temperature); protective functions (hair provides a physical barrier for the skin, protecting it from external damage); and cosmetic functions, as hair has significant aesthetic value and a remarkable social function.

[0012] Hair follicles undergo cyclical growth, and their condition has a decisive influence on hair growth. Hair does not grow continuously but rather through a cyclical process of growth and shedding. The three main phases of normal hair follicle cyclical replacement and how they affect hair growth are: 1) Anagen (growth phase): accounting for 85%, lasting 2-8 years. This is the initial stage of hair growth, a period of active hair growth that can last for several years; during the anagen phase, the hair follicle...

[0013] Matrix cells proliferate rapidly, differentiate very actively, and push hair fibers upward, resulting in increased hair length; the duration of the growth phase determines the maximum length that hair can reach.

[0014] 2) Regression phase: accounting for 1-2%, lasting 2-4 weeks. This is the late stage of hair growth, a transitional phase that lasts for several weeks. During this phase, the hair follicle begins to shrink, and hair growth stops. During the regression phase, the structure of the hair follicle changes, and the dermal papilla gradually separates from the hair follicle matrix. At the end of the regression phase, the hair matrix cells stop producing hair, and the hair becomes a specific shape, namely a rod. The end of this phase marks the beginning of the next phase of the hair growth cycle.

[0015] 3) Telogen effluvium: accounting for 10-15%, lasting 2-3 months. The telogen effluvium is the final stage of the hair growth cycle. During this period, the hair follicles completely stop activity and shrink, lasting for several months. In this stage, the hair follicles are almost inactive, and the old hair fibers gradually separate from the hair follicles and eventually fall out. After the telogen effluvium ends, the hair follicles will re-enter the growth phase, and new hairs will begin to grow, replacing the fallen hairs.

[0016] The effects of the cyclical turnover of hair follicles on hair growth include:

[0017] 1) Hair density: The shedding of telogen effluvium hairs and the growth of new hairs during the anagen phase together determine hair density.

[0018] 2) Hair length: The duration of the growth phase, which affects the maximum length that hair can grow to.

[0019] 3) Hair color: As we age, the activity of melanocytes in hair follicles may decrease, causing hair color to lighten.

[0020] 4) Hair quality: During the periodic replacement process, the health of the hair follicles affects the thickness and elasticity of the hair.

[0021] 5) Hair growth pattern: Different individuals have different hair follicle cyclical replacement patterns, which may lead to different hair growth patterns, such as sparse or dense hair.

[0022] Currently, stem cell transplantation is one of the available strategies for achieving hair follicle function regeneration. Stem cell therapy, as a potential novel treatment, has received considerable attention. Its principle in preventing AGA (Alopecia Areata Growth) 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 injecting 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, thus maintaining a stable stem cell population within the hair follicle; however, this ability weakens with age.

[0023] Hair follicles contain various cellular resources, such as melanocytes, epithelial cells, and stem cells from different developmental origins, which can continuously renew and regulate hair growth and homeostasis. There are two main types of stem cells in hair follicles: the first is HFSCs, which are located within the outer root sheath (ORS) of the arrector pili muscle attachment area and the proximal isthmus region, both areas being referred to as "protrusions." The second is dermal papillary cells (DPCs), which are responsible for controlling hair induction and growth and participate in the formation of new hair follicles. Androgenetic alopecia (AGA) is a non-scarring form of hair loss, where HFSCs are undamaged, only progenitor cells are. This fact suggests that androgenetic alopecia may be reversible. In scalps affected by androgenetic alopecia, the replication potential of DPCs is reduced, and their shape, size, and characteristic markers are also easily lost. In recent years, some clinics have begun performing simple stem cell culture and transplantation. Due to the regenerative potential of stem cells, stem cell therapy has attracted considerable attention from doctors and patients with androgenetic alopecia; although studies on the efficacy of stem cell therapy in AGA have been published, the treatment efficacy of this therapy for AGA still requires further observation.

[0024] Studies and surveys have shown that hair follicle stem cells contain hair follicle epithelial stem cells, which have the function of regulating the hair cycle (see JID Symposium Proceedings, 8:28-38 (2003)); reports indicate that the interaction between epithelium and mesenchyme is a key factor in hair follicle formation. Hair transplantation is one of the effective methods for solving baldness, which involves taking hair roots from a still functional donor area (e.g., along the back and sides of the head) and then transplanting them to a scalp area where the hair roots have died; surgical procedures, such as scalp reshaping surgery, include, for example, scalp reduction surgery, scalp flap surgery, and tissue extension or tissue expansion.

[0025] Drug therapy includes the use of minoxidil and finasteride. Finasteride and minoxidil are the only two FDA-approved drugs for hair regrowth; the main drawback of these drug therapies is that they have little effect on hair loss treatment after discontinuation, therefore these treatments are only temporary, not permanent, measures for treating hair loss.

[0026] Currently available alternatives include, for example, hair transplantation and scalp reshaping surgery, as well as various pharmacological (or drug) therapies. Although there has been much research and development on hair growth methods, unfortunately, these methods are still relatively cumbersome, inconvenient to use, and have unstable effects. There is currently no effective method that can fundamentally prevent or treat hair loss.

[0027] On the other hand, various decellularization techniques, including physical, chemical, and biological methods, are used 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. Extracellular matrix (ECM) materials made from homologous or xenogeneic tissues after the removal of immunogenic substances are also called decellularized product formulations, or by some technicians, decellularized patches or tissue regeneration scaffolds. Currently, these products (ECM) are widely used in tissue engineering and clinical regenerative medicine, such as for the repair and reconstruction of heart valves, blood vessels, nerves, tendons, bones, cartilage, skin, esophagus, trachea, abdominal wall, and many other tissues. Sufficient research has shown that by using appropriate decellularization processes, it is possible to prepare ECM with low levels of immunogenic substances while retaining 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 effect of ECM products is closely related to the source of the material tissue and the decellularization process. Currently, porcine small intestinal submucosa (SIS) is a research hotspot and focus, as it has the advantages of being easy to obtain, having abundant sources, not raising ethical issues, and having a low risk of virus transmission. Furthermore, it is also characterized by its ability to be mass-produced, making it highly favored by both industry technology leaders and academic experts. For many years, our company's technical team has been using porcine small intestinal submucosa (SIS) as the main raw material to conduct repeated research and optimization of the decellularization process and to carry out further deep processing, thereby developing, producing, and applying multiple series of Class III medical devices.

[0028] Although decellularized matrix (ECM) formulations are currently available for the prevention and treatment of various types of hair loss in adults, showing considerable potential and interest, ECM is composed of various 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:

[0029] 1. Structural support: ECM provides a stable support structure for hair follicles, maintaining their shape and integrity, and contributing to normal hair growth.

[0030] 2. Cell signaling: ECM interacts with cell surface receptors, triggering intracellular signaling and affecting the behavior of hair follicle stem cells and the hair growth cycle.

[0031] 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.

[0032] 4. Nutrient Delivery: ECM helps deliver nutrients and oxygen, providing the necessary nutrition and environment for hair growth.

[0033] 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.

[0034] 6. Immune regulation: ECM can regulate local immune responses, affect the inflammatory state of hair follicles, and thus affect hair growth.

[0035] 7. Regulation of the hair growth cycle: The ECM is closely related to each stage of the hair growth cycle, including the anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase.

[0036] 8. Wound healing and regeneration: After skin injury, the repair and regeneration of the ECM is crucial for the recovery of hair follicles and hair growth.

[0037] ECM promotes hair care and growth in the following ways: First, it improves the hair growth cycle, prolongs the growth phase, and shortens the regression or resting phase to allow timely entry into the growth phase; second, it promotes an increase in hair density and thickness; and third, it enhances hair elasticity and breakage resistance.

[0038] ECM plays a multifaceted role in promoting hair growth; however, current treatment effects are not satisfactory. One important reason may be that the entire preparation process of decellularized matrix, including timely and proper pretreatment of the target tissue after ex vivo (S1), and the varying degrees of damage to the natural three-dimensional structure of ECM caused by different decellularization processes (sometimes overly harsh decellularization conditions, high reagent concentrations, long durations, and repeated treatments), as well as the different effects of different processing techniques on the naturally occurring bioactive components and / or growth factors in ECM, leading to significant loss or destruction of these components, reduced or even complete loss of their activity in promoting tissue regeneration. This directly affects the hair growth-promoting efficacy of ECM preparations. In practical use, it has poor induction ability for various hair stem cells or related hair regeneration cells, including a lack of sufficient corresponding cell growth factors and cell channel signaling support, thus failing to effectively promote the regeneration of injured or damaged hair tissue; ultimately, it is difficult to demonstrate a significant effect in promoting hair regeneration.

[0039] The main references are as follows:

[0040] Non-patent articles

[0041] Recent advances in research on the role of the Wnt signaling pathway in the induction of hair follicle formation and growth by pilosebaceous cells.

[0042] Chinese Journal of Cell Biology 2021, 43(7):1550–1560

[0043] Author and Affiliation: Yuan Mu, Department of Stem Cell and Regenerative Medicine, Daping Hospital, Army Medical University

[0044] Research progress on stem cell therapy for androgenetic alopecia

[0045] Journal of China Pharmaceutical University, 2023, 54(3): 372-379

[0046] Author and Affiliation: Yan Wenjing, School of Life Sciences and Technology, China Pharmaceutical University

[0047] "A study on the mechanism of action of basic fibroblast growth factor in treating chemotherapy-induced hair loss"

[0048] Doctoral dissertation from China Medical University, 2021, 82 pages

[0049] Author and Affiliation: Qin Jie, The First Affiliated Hospital of China Medical University

[0050] Wnt Ligands Secreted by Subepithelial Mesenchymal Cells Are Essentialfor the Survival of Intestinal Stem Cells and Gut Homeostasis.

[0051] Valenta et al.,2016,Cell Reports 15,911–918

[0052] “MAP3K2-regulated intestinal stromal cells define a distinct stemcell niche”

[0053] 《Nature》volume 592,pages606–610(2021)

[0054] Author and Affiliation: Su Bing, Shanghai Jiao Tong University School of Medicine and Shanghai Institute of Immunology

[0055] This study discovered a novel type of intestinal stromal cell called MRISC at the base of intestinal stem cells. It revealed the role and mechanism of MRISC cells in the repair of intestinal epithelial tissue damage by specifically regulating the microenvironment signal of intestinal stem cells during intestinal inflammation and injury, providing new insights for intestinal repair and regeneration.

[0056] Existing patent technology literature:

[0057] US20140086867A1;

[0058] US20210299036A1;

[0059] CN201610511579.7;

[0060] Regarding the decellularization protection solution, CN201610511580.X (abbreviated as D4), invention title: An optimized antioxidant decellularization protection solution; BioDexel (Beijing) Biotechnology Co., Ltd. (inventors: Shi Zhen and Shi Weiyun) discloses the invention patent; the protection solution has a specific pH value, specific crystal and colloidal osmotic pressure, and appears as a light red liquid.

[0061] It is obvious that the main function of the protective solution of this invention is at least anti-oxidation; in addition to using DMEM cell culture medium powder, the formula also contains nine other ingredients; as follows: (2) L-histidine hydrochloride 2.87-3.83g / L; (3) chondroitin sulfate 25-30g / L; (4) low molecular weight dextran 20-35g / L; (5) hydroxypropyl methylcellulose 2-8g / L; (6) allopurinol 0.5-0.8g / L; (7) HEPES buffer 20-25ml / L; (8) dexamethasone hydrochloride 1-2mg / L; (9) reduced glutathione 1.5-3g / L; (10) levofloxacin 0.1-0.2g / L.

[0062] Firstly, the protective solution uses DMEM cell culture medium powder. DMEM is a widely used cell culture medium, modified by Dulbecco from Eagle's Minimum Essential Medium (MEM). The components 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 and macro elements like calcium, phosphorus, potassium, magnesium, and iron); glucose (as an energy source 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 acidity and alkalinity of the culture medium). DMEM primarily provides the necessary nutrients to support cell growth in vitro; however, this is actually unnecessary in the protective solution invented by D4, seemingly redundant, and deviates from the original purpose and meaning of the D4 invention; it also strays far from the invention's subject matter: "antioxidant decellularization protective solution."

[0063] Secondly, because DMEM is a cell culture medium, its composition is inherently complex, as mentioned above. Consequently, the decellularization protective solution of this invention is actually a formula on top of a formula, exhibiting obvious dual complexity. It aims to promote cell growth first, and then decellularize the cells later. This makes the ultimate purpose of using the protective solution somewhat contradictory, or at least inconsistent, or even completely opposite.

[0064] Thirdly, in the D4 protective solution, on the one hand, there is a crystal osmotic pressure, between 330-380 mOsm / kgH2O, derived from histidine hydrochloride; in Example 1, each liter of protective solution contains 2.87 grams of histidine hydrochloride; on the other hand, there is a colloidal osmotic pressure, between 310-350 mOsm / kgH2O, derived from chondroitin sulfate, hydroxypropyl methylcellulose, and low molecular weight dextran. The molecular structures and molecular weights of these three components themselves vary considerably.

[0065] Chondroitin sulfate sodium (CS) is an acidic mucopolysaccharide composed of D-glucuronic acid and 2-acetamino-2-deoxy-sulfate-D-galactose, with the molecular formula (C14H21NO14S)n. Depending on the position of the sulfate group, if it is at position 4 of the galactose, it is called chondroitin sulfate A; if it is at position 6, it is called chondroitin sulfate C. Crucially, the molecular weight of chondroitin sulfate varies considerably, ranging from 10,000 to 50,000 Daltons. Furthermore, the properties of chondroitin sulfate also differ significantly depending on its source and extraction method.

[0066] Hydroxypropyl methyl cellulose (HPMC), as listed in the 2005 edition of the Chinese Pharmacopoeia, has a methyl substitution degree of 1.0–2.0 and a hydroxypropyl substitution degree of 0.1–0.34, with a relative molecular mass generally ranging from 10,000 to 150,000. Therefore, the specific molecular weight of HPMC can vary significantly due to the different degrees of hydroxypropyl and methyl substitution.

[0067] The molecular weight of low molecular weight dextran ranges from 25,000 to 45,000, resulting in significant fluctuations in molecular weight.

[0068] Moreover, these three components can generate colloidal osmotic pressure. DosageThe dosage of HPMC in D4 protective solution varies considerably. For example, the lower and upper limits of HPMC dosage differ by a factor of four (from 2 g / L to 8 g / L). In reality, these three components have two main characteristics: firstly, the molecular weight of each component varies significantly; secondly, the dosage of each component also varies considerably. Therefore, in actual operation, even these three components, which generate colloid osmotic pressure, are subject to numerous variable factors. This directly affects the colloid osmotic pressure parameters in D4 protective solution, resulting in many uncertainties. Furthermore, to achieve the predetermined target colloid osmotic pressure value, unnecessary adjustments and multiple verifications may be required.

[0069] Fourthly, it is particularly noteworthy that the osmotic pressure in the D4 protective solution is actually composed of both crystal osmotic pressure and colloidal osmotic pressure, which can reach at least 600 mOsm / kgH2O. This does not effectively match the intended protective purpose of the protective solution and may even produce the opposite or significantly adverse side effects.

[0070] Fifthly, the protective solution invented by D4 is actually very complex, with nearly twenty different components. Because of the large number of components, the actual operation is more difficult and complex in terms of the components and corresponding dosages required to prepare the decellularization protective solution, adding more uncertainty and thus making it easier to cause more fluctuations and changes in the effect.

[0071] Sixthly, the cell-protecting solution invented by D4 uses at least two reagent-grade products for cell culture; one is the aforementioned DMEM cell culture medium powder, and the other is HEPES buffer (a non-ionic amphoteric buffer with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid); these two components alone are not cheap, for example, Thermo Fisher's HEPES costs more than 400 yuan per 20ml, which directly leads to the high cost of using this antioxidant cell-protecting solution.

[0072] Seventhly, D4 uses an antioxidant decellularization protectant throughout the entire process; however, in reality, the cell membrane is completely ruptured during the decellularization process, rendering the D4 protectant ineffective in providing the claimed protective effect. Summary of the Invention

[0073] This invention was developed by senior American R&D personnel within the company. They extensively reviewed numerous domestic and international literatures on stem cells, hair development, and hair follicle stem cells, carefully studied authoritative articles, meticulously researched the principles of hair growth and hair loss at various levels, explored in depth, and understood the mechanisms of action between hair loss-related factors, identifying key molecules or pathways.

[0074] Studies have found that stem cells can proliferate in various tissues, driving tissue regeneration. This mainly depends on signal transduction on the cells, but requires the provision of good tissue regeneration factors and an external microenvironment. However, this problem has not been truly solved.

[0075] Hair follicle stem cells are adult stem cells that sometimes remain dormant in vivo. Only under specific conditions can they exhibit strong proliferative capacity, subsequently differentiating into the epidermis, hair follicles, and sebaceous glands, participating in the hair regeneration process. Hair follicle stem cells live within each hair follicle and are rapidly activated and divide during a new hair growth cycle. Current literature reports the use of decellularized matrix to activate hair follicle stem cells in vitro or in vivo, thereby promoting hair growth and contributing to hair loss prevention and cosmetic applications. The technical problem solved by this invention is as follows:

[0076] The first technical problem is that the current decellularization process does not use tissue protection solutions, or only provides antioxidant protection, or the composition is too complex. There is no effective protective solution specifically for ECM before decellularization, or the protection system for ECM is not comprehensive, especially in preventing protease degradation.

[0077] The second technical problem is that the decellularization process is demanding. For example, if the concentration is high or the decellularization process takes a long time, it will result in a large loss of active ingredients and serious damage.

[0078] The third technical problem is that, among similar products, the active ingredients for promoting hair growth are low or there are few cell growth factors, making it difficult to stimulate the regenerative vitality / activity of hair follicle stem cells (DPC) and / or HFSC, resulting in an insignificant and unsustainable effect on promoting hair growth.

[0079] In order to solve the above-mentioned technical problems and overcome the shortcomings of the prior art, the present invention discloses the following technical solutions.

[0080] The first objective of this invention is to develop a medical protective solution, namely a tissue structure or extracellular matrix (ECM) protective solution, specifically a medical xenogeneic animal tissue protective solution, which is used for soaking in low temperature conditions before decellularizing the target tissue.

[0081] The protective liquid includes at least two types of components: isotonic solution and lysosomal stabilizer, and can be referred to as F2 solution;

[0082] In practice, F2 solution is essentially an isotonic solution containing at least a small amount of lysosomal stabilizer.

[0083] The protective solution is used to prevent the degradation or destruction of active ingredients and / or various cell growth factors in the extracellular matrix (ECM);

[0084] The low temperature condition is 2-8℃.

[0085] Furthermore, the aforementioned isotonic solution is a commercially available isotonic whole-enteric irrigation solution; the isotonic solution contains, per liter of protective solution, 60 grams of polyethylene glycol, 5.68 grams of anhydrous sodium sulfate, 1.46 grams of sodium chloride, 0.74 grams of potassium chloride, 1.68 grams of sodium bicarbonate, with the remainder being deionized water or double-distilled water.

[0086] The polyethylene glycol is pharmaceutical grade PEG4000 (2020 edition of the Pharmacopoeia).

[0087] Furthermore, the isotonic whole-enteric lavage solution is a compound polyethylene glycol electrolyte powder (II), the detailed composition of which is as follows: ： The concentrations of Na ions are 125 mmol / L, K ions are 10 mmol / L, HCO3- ions are 20 mmol / L, SO42- ions are 40 mmol / L, and Cl ions are 35 mmol / L. The osmotic pressure can also be adjusted to 280-320 mOsmol / L.

[0088] Isotonic whole bowel lavage solution is a relatively mild laxative, mainly used for bowel cleansing before intestinal endoscopy or intestinal surgery; it can also be used to treat functional constipation.

[0089] In this invention, the applicant (isotonic whole colonic irrigation solution) is used for new purposes; that is, in different technical fields, to solve different technical problems, and to achieve different technical objectives and effects.

[0090] Furthermore, the aforementioned lysosomal stabilizer may be one or a combination of hydrocortisone, prednisone, prednisolone, methylprednisolone, and triamcinolone; further, the aforementioned lysosomal stabilizer is hydrocortisone.

[0091] Furthermore, the concentration of hydrocortisone in the protective solution is 1 to 10 mg per liter.

[0092] Furthermore, the aforementioned lysosomal stabilizer may be more preferably a nonsteroidal anti-inflammatory drug (NSAID), including sodium or potassium salts of salicylates, arylpropionic acids, anilines, indoles, arylaceous acids and their derivatives, or a combination thereof. The NSAID is selected from aspirin, diclofenac, loxoprofen, ibuprofen, acetaminophen, celecoxib, etodoxacin, pranoprofen, flurbiprofen ester, lornoxicam, thiamide, tramadol and zaltoprofen, and a combination thereof of pharmacologically acceptable sodium or potassium salts of these components.

[0093] Furthermore, salicylic acid is preferred as the lysosomal stabilizer;

[0094] Aspirin is a more preferred lysosomal stabilizer;

[0095] Furthermore, the concentration of aspirin in the protective solution is 0.1 to 1 gram per liter.

[0096] Specifically, in this patent embodiment, preferably, the lysosomal stabilizer is aspirin at a concentration of 0.2 g / L (protective solution).

[0097] Furthermore, the target tissue was immersed in F2 protective solution for at least one hour under low temperature (2-8°C) conditions.

[0098] The second objective of this invention is to provide a medical decellularized matrix (ECM) in which the ECM source tissue is immersed in the aforementioned F2 protective solution after the ECM source tissue leaves the living body, i.e. after warm ischemia and before decellularization.

[0099] The purpose of soaking ECM source tissue in a protective solution is mainly to reduce the degradation or damage of effective components and natural structures in ECM after it leaves the living tissue by adverse external factors. This can significantly increase the content of various active ingredients that can activate stem cells in the final ECM preparation, thereby further enhancing the tissue and cell regeneration performance and vitality of ECM.

[0100] Furthermore, the ECM source tissue is one or more of the following: dermis, pericardium, visceral membrane, peritoneum, bladder membrane, submucosa of small intestine, submucosa of stomach, or other membrane tissues or important organs.

[0101] Furthermore, the ECM source tissue may be the submucosa of the porcine small intestine;

[0102] Furthermore, the pigs mentioned are commercially available Duroc-Landrace-Large White crossbred commercial meat pigs.

[0103] Furthermore, the pigs are Duroc-Landrace-Large White crossbred commercial meat pigs; weighing 30-100 kg; preferably feeder pigs, weighing 30-60 kg; and aged 80-120 days.

[0104] Furthermore, the decellularized matrix contains one or a combination of Wnt2b and bFGF.

[0105] After rapid impurity removal and rinsing, immediately place the sample in the aforementioned F2 protective solution and soak for at least one hour until decellularization is performed.

[0106] The third objective of this invention is to provide a method for preparing an injectable decellularized matrix microparticle formulation.

[0107] The method for preparing the injectable decellularized matrix microparticle formulation includes the following three steps:

[0108] Step 1: Preparation of decellularized matrix microparticles

[0109] 1) Protective solution immersion and pretreatment: The target tissue removed from the living organism is immersed in the aforementioned protective solution (F2) at a low temperature (2-8 degrees Celsius).

[0110] In liquid, to protect the active ingredients and / or various cell growth factors in the extracellular matrix from degradation and destruction; pretreatment, such as removing impurities and lipids.

[0111] 2) Inactivation, decellularization, DNA removal, and freeze-drying;

[0112] 3) Particle preparation: The freeze-dried ECM is frozen with liquid nitrogen and then crushed and sieved to obtain particles with a diameter between 50μm and 400μm.

[0113] 4) Packaging and sterilization.

[0114] Step 2: Preparation of suspending agent

[0115] A suspending agent is prepared by mixing a liquid containing glycerol, sodium hyaluronate, and phosphate buffer solution. The phosphate buffer solution is used to adjust the pH and osmotic pressure of the suspending agent.

[0116] Dispense the suspending agent into containers that match its sterilization method, seal, and sterilize by moist heat.

[0117] Step 3: The decellularized matrix microparticles and suspending agent are individually packaged and sterilized separately. Before use, they are thoroughly mixed using a three-way tube. The mixture is then prepared into an injectable decellularized matrix microparticle formulation with good tissue repair and / or regeneration functions.

[0118] Furthermore, the specific details include the following three steps:

[0119] Step 1: Preparation of decellularized matrix microparticles

[0120] 1) Pretreatment: Fresh pig small intestine or the submucosa of pig small intestine after the mucosal layer / muscle layer has been removed is immersed in F2 protective solution at low temperature (2-8℃) for at least one hour to protect the active ingredients and various cell growth factors in the extracellular matrix from degradation and destruction.

[0121] 2) Inactivation: Take the submucosa of pig small intestine and inactivate it by treating it with a mixed solution of peracetic acid and ethanol under ultrasonic conditions at room temperature.

[0122] 3) Decellularization: After inactivation, SIS was first washed with water under sonication, aliquoted into small packages, and rapidly frozen overnight. The next day, the freeze-thaw cycle of "washing-aliquoting-freezing-thawing" was repeated. The mixture was treated with a solution containing 0.05% trypsin and 0.05% EDTA at 36±2℃, followed by sonication with PBS; then treated with a 1.5% sodium chloride hypertonic solution at 36±2℃, followed by washing with water; then treated with a 20mM NaOH aqueous solution at 36±2℃; washed with water until neutral; and finally treated with DNase to remove residual DNA.

[0123] 4) Drying: Fix the decellularized SIS slides onto a mold and freeze-dry them.

[0124] 5) Microparticle preparation: The lyophilized SIS tablets were cut into small pieces, frozen in liquid nitrogen, and then pulverized. The pulverized particles were sieved using stainless steel sieves of different mesh sizes to obtain decellularized matrix microparticles. The obtained decellularized matrix microparticles had a diameter ranging from 50 μm to 400 μm.

[0125] 6) Dispensing: Dispense the decellularized matrix microparticles into syringes and seal them.

[0126] 7) Sterilization: Sterilize the pre-sealed decellularized matrix microparticles in the syringe. Select a suitable sterilization method for the decellularized matrix and its packaging, such as cobalt sterilization.

[0127] Sterilization can be performed using either electron beam radiation or ethylene oxide.

[0128] Step 2: Preparation of the suspending agent. Prepare a suspending agent containing at least glycerol, sodium hyaluronate, and phosphate buffer, wherein the concentration of glycerol is 0.5% and the concentration of sodium hyaluronate is 0.2%, with a molecular weight in the range of 1,000,000 to 1,600,000 Daltons.

[0129] Step 3: The decellularized matrix microparticles and suspending agent are individually packaged and sterilized separately. Before use, they are thoroughly mixed using a three-way tube. The mixture is then prepared into an injectable decellularized matrix microparticle formulation with excellent tissue repair and regeneration functions.

[0130] The fourth objective of this invention is the medical use of an acellular matrix prepared for the aforementioned objective two, which is for repairing tissue and / or organ defects; the tissues and organs are defects in the following areas: head appendages, hair, ear, face, periosteum, nerves, dura mater, spinal dura mater, tendons, ligaments, urinary tract, bladder, ureter, inguinal hernia, hiatal hernia, abdominal wall hernia, breast or uterine mucosa, oral soft tissue, joints, menisci, etc.

[0131] Furthermore, the medical use of the decellularized matrix is ​​for application to the head to prevent hair loss and to help promote hair follicle growth and hair regeneration.

[0132] Furthermore, the head application method includes external application to the scalp in the hair loss area, assisted introduction using microneedles, nanochips, ultrasonic ions, etc., subcutaneous injection into 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 cells.

[0133] The head application methods include: external application to the scalp of the hair loss area; assisted introduction of the hair loss area to the scalp using microneedles, nanochips, ultrasonic ions, etc.; scalp injection to the hair loss area; microneedle patches to the scalp of the hair loss area; or use as an in vitro culture medium for hair follicle-related tissues and cells.

[0134] Furthermore, the method of application is to inject the mixture into the scalp of the hair loss area. The injected preparation is a mixture of decellularized matrix microparticles and a suspending agent. The mixing ratio is microparticles: suspending agent = 50mg-200mg: 1ml. The microparticle size is in the range of 50μm-400μm.

[0135] The hair growth promoting ECM preparation provided by this invention can be composed of ECM microparticles and a suspension, which are individually packaged and sterilized separately. They are mixed before use. After mixing, the microparticles do not aggregate or settle, are evenly and stably dispersed, and have low fluidity, so they can be smoothly injected through small diameter needles (27G and 30G) for minimally invasive treatment.

[0136] The ECM microparticles are prepared by using ECM from the submucosa of the small intestine or other sources as raw materials, and through processes such as inactivation, decellularization, freeze drying, granulation, packaging, and sterilization.

[0137] An ideal tissue regeneration agent should have good biocompatibility (safety), natural and long-lasting effects, and be easy to use (e.g., convenient injection, or flexible application to the head via non-invasive, minimally invasive, or microneedle methods).

[0138] ECM has unique advantages, but there are still some challenges in preparing it into ECM, or further into hair growth promotion products, as follows:

[0139] 1) What kind of decellularization process should be adopted so that ECM can effectively remove immunogenic substances while ensuring that the components and structure of ECM are relatively intact, so that ECM retains its original active ingredients and various cell growth factors, which is conducive to safely and effectively exerting endogenous induced regeneration after implantation.

[0140] 2) How to minimize or reduce the loss or degradation of various active components in the extracellular matrix during the decellularization process?

[0141] 3) How can ECM materials be conveniently, effectively, and efficiently used to promote tissue regeneration, including hair growth?

[0142] 4) How to prepare highly efficient, highly active, and stable ECM formulations?

[0143] 5) How can ECM be used scientifically and rationally to promote tissue regeneration, as well as hair care and hair growth, such as in microparticle form or microneedling?

[0144] 6) How to inject ECM microparticles (powder): Before use, they should be mixed with the suspension solution before being injected into the scalp.

[0145] Technical terms and their related functions or principles are mainly applicable to the technical field and technical effects of this invention.

[0146] Note: Although Chinese and English nouns or terms may appear different, their purpose and use are essentially the same; unless otherwise specified, the connotations of the above terms are substantially equivalent. Terms / nouns should first be understood and used according to the following textual descriptions, unless otherwise detailed; other terms should be understood according to the level of a person skilled in the art; for example, in this patent, isotonic agent and isotonic solution can generally be regarded as having the same or equivalent meanings, unless there is a specific detailed description or sufficient evidence, in which case they cannot be regarded as the same or conditionally equivalent.

[0147] Warm ischemia time: The period from when blood supply to a tissue or organ stops to when cold preservation begins. This period is when the damage to the tissue or organ 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. At this time, due to the lack of oxygen and various metabolic substrates, the metabolic level of the tissue or organ remains high, so ischemic damage occurs quickly and is severe. Furthermore, even after oxygen is depleted, anaerobic metabolism can still occur, but metabolic products cannot be cleared, which can cause acidosis. The nutrients and enzyme systems necessary for metabolism are still being consumed.

[0148] Technical terms related to decellularized matrix:

[0149] 1) Low temperature: refers to 2-8℃; similar to the refrigeration temperature of a household refrigerator; protective liquid and preservation liquid, freshness preservation liquid can be substantially equivalent; decellularized matrix preparations, extracellular matrix preparations and extracellular-free preparations or combinations thereof, unless otherwise specified, can generally be used interchangeably in this invention, regarded as equivalent or similar, and can all be regarded as substantially the same.

[0150] 2) Extracellular matrix (ECM): It is a non-cellular component present in all tissues and organs. It not only provides the necessary physical support for cellular tissues and provides a suitable site and microenvironment for the normal physiological activities of various cells, but also plays an important lever regulatory 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 cellular function is determined by the external microenvironment created by the extracellular matrix (ECM). The ECM is composed of structural proteins such as collagen, elastin, and fibrils as its main framework, with functional proteins such as fibronectin (FN) and laminin (LN) attached to it. It also carries various cell growth factors, such as fibroblast growth factor (FGF), especially bFGF, as well as transforming growth factor (TGF) and vascular endothelial growth factor (VEGF). It may also contain trace amounts of the 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.

[0151] 3) Exosomes: These are tiny vesicles secreted by cells, approximately 30-200 nm in diameter, with a cup-shaped morphology and a double-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, nerve cells, stem cells) can produce and release exosomes. Exosomes contain cell-derived proteins, rRNA, and microRNA. 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, participating in intercellular communication. Exosomes play a crucial role in physiological processes such as immune responses, inflammatory responses, angiogenesis, apoptosis, and coagulation. The RNA and protein components contained in exosomes from different cell sources vary. Existing literature indicates that exosomes from the ECM can enhance angiogenesis and promote better blood supply to nourish hair follicles. ECM exosome-based therapies 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 demonstrate that ECM exosomes have the ability to stimulate hair growth, improve hair follicle function, and reduce inflammation associated with certain types of hair loss. The dermal papilla cells (DPCs) in the hair bulb at the base of the hair follicle play a crucial role in hair follicle development; without DPC division, hair follicles cannot form, and the three hair growth cycles cannot occur. 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 an important reason for using F2 protective solution and a dual low-concentration gentle decellularization method in this invention, one of the purposes of which is to preserve more and better exosomes from the ECM.

[0152] 4) Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are a type of heteropolysaccharide. They are mainly found in animal connective tissue and are important raw materials for normal tissue physiological activities and tissue repair and regeneration. They are also important components of ECM. According to the type of monosaccharide residues, the type of inter-residue bonds, and the number and position of sulfate groups, glycosaminoglycans can be divided into 5 main categories: hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratin sulfate (KS), heparan sulfate, and heparin (HP).

[0153] 5) Hyaluronic acid (HA), also known as hyaluronic acid or hyaluronic acid salt, is a glycosaminoglycan and a major component of the extracellular matrix (ECM). It participates in tissue reconstruction, intercellular space expansion, inflammatory responses, and many other cellular physiological and tissue repair processes. Studies have shown that hyaluronic acid has significant cellular effects, significantly contributing to cell chemotaxis and migration, as well as cell proliferation. This mechanism occurs through the binding of HA-binding proteins to HA on the cell surface. Adhesion molecule CD44 is the cell surface receptor for HA and is a widely distributed cell surface glycoprotein involved in specific cell-cell and cell-matrix adhesions. Ru-Ming Liu, et al., in *Experimental Cell Research*...

[0154] An article published in Volume 345, Issue 2, July 15, 2016, Pages 218-229 indicates that hyaluronic acid can also promote mesenchymal stem cell proliferation by activating the Wnt / β-Catenin signaling pathway.

[0155] 6) Acellular Tissue Matrix (ACTM): This refers to the process of removing or inactivating cells, viruses, DNA, and other components in animal organs or tissues that would cause an immune rejection response, using specific reagents and treatment methods, while preserving the integrity of the original natural three-dimensional structure to the greatest extent possible, and retaining as many cell growth factors and active functional components as possible in the original matrix. Due to its natural three-dimensional (3D) structure, the presence of bioactive factors, the ability to be degraded by the host, and the ease with which it can induce the migration and differentiation of recipient stem cells, acellular matrix is ​​widely used in clinical practice for tissue repair and regeneration (congenital defects and acquired trauma). Acellular matrix is ​​a novel tissue regeneration and repair material with excellent biological scaffold properties.

[0156] 7) Decellularized regenerative repair biomaterials from xenogeneic animals, including but not limited to bio-derived materials for various superficial or deep injuries (whether caused by internal or external factors, or acute or chronic). In this patent, it mainly refers to the ECM three-dimensional structure remaining after processing animal-derived tissues through decellularization processes, containing various cytokines and bioactive components. The animal-derived tissue raw materials mentioned in this patent can be derived from the dermis, submucosa of the small intestine, bladder matrix, pericardium, or other tissues. The physical appearance of the decellularized matrix preparation can be one or a combination of powder, granules, gel, paste, and sheet.

[0157] Technical terms in the subfield of hair

[0158] 1) Dermal Papilla Cells (DPCs) are a group of dermal cells located at the base of the hair follicle, playing a leading role in hair follicle growth and development, cycle regulation, and maintaining hair growth. Abnormalities in the structure or function of DPCs are the main initiating factors leading to hair follicle growth imbalance. The concealed spatial location of the dermal papilla presents considerable challenges for its isolation and culture, but DPCs have crucial physiological functions, and research on them has been ongoing, gradually becoming a hot topic in the study of hair follicle growth, development, and cycle regulation. In a cellular sense, DPCs can be classified as mesenchymal stem cells, belonging to the category of pluripotent stem cells. Pluripotency means that they can differentiate into stem cells with various tissue structures. Cultured DPCs in vitro resemble fibroblasts morphologically, but are fundamentally different. The most prominent characteristic of cultured DPCs in terms of growth pattern is the formation of multilayered cell clumps before cell fusion, i.e., the characteristic of agglutinative growth. However, the agglutinative growth characteristic of dermal papilla cells gradually weakens with increasing cell passage number. Generally, dermal papilla cells after the 7th passage will lose their agglutinative growth ability. Agglutinative growth is one of the important biological characteristics of dermal papilla cells and is closely related to their biological functions. 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 an important reason for using F2 protective solution and employing a dual low-concentration, gentle decellularization method in this invention; one of its purposes is to preserve more and better exosomes from the ECM.

[0159] 2) Hair follicle stem cells (HFSC) are the primitive cells in hair follicles. Hair and body hair grow from hair follicles.

[0160] However, hair follicle stem cells responsible for hair growth are usually in a dormant state, but they will rapidly activate and divide in a new hair growth cycle. When adjacent areas are damaged, hair follicle stem cells can migrate from their original location in the ridge and participate in the repair of the damaged area. The hair follicle is the basic unit of hair, and hair grows and differentiates from cells within the hair follicle. Each hair follicle contains a group of hair follicle stem cells, providing new cells to the growing hair follicle. Human hair follicle stem cells are a type of adult stem cell and share the common characteristics of adult stem cells. Many tissue functions in the human body depend on the healthy activity of stem cells, and damage to or reduction in the number of stem cells can lead to aging.

[0161] Technical terms related to stem cell regeneration

[0162] 1) Wnt signaling is closely related 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 determines cell differentiation fate during development. This developmental cascade integrates signaling from other pathways, including basic fibroblast growth factor (bFGF) and transforming growth factor.

[0163] β (TGF-β) and bone morphogenetic protein (BMP) are present in various cell types and tissues; Wnt peptides form a highly conserved family of secretory signaling molecules that regulate cell-cell interactions during tissue regeneration; Wnt family factors are signaling molecules that function in multiple cellular pathways and cell-cell interactions; Wnt signaling and tissue regeneration, the terms “Wnt” or “Wnt gene product” or “Wnt peptide” as used herein include native Wnt peptide sequences, Wnt peptide fragments, chimeric Wnt peptides, or functionally active variants of the aforementioned types. The terms “WNT” and “Wnt” are used interchangeably herein. As used herein, “bioactivators that activate the Wnt pathway” can be, for example, bioactivators that activate the classical Wnt pathway (also known as the β-linkin pathway) or bioactivators that activate non-classical Wnt pathways (planar cell polarity pathway; PCP)

[0164] Bioactivators of the Wnt pathway (also known as the Ca2+ pathway). Examples of typical bioactivators that can activate the Wnt pathway, as well as some non-classical bioactivators that activate the Wnt pathway.

[0165] 2) Basic fibroblast growth factor (bFGF, or fibroblast growth factor-2, FGF-2) has been reported in the literature to have effects on hair follicle proliferation and cycle transition in hair follicle organoid 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 the hair follicle cycle during the hair loss process. In mouse models of hair loss, some growth factors, individually or synergistically, promote hair growth, with bFGF showing potential therapeutic effects for hair loss. Research has confirmed that FGF can induce β-Catenin and the Shh pathway in mice, accelerating the transition of hair follicles from the telogen phase to the anagen phase. bFGF plays an important role in hair follicle cell proliferation and cycle transition after hair loss and helps enhance the Shh signaling pathway.

[0166] 3) The Sonic hedgehog (Shh) pathway plays a crucial role in hair follicle growth, development, and cycle transition. Shh is a key gene in this pathway, upregulated in the early anagen phase, and is one of the factors regulating hair follicle growth and cycle transition. Although a direct link between inducing hair follicle cell apoptosis and Shh has not yet been found, disruption of Shh signaling has been identified as a key event in the mechanism of hair loss.

[0167] Additional explanation: The decellularized matrix formulation of this invention, when used as a hair growth promoter, can promote the production of growth factors in dermal papilla cells; for example, it stimulates 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 this invention can also be called a dermal papilla cell growth factor production promoter, a dermal papilla cell vascular endothelial growth factor (VEGF) production promoter, a dermal papilla cell fibroblast growth factor (FGF) production promoter, and a dermal papilla cell insulin-like growth factor-1 (IGF-1) production promoter. In summary, throughout the entire preparation process of the xenogeneic animal decellularized matrix, including the immediate period after removal from the living organism, is the target tissue subjected to targeted pretreatment, and are professional, targeted, or specific (tissue or membrane) protective solutions used?

[0168] Immediately after the target tissue leaves the living body, i.e. in the early stage (after being kept at a constant temperature after leaving the living body) or in the early stage, can the tissue be preserved or pretreated in a non-destructive or minimally destructive manner, i.e., whether it is immersed in a low-temperature and high-quality protective solution?

[0169] Is the entire decellularization process gentle, minimally invasive, rapid, efficient, and suitable for the corresponding target tissue?

[0170] And whether the subsequent deep processing (freeze-drying and granulation) and sterilization processes of ECM are appropriate and suitable.

[0171] These key or important technical details directly affect whether the natural three-dimensional structure and various effective active ingredients (such as Wnt and bFGF bioactive factors) in the final ECM product can be adequately protected and preserved. In other words, whether the natural biological structure, effective ingredients, and bioactive factors of ECM can be effectively protected in a three-dimensional and comprehensive manner, achieving high-quality protection in terms of quality, quantity, and integrity (equivalent to four protections: preservation, good condition, preservation, and integrity).

[0172] Therefore, this technical field needs to develop an ECM formulation that is biocompatible, has natural biological components and structure, can endogenously induce regeneration, including promoting hair regeneration, has a controllable duration of effect, can be minimally invasively injected (with appropriate particle size), is convenient for clinical use, has a wide range of applications, and is suitable for large-scale production.

[0173] To further elaborate on the purpose of this invention, an extracellular matrix or tissue protection solution has been developed.

[0174] Of course, those skilled in the art, whose IPC main classification number is A61L27, can also reasonably deduce that the protective solution of this invention is also applicable to other equivalent or similar tissues to be decellularized. For example, when the protective solution is used to soak target tissues such as dermal membrane, bladder membrane, pericardium, peritoneum, and amnion at low temperature (2-8 degrees Celsius), the protective solution can prevent cell membranes and lysosomes from rupturing and prevent the degradation and destruction of various active ingredients and growth factors in the extracellular matrix (ECM).

[0175] Furthermore, the target tissue disclosed in this embodiment is small intestinal submucosal tissue or ECM from other sources. That is, the present invention discloses a protective solution for small intestinal submucosal tissue (SIS) or other extracellular matrix from other sources.

[0176] Furthermore, the protective solution for the submucosa of the small intestine or other ECM sources is a membrane that protects the ECM in the small intestinal tissue or other sources. Before pretreatment or decellularization, the protective solution can protect the cell membrane and 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, etc.

[0177] Furthermore, the protective solution of the present invention is a protective solution for cell membranes and lysosomal membranes, and it can also be an isotonic protective solution for the extracellular matrix with high sodium and low potassium; it is completely different from the antioxidant protective solutions in the prior art and does not belong to the same inventive concept.

[0178] Furthermore, the extracellular matrix protection solution disclosed in this invention comprises at least the following two types of components: isotonic solution and lysosomal stabilizer.

[0179] The first type of component: isotonic solution, which may also include buffers such as lactate-sodium lactate, citrate-sodium citrate, and succinate-sodium succinate.

[0180] Furthermore, in the F2 solution, the sodium ion concentration was 145 mmol / L ± 10%, and the potassium ion concentration was 4 mmol / L ± 10%; the pH was adjusted to 7.4, which is weakly alkaline, basically consistent with the pH of small intestinal fluid (7.6), both being weakly alkaline. (Cryogenic cells)

[0181] The second category of components includes lysosomal stabilizers, specifically glucocorticoids (such as hydrocortisone), prostaglandins (PGI, PGE, and cathepsin inhibitors; anisodamine can inhibit Ca2+ influx. (Note: Promoting the stability of lysosomal membranes within body tissue cells is considered a mechanism of the anti-inflammatory effects of corticosteroids; cortisone is a 21-carbon steroid hormone, one of the main hormones released by the adrenal glands in stress response; it can control and regulate the release of lysosomal enzymes into the cytosol.)

[0182] Furthermore, it can specifically include cortisone, hydrocortisone, prednisolone, prednisolone, methylprednisolone, etc.

[0183] Furthermore, the aforementioned lysosomal stabilizer may preferably be a nonsteroidal anti-inflammatory drug (NSAID), including sodium or potassium salts of salicylates, arylpropionic acids, anilines, indoles, arylaceous acids and their derivatives, or a combination thereof. The NSAID is selected from aspirin, diclofenac, loxoprofen, ibuprofen, acetaminophen, celecoxib, etodoxacin, pranoprofen, flurbiprofen ester, lornoxicam, thiamide, tramadol, and zaltoprofen, as well as a pharmacologically acceptable sodium or potassium salt of these components, or a combination thereof.

[0184] Furthermore, salicylic acid is preferred as the lysosomal stabilizer;

[0185] Aspirin is a more preferred lysosomal stabilizer;

[0186] Furthermore, the concentration of aspirin in the protective solution is 0.1 to 1 gram per liter.

[0187] In addition, the following components may be added to the F2 liquid of the present invention as needed, such as: preservatives (ethylparaben, butylparaben, chlorphenesin, phenoxyethanol, etc.); anti-inflammatory agents (e.g., glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, physalicylic acid, zinc oxide, allantoin, etc.).

[0188] Of course, based on the present invention, antioxidants can also be added to the F2 protective solution.

[0189] Furthermore, it can also specifically include plant-derived antioxidants, animal-derived antioxidants, and / or allopurinol and other antioxidants that combat free radicals.

[0190] Furthermore, this can specifically include N-acetylcysteine ​​(NAC), an N-acetylated derivative of cysteine. NAC has good antioxidant and anti-inflammatory effects and can generate glutathione (GSH) in vivo. The presence of SH helps scavenge H₂OOH and HOCl. NAC readily deacetylates to become cysteine, which then forms glutathione. Additionally, NAC can reduce pro-inflammatory mediators. Based on its current pharmaceutical uses, NAC's powerful expectorant effect has three mechanisms: first, disrupting disulfide bonds in sputum mucoproteins and reducing sputum viscosity; second, enhancing the airway clearance function of respiratory cilia; and third, increasing alveolar surfactant. Traditionally, NAC is a commonly used expectorant, suitable for respiratory diseases such as bronchitis and bronchiectasis accompanied by hypersputum secretion. Currently, there is new medical understanding of its efficacy and pharmacological mechanisms, expanding its pharmacological effects and developing new dosage forms, such as oral formulations (tablets, granules) and nebulized solutions. More importantly for this invention, NAC also possesses antioxidant and anti-inflammatory effects, and can generate glutathione (GSH); NAC can scavenge oxygen free radicals. Furthermore, some researchers have discovered that NAC has a synergistic antibacterial effect, enhancing the antibacterial activity of piperacillin and ciprofloxacin against pathogens. The synergistic antibacterial mechanism of NAC includes: 1. NAC can reduce the number of glycolipids in epithelial cells, inhibiting pathogen adhesion; 2. NAC can disrupt bacterial biofilms, enhancing the bactericidal effect of antibacterial drugs and reducing bacterial resistance. Therefore, in addition to its expectorant effect, NAC also possesses antioxidant, anti-inflammatory, and synergistic antibacterial pharmacological effects; its applications are further broadened. This is an additional inventive point added to the F2 solution of this invention, and is also the main reason for choosing NAC as an important antioxidant, which can be attributed to a second inventive point outside of this invention.

[0191] Further preferably, the NAC concentration of N-acetylcysteine ​​is 0.2–1 g / L; more preferably, it is 0.6 g / L.

[0192] On the other hand, antioxidants such as phosphoric acid, citric acid, ascorbic acid, succinic acid, fumaric acid, phospholipids, hexametaphosphate (esters), etc., can be added on or in addition to the original basis of this invention; as well as antibiotics (such as ciprofloxacin and / or streptomycin and / or chlortetracycline) and anti-inflammatory drugs (such as tranexamic acid, thiotaurine, taurine, etc.).

[0193] The inventor would like to emphasize that while the original technical concept of this invention did indeed partially reference or draw inspiration from organ transplant preservation solutions (such as the University of Wisconsin-UW solution and HTK solution), and there are certain similarities, the two are fundamentally different and significantly distinct in terms of specific technical details or underlying mechanisms of action.

[0194] Common organ transplant preservation solutions include:

[0195] UW solution is characterized by high potassium (125 mmol / L) and low sodium (29 mmol / L), similar in composition to intracellular fluid. The high concentration of K+ ions inhibits the outflow of K+ from the cell down its concentration gradient; it also contains hydroxyethyl starch (HES), a colloidal carrier that retains the fluid within the vascular space, preventing extracellular swelling; lactobionic acid and gorsose, both membrane-impermeable substances, reduce tissue edema; glutathione, as a reducing agent, scavenge oxygen free radicals and maintain cell membrane integrity; adenosine, a substrate for the synthesis of adenosine triphosphate (ATP), provides energy support for organ metabolism; and allopurinol, a xanthine oxidase inhibitor, has a protective effect during ischemia. UW solution is primarily used for organ preservation before liver, pancreas, and kidney transplantation; it is used to preserve the viability and / or activity of tissue cells.

[0196] HTK solution is mainly composed of histidine, with added tryptophan and ketoglutarate to promote ATP production. The low potassium content reduces calcium overload, and the addition of glycine, alanine, and LK614 (iron chelating agent) can reduce damage from oxygen free radicals.

[0197] Celsior solution, with lacturonic acid as the osmotic pressure component and histidine as the buffer system, is a high-sodium, low-potassium extracellular fluid type of protective solution. The low potassium content restricts the opening of voltage-dependent calcium channels, limits 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.

[0198] HCA solution is a hypertonic citrate-purine solution, high in potassium and low in sodium (intracellular fluid type), with histidine as a buffer system and dextran to maintain osmotic pressure. It is mainly used as a preservation solution during kidney transplantation.

[0199] Firstly, UW fluids are mainly used for organ preservation before transplantation; while the F2 fluid of this invention does not need to preserve the viability and / or activity of tissue cells; the F2 fluid mainly maintains the osmotic pressure of extracellular fluid and prevents various types of cell membrane rupture.

[0200] Secondly, F2 solution does not need to provide energy levels similar to those in organ preservation solutions, meaning it does not require the supplementation of carbohydrates (such as glucose, lactose, sucrose, etc.) to maintain the vitality and survival of cells in tissues / organs. In other words, the purpose of F2 solution is mainly to maintain cell membrane stability and prevent its rupture, and further to prevent the rupture of lysosomal membranes and the release of proteases from cells, which could cause rapid degradation and severe damage to the extracellular matrix.

[0201] Thirdly, the present invention sets the osmotic molar concentration of the F2 protective solution to 280-300 mOsmol / kg, which is a low-temperature osmotic pressure, slightly lower than that of organ transplant preservation solutions. This is mainly because the temperature of the F2 protective solution is maintained at 2-8℃. At the same time, the pH is adjusted to be slightly alkaline, about 7.2-8.2, preferably 7.6. This is mainly because the original tissue, namely the small intestinal tissue, especially the digestive juice 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 rupture).

[0202] Fourthly, UW solution (University of Wisconsin solution) refers to the preservation solution used in organ transplantation to preserve donor organs. It primarily contains lactobionic acid, a major non-permeable anion with a relatively large molecular weight, which can reduce cell swelling during refrigeration. It also contains raffinose, hydroxyethyl starch, and adenosine. It mainly uses hydroxyethyl starch (50 g / L) to maintain (colloidal) osmotic pressure. Hydroxyethyl starch (HES) is a widely used synthetic colloidal solution in clinical practice and is also a natural polysaccharide. Hydroxyethyl starch (HES) is a high-molecular-weight complex formed by the hydroxyethylation of the glucose rings of amylopectin in corn or potatoes. Natural starch cannot be used as a plasma substitute because it is unstable and easily hydrolyzed by endogenous amylases. Hydroxyethylation of starch can delay its breakdown and elimination in the blood, significantly prolonging its residence time in blood vessels. HES has the effect of expanding blood volume; however, this invention does not use hydroxyethyl starch products, i.e., it does not contain colloidal solutions.

[0203] In this invention, the target tissue is soaked in a protective solution (F2 solution) before decellularization, and then decellularized using a double low-concentration solution to prepare ECM. Furthermore, the method for preparing the hair-growth-promoting ECM microparticles involves using porcine small intestinal submucosa or ECM from other sources as raw materials, and soaking them in a low-temperature F2 solution to protect the active ingredients in the extracellular matrix from degradation and destruction.

[0204] Other process steps, such as inactivation, decellularization, lyophilization, granulation, dispensing, and sterilization, are basically the same as conventional methods. The difference lies in the decellularization reagent and concentration; this invention uses a mixed solution of 0.05% trypsin and 0.05% EDTA for decellularization (i.e., a dual low-concentration, gentle combined method) to prepare the decellularized matrix.

[0205] The mechanism of action of this invention is as follows: by using F2 protective solution, on the one hand, it prevents the cell membrane in the target tissue from being pathologically or physiologically ruptured by various external non-physiological environmental factors after leaving the living tissue, and strives to maintain the integrity of the cell membrane structure.

[0206] Furthermore, F2 solution can also prevent intracellular lysosomes from becoming independent after cell membrane rupture, which would make lysosomes easily break down and release various proteases from the lysosomes.

[0207] F2 protective solution can prevent lysosome rupture, thereby inhibiting / reducing the release of various proteases and preventing their degradation of ECM, including WNT pathway active factors (WNT ligands and / or agonists, including Wnt2b); thus maximizing the preservation of the activity of various active ingredients and cell growth factors in ECM powder; this type of ECM can better promote the activation or regeneration of various stem cells, such as promoting the activation and regeneration of hair follicle stem cells, thereby achieving the medical purpose of hair care and hair regeneration.

[0208] Compared with the prior art, the present invention has the following beneficial effects:

[0209] 1) The protective solution provided by this invention has a simple formula, which can protect both cell membranes and lysosomal membranes, and also inhibit the protease activity of lysosomes; thus achieving the effects of double membrane protection and increasing Wnt2b content.

[0210] 2) The protective liquid provided by this invention uses readily available raw materials, which are all commercial products and inexpensive.

[0211] 3) The decellularized matrix preparation prepared by this invention simply adds a low-temperature immersion protection treatment before the original ex vivo tissue is decellularized; it is simple, convenient, easy to master, easy to operate, and highly feasible.

[0212] 4) The ECM products provided by this invention, especially the ECM microparticles, have the advantage that they can fully remove the immunogenic components in SIS, have good biocompatibility, and retain the natural three-dimensional structure of SIS and active ingredients, including collagen and various growth factors, to the greatest extent.

[0213] 5) The ECM preparation prepared by this invention can be applied to the head or scalp to prevent hair loss and / or induce new hair growth.

[0214] 6) The hair growth preparation of the present invention is richer in various growth factors; on the one hand, it promotes scalp blood circulation and increases blood supply to the hair papilla; on the other hand, it can improve hair follicle nutrition, slow down the process of hair follicle miniaturization, increase the number of hair follicles, and promote hair growth; thus achieving the dual effect of preventing hair loss and protecting hair growth.

[0215] 7) Compared to several clinical hair growth agents (minoxidil, finasteride, including some plant extracts and plant extract hair growth products, although they have been used for many years, they still have some side effects, and their hair growth effect is short-lived and will gradually disappear over time; compared to single-ingredient products that cannot or have difficulty inducing the regeneration of hair HFSC / secretion of collagen and other hair functional ingredients, which will completely degrade and disappear after a period of use; the ECM preparation of this invention has medium and long-term efficacy.

[0216] 8) Consumers only need a few injections, and the hair regrowth is done in the original area (without involving complicated procedures such as scalp transplantation); thus reducing unnecessary trouble and financial burden for consumers; it can effectively and lastingly induce the regeneration of autologous hair HFSC, promote DPC activation and growth and development; the effect is stable and long-lasting, requiring only a limited number of re-injections.

[0217] In this patent, the term "comprising" (and related terms such as "comprising," "having," or "including") includes embodiments, such as compositions, methods, or processes of any substance, that are "consisting of the described features" or "consisting substantially of the described features." Regarding isotonic agents and isotonic solutions, the applicant needs to clarify that, in this patent, when referring to numbers or numerical ranges, the use of data terminology means that the referred numbers or numerical ranges are subject to experimental variation or variability or statistical experimental error, and point values ​​of data can also be considered approximate values; that is, the numbers or numerical ranges can vary, typically from -10% to +10% of the stated numbers or numerical ranges.

[0218] In addition, unless otherwise specified, the numerical values ​​in the test results are usually the average of the test results of at least three samples or examples.

[0219] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any 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 commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of the present invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, used, or similar to those described in the embodiments of the present invention may be used to implement the present invention.

[0220] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0221] Before describing 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 embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0222] The 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 should not be limited thereto; the embodiments are not intended to limit the scope of the present invention in any way. In the following embodiments, various processes and methods not described in detail are methods of common knowledge used in the art; it may also include methods and techniques commonly used in the field of medical biomaterials that have been omitted. Detailed Implementation

[0223] Example 1: Preparation of F2 Protective Solution and Suspension Solution (One of the key points of this invention)

[0224] 1) F2 protective solution, which includes two types of components: isotonic agent and lysosomal stabilizer.

[0225] Step 1: Prepare an isotonic solution. In this invention, a commercially available isotonic whole colonic irrigation solution (from Shenzhen Wanhe Pharmaceutical Co., Ltd., trade name: He Shuang; generic name: Compound Polyethylene Glycol Electrolyte Powder (II), namely, 60g of polyethylene glycol 4000, 5.68g of anhydrous sodium sulfate, 1.46g of sodium chloride, 0.75g of potassium chloride, and 1.68g of sodium bicarbonate) by adding water to make a solution of 125 mmol / L sodium ions, 10 mmol / L potassium ions, 20 mmol / L bicarbonate ions, 40 mmol / L sulfate ions, and 35 mmol / L chloride ions) for the whole colonic irrigation. After preparation, mix thoroughly and adjust the pH value to 7.4.

[0226] Step 2: Add a small amount of lysosomal stabilizer, i.e., an NSAID drug. Specifically, choose aspirin, adding 0.2 grams (w / v) of aspirin per liter of protective solution to the solution prepared in Step 1 (isotonic whole bowel lavage solution). Stir thoroughly and store at 4 degrees Celsius until use. II) Preparation of the suspension: Use 0.5% (w / v) glycerol and 0.2% (w / v) sodium hyaluronate, with a molecular weight of 120-150 million Daltons. Use phosphate buffer to adjust the pH of the suspension to 7.10 and the osmotic pressure to 330 mOsmol / L. Store at room temperature until use.

[0227] Example 2: Preparation of conventional ECM microparticles (i.e., control group ECM, without immersion in protective solution).

[0228] (1) Inactivation: Take the submucosa of pig small intestine and use a mixed aqueous solution of 1% peracetic acid and 20% ethanol to inactivate it by soaking at room temperature for 60 minutes under ultrasonic conditions.

[0229] (2) Decellularization: The inactivated SIS was first washed with water under sonication, aliquoted into small packages, and rapidly frozen overnight. The next day, the freeze-thaw cycle of "washing-aliquoting-freezing-thawing" was repeated. The mixture of 0.05% trypsin and 0.05% EDTA was used for treatment under sonication at 36±2℃, followed by sonication washing with PBS. The mixture was then treated in a 15% sodium chloride hypertonic solution under sonication at 36±2℃, followed by washing with water. The mixture was then treated in a 25mM NaOH aqueous solution under sonication at 36±2℃, followed by sonication washing with water until neutral. Finally, DNase was added to remove residual DNA.

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

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

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

[0233] (6) Sterilization: Cobalt-60 sterilization method is selected, with a radiation dose of 25kGy-30kGy.

[0234] Example 3: Preparation of ECM microparticles (i.e., preparation of highly active ECM by immersion in a low-temperature F2 protective solution).

[0235] The process steps are basically the same as in Example 2, except that before or during pretreatment, i.e. after the target tissue leaves the living organism (animal living tissue is usually between 35-39°C, more commonly 36-38°C), the F2 protective solution prepared in Example 1 is immediately applied to the target tissue (which may also include the target organ), specifically the submucosa of the porcine small intestine (SIS). (Note: Of course, those skilled in the art, with IPC main classification number A61L27, can reasonably deduce that the protective solution of this invention is also applicable to other equivalent or similar tissues or organs to be decellularized). Using the protective solution of this invention, the tissue to be decellularized is soaked at a low temperature (2-8°C) for at least one hour to protect the active ingredients and / or various cell growth factors in the extracellular matrix from degradation and destruction.

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

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

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

[0239] It is important to note that the pretreatment of the ECM samples prepared in both examples before detection should use urea-heparin solution (non-enzymatic degradation method) and be performed according to the literature method. Regarding the detection of relevant active components (growth factors) in the ECM prepared in Examples 2-3, this invention primarily targets typical regenerative growth factors, which are also cytokines closely related to stem cell regeneration, such as hair growth, namely basic fibroblast growth factor (bFGF), using ELISA. The detection results are as follows:

[0240] In Example 2, the bFGF content in the ECM was 12.76 + / - 0.71 pg / mg, and the Wnt2b content was 48.79 + / - 0.18 pg / mg.

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

[0242] In summary: After the target tissue is removed from the living body (36-38℃), it is soaked in a cryoprotectant solution before undergoing subsequent processing, including decellularization. This type of cryoprotectant soaking 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 this invention and actual test results, it can be logically and scientifically deduced that the ECM prepared by this invention (immersed in preservation solution) can better retain regeneration-related active components (such as Wnt protein, bFGF, etc.) in the ECM. This indicates that the ECM prepared by this invention has a better natural ECM structure, can better bind to integrins on the surface of target cells or stem cells, promote the activation of stem cell signals or tissue regeneration at the treatment site, stimulate the activity of major stem cells, and initiate the repair and regeneration of various target tissues. In this patent, it particularly stimulates tissues related to hair regeneration, 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: Hair regrowth effect test of two types of ECM mixtures

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

Claims

1. A medical protective solution, used for immersion in low-temperature conditions before decellularization of mammalian target tissues, characterized in that, The protective solution includes an isotonic solution and a lysosomal stabilizer, referred to as F2 solution; the protective solution is used to prevent the degradation or destruction of active ingredients and / or various cell growth factors in the decellularized matrix (ECM); the low temperature condition is 2-8℃.

2. The isotonic solution according to claim 1, characterized in that, The isotonic solution is a commercially available isotonic whole colonic irrigation solution; the isotonic solution contains, per liter of protective solution, 60 g of polyethylene glycol, 5.68 g of anhydrous sodium sulfate, 1.46 g of sodium chloride, 0.74 g of potassium chloride, 1.68 g of sodium bicarbonate, with the remainder being deionized water; the polyethylene glycol is pharmaceutical grade PEG4000 as specified in the 2020 edition of the Pharmacopoeia.

3. The lysosomal stabilizer according to claim 1, characterized in that, The lysosomal stabilizer is a nonsteroidal anti-inflammatory drug (NSAID), including sodium or potassium salts of salicylates, arylpropionic acids, anilines, indoles, arylaceous acids and their derivatives, or combinations thereof. The NSAID is selected from aspirin, diclofenac, loxoprofen, ibuprofen, acetaminophen, celecoxib, etodoxacin, pranoprofen, flurbiprofen ester, lornoxicam, thiamide, tramadol, and zaltoprofen, as well as pharmacologically acceptable sodium or potassium salts of these components, or combinations thereof.

4. The lysosomal stabilizer according to claim 1, characterized in that, The lysosomal stabilizer is aspirin, with a final concentration of 0.1-1 gram per liter of protective solution.

5. A medical decellularized matrix, characterized in that, The decellularized matrix, whose source tissue is immersed in the protective solution of claim 1 after leaving the living tissue and before decellularization treatment, significantly increases the content of active ingredients in the ECM.

6. The decellularized matrix according to claim 5, characterized in that, The decellularized matrix is ​​the submucosa of the porcine small intestine, containing one or a combination of Wnt2b and bFGF.

7. An injectable medical decellularized matrix preparation, characterized in that, The formulation consists of a decellularized matrix and a suspending agent, individually packaged and sterilized separately, and mixed before use; the decellularized matrix consists of particles with a diameter between 50 μm and 400 μm; the suspending agent contains glycerol, sodium hyaluronate, and phosphate buffer; the source tissue of the decellularized matrix, after leaving the living organism, is first immersed at low temperature in the protective solution described in claim 1, and then gently decellularized using a mixed solution of 0.05% trypsin and 0.05% EDTA (i.e., a dual low-concentration method).

8. A method for preparing an injectable decellularized matrix formulation as described in claim 7, characterized in that, It includes the following three steps: Step 1: Preparation of decellularized matrix microparticles (a) Protective solution immersion and pretreatment: The target tissue that has been removed from the living body is immersed in the protective solution described in claim 1 under low temperature conditions to protect the active ingredients and / or various cell growth factors in the extracellular matrix from degradation and destruction. Pretreatment refers to the removal of impurities and grease; (b) Inactivation, decellularization, DNA removal, and freeze-drying; (c) Microparticle preparation: freeze-dried ECM is frozen with liquid nitrogen, then crushed and sieved to obtain microparticles with a diameter between 50μm and 400μm. (d) Dispensing and sterilization; Step 2: Preparation of suspending agent 1) Prepare a liquid containing glycerol, sodium hyaluronate, and phosphate buffer, wherein the glycerol concentration is 0.5%; The sodium hyaluronate concentration is 0.2%, and the molecular weight is between 1 million and 1.6 million Daltons; phosphate buffer is used to adjust the pH and osmotic pressure of the suspending agent; 2) Dispense the suspending agent into containers that match its sterilization method, seal, and sterilize by moist heat; Step 3: Decellularized matrix microparticles and suspending agents are individually packaged and sterilized separately. Before use, they are thoroughly mixed using a three-way tube. The mixture is then prepared into an injectable decellularized matrix microparticle formulation with good tissue repair and / or regeneration functions.

9. The medical use of a decellularized matrix according to claims 5 to 6, characterized in that, The medical purpose is to repair defects in tissues and / or organs; the tissues and organs are defects in the following areas: head appendages, hair, ear, face, periosteum, nerves, dura mater, spinal dura mater, tendons, ligaments, urinary tract, bladder, ureter, inguinal hernia, hiatal hernia, abdominal wall hernia, oral soft tissues, joints, menisci, etc.

10. The medical use of the decellularized matrix preparation according to claims 7 and 8, characterized in that, The medical application is for scalp use to prevent various types of hair loss in adults and to help promote hair follicle growth. The scalp application methods include external application to the scalp in the hair loss area, assisted introduction using microneedles, nanochips, ultrasonic ions, subcutaneous injection, microneedle patches on the scalp in the hair loss area, or as an in vitro culture medium for hair follicle-related tissues and cells. The medical purpose is to activate the growth of hair follicle stem cells. The hair follicle stem cells refer to one or a combination of DPC and HFSC from scalp hair follicles.