A cold preservation solution and application of a combined traditional Chinese medicine compound biological stent drug delivery system in tympanic membrane repair

Abstract

The application relates to the field of biological medicine, and particularly relates to a cold storage solution and application of a combined traditional Chinese medicine compound biological stent drug delivery system in eardrum repair. The cold storage solution comprises a basic solution and L-alanyl-L-glutamine; the basic solution comprises a STEEN solution. The cold storage solution can promote metabolism and synthesis of glutathione of fat grafts, regulate ROS mitochondrial oxidative stress, reduce cold storage damage of the fat grafts, and improve the cold storage quality of the fat grafts. Meanwhile, the application uses Shengji Yuhong collagen sponge which contains an important classical prescription for promoting repair, forms a functional biological material with supporting and pro-angiogenic effects, forms a linkage mode of "double-stage regulation before and after transplantation", closely combines the actual difficulties of cell damage of fat groups and reduction of repair success rate caused by long eardrum repair time in the clinic, explores a brand-new research mode for improving the eardrum repair scheme, and has far-reaching clinical significance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a cryopreservation solution and its combination of traditional Chinese medicine compound biological scaffold drug delivery system in tympanic membrane repair. Background Technology

[0002] Tympanic membrane perforation is a common disease in otolaryngology, often triggered by ear trauma or inflammation. Common symptoms after tympanic membrane perforation include ear pain and discomfort, dizziness, hearing loss, external auditory canal bleeding, and external auditory canal lacerations, making it a common emergency case in otolaryngology. Hearing loss can manifest as mild to moderate conductive hearing loss; without timely intervention, it may lead to permanent perforation and irreversible hearing loss. Most minor acute perforations tend to heal spontaneously, while large acute tympanic membrane perforations and most chronic perforations do not heal on their own. Currently, international research on tympanic membrane perforation is limited, mainly focusing on the diagnosis and surgical sampling; perioperative research and the use of traditional Chinese medicine preparations are still lacking. Surgical repair is currently the primary treatment for tympanic membrane perforation. However, during tympanic membrane perforation surgery, it is routine to remove tissue from the perforation edge before transplantation, creating a fresh wound that can lead to postoperative inflammatory reactions. Due to the unique nature of the packing and bandaging after tympanic membrane repair, dressing changes cannot be performed as routinely as for open wounds on the body surface. Repeated removal and packing of the external auditory canal filler can alter the position of the repair graft, leading to repair failure. As a result, the opportunity to administer medication to control inflammation and prevent infection after repair is missed, and progressive inflammation reduces the survival rate of the tympanic membrane graft.

[0003] The three-dimensional structure and survival rate of tympanic membrane graft materials are key factors for successful tympanic membrane repair. In modern clinical practice, temporalis fascia and tragus cartilage are the most commonly used grafts in tympanic membrane repair. However, both require large incisions inside, outside, or behind the ear for harvesting, and their regeneration is slow and the supply is limited, restricting their application in tympanic membrane repair. The inability of tympanic membrane repair materials to survive is a direct cause of repair failure. Subsequent secondary repairs not only undermine patient confidence and delay treatment but also increase the patient's financial burden and weaken the repair effect. In contrast, fat pads are often chosen as repair materials for small tympanic membrane perforations, leveraging their ability to repair the tympanic membrane, showing promising potential. Fat pads also contain abundant adipose-derived stem cells and extracellular matrix (mainly composed of collagen, elastin, and mucopolysaccharides), possessing a certain degree of shape-forming ability. Adipose-derived stem cells have also been shown to have the potential to differentiate into various cell types, including chondrocytes, vascular endothelial cells, and nerve cells. Proper induction offers unlimited potential for promoting tympanic membrane healing and improving hearing loss.

[0004] The survival and efficacy of tympanic membrane transplant materials depend on angiogenesis and epithelial formation after transplantation. The time between graft removal and transplantation varies from one to several hours. After removal, the graft is often stored on gauze soaked in physiological saline; or, to achieve greater shape flexibility, it may be allowed to dry and dehydrate. After normal tissue is removed, ischemia and hypoxia disrupt normal metabolism. Lysosomes released from broken cells and the proliferation of microorganisms in the surrounding environment gradually erode normal cells. The accumulation of metabolic waste and the fragmentation of intracellular DNA lead to irreversible cell degeneration. Although removal during surgery does not cause complete tissue necrosis, it still cannot prevent the reduction in cell viability, inevitably negatively impacting the subsequent recruitment of angiogenesis factors and the initiation of the repair process. Cryopreservation is a common method for preserving transplant donors, developed over nearly half a century and widely used clinically. Rapid cryopreservation allows the graft to enter a dormant state in the shortest possible time, reducing cell metabolism and death. Common cryopreservation conditions are 50% oxygen and 4°C, but the associated problems of cryopreservation damage and cold shock are also key limitations on long-term graft preservation. Graft resuscitation after cryopreservation activates mitochondrial oxidative stress in cells, leading to the production of large amounts of reactive oxygen species (ROS), causing mitochondrial damage and cell death. Simultaneously, inflammatory pathways are activated, producing a large number of inflammatory factors, all contributing to graft damage. STEEN solution is one of the commonly used cryopreservation solutions for in vitro organ perfusion, composed of human serum albumin, dextran 40, glucose, calcium chloride, magnesium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, sodium chloride, and water for injection. However, current research on STEEN solution is limited to the field of in vitro organ perfusion.

[0005] Tympanic membrane repair requires disrupting the old tympanic membrane fibrous annulus to create a fresh wound as a repair bed. Like other wounds, the repair process of the tympanic membrane after being covered and packed with a graft involves six processes: coagulation, inflammation, matrix synthesis and deposition, angiogenesis, fibrous tissue formation, and wound contraction and remodeling. Interference with any of these steps will prolong the healing process, leading to poor wound healing or even graft detachment. The use of medicated collagen offers unlimited possibilities for sustainable medication during and after tympanic membrane repair, especially with topical Chinese medicine preparations, paving the way for improved clinical efficacy and laying a methodological foundation for the inheritance and development of traditional Chinese medicine. Furthermore, compared to the negative effects of inflammatory factors during cryopreservation, inflammation plays a positive role in the early stages of wound repair. For example, interleukin-8 (IL-8) is released at the onset of inflammation, traveling directly from fibrin clots to the damaged tissue. Strong signals recruit working cells to the wound, where neutrophils are the main force in the fight, removing bacteria, foreign bodies, and damaged tissue, preventing wound infection. Furthermore, IL-8 has a strong pro-angiogenic effect, opening new oxygen and blood supply channels for the graft, thus promoting graft survival in multiple ways. Besides inflammation, angiogenesis is the most crucial stage of repair. The blood and nutrients transported by newly formed blood vessels to the fat pad are the material basis for later dermal repair. Chronic wounds such as tympanic membrane perforations are prone to skin defects and scarring due to repeated non-healing, resulting in poor local blood supply and increasing the difficulty of healing. Therefore, promoting angiogenesis is an important means to help heal refractory wounds. To reduce donor damage caused by cryopreservation, current research has explored optimizing cryopreservation temperature, oxygen concentration, and adding oxygen-carrying agents and antitrypsin to the cryopreservation solution, but an ideal solution has not yet been found.

[0006] The traditional Chinese medicine compound biological scaffold drug delivery system (hereinafter referred to as "this drug delivery system") described in this article is a combination of 95% ethanol extract of traditional Chinese medicine compound and collagen. The pharmacological effects of this drug delivery system in inhibiting inflammation of chronic ulcer wounds, promoting angiogenesis, and promoting cell epithelialization, thereby accelerating healing, have been verified in previous experiments. It can be used for external auditory canal packing in tympanic membrane repair to promote the healing and filling of tympanic membrane grafts and transplant beds.

[0007] This application uses the drug delivery system combined with cryopreservation solution to pack the external auditory canal of patients after tympanic membrane repair surgery, and observes its effects on transplant success rate, changes in wound inflammation response, average score of post-transplant neovascularization and epithelial formation, and inner ear damage such as hearing loss in tympanic membrane repair surgery of different degrees. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of a cryopreservation solution and its combination with traditional Chinese medicine compound biological scaffold drug delivery system in tympanic membrane repair.

[0009] To address the aforementioned technical problems, this invention discloses the application of a cryopreservation solution and its combined traditional Chinese medicine compound biological scaffold drug delivery system in tympanic membrane repair. The specific technical solution is as follows: In a first aspect, the present invention provides a cryopreservation solution comprising a base solution and L-alanyl-L-glutamine; wherein the base solution comprises STEEN solution. The STEEN solution is a lung perfusion solution. The cryopreservation solution is prepared by adding L-alanyl-L-glutamine to the base solution.

[0010] In the aforementioned cold preservation solution, the concentration of L-alanyl-L-glutamine is 4~16 mM. In some embodiments of the present invention, the concentration of L-alanyl-L-glutamine is 8 mM.

[0011] In some embodiments of the present invention, the formulation (500 mL) of the cryopreservation solution is specifically as follows: 2.5 g sodium chloride, 175 mg potassium chloride, 110 mg calcium chloride dihydrate, 121 mg magnesium chloride hexahydrate, 82 mg sodium dihydrogen phosphate monohydrate, 53 mg sodium bicarbonate, 1 g glucose, 2.5 g dextran-40, 35 g human serum albumin and 4~16 mM L-alanyl-L-glutamine, with PBS as the solvent.

[0012] Secondly, the present invention provides the application of the cryopreservation solution described in the first aspect in the cryopreservation of fat grafts. In some embodiments of the present invention, the fat grafts include, but are not limited to, adipocytes and / or adipose tissue.

[0013] The applications include any one or more combinations of the following (1) to (4): (1) Improve the activity of fat grafts; (2) Regulate the levels of inflammatory factors and / or vascular endothelial growth factor; In some embodiments of the present invention, the inflammatory factors include IL-8, IL-6, IL-1β and TNF-α; (3) Regulate the content of reactive oxygen species (ROS); (4) Increase glutathione (GSH) content.

[0014] The cold storage is carried out for 1-4 hours at a temperature of 2-8°C. In some embodiments of the present invention, the oxygen content in the cold storage environment is 48%-52%.

[0015] Thirdly, the present invention provides the application of the cryopreservation solution described in the first aspect in the cryopreservation of fat grafts in tympanic membrane perforation repair materials. The fat grafts are placed in the cryopreservation solution for cryopreservation to repair tympanic membrane perforations.

[0016] The tympanic membrane perforation repair materials include fat grafts and traditional Chinese medicine compound biological scaffold drug delivery systems.

[0017] The herbal compound biological scaffold drug delivery system is a Shengji Yuhong collagen sponge. In some embodiments of the present invention, the Shengji Yuhong collagen sponge is prepared by immersing a collagen sponge in a Shengji Yuhong drug solution with a concentration of 50-200 μg / mL. In some embodiments of the present invention, the Shengji Yuhong drug comprises the following components by mass fraction: 6 parts Angelica sinensis, 6 parts Glycyrrhiza uralensis, 6 parts Angelica dahurica, 6 parts Lithospermum erythrorhizon, 2.4 parts Dragon's Blood, 6 parts Terminalia chebula, and 6 parts Hibiscus rosa-sinensis flower. In other embodiments of the present invention, the Shengji Yuhong collagen sponge is prepared by immersing a collagen sponge in a Shengji Yuhong drug solution with a concentration of 100 μg / mL, wherein the 100 μg / mL Shengji Yuhong drug solution is obtained by extracting 10 mg of Shengji Yuhong drug and then adjusting the volume to 100 mL. In some other embodiments of the present invention, the regenerating Yu Hong collagen sponge is prepared by immersing a collagen sponge with a length and width of 50 mm and a height of 1 mm in 12.5 mL of a 100 μg / mL regenerating Yu Hong drug solution.

[0018] In this process, the fat graft is placed in a cryopreservation solution for cryopreservation and then transplanted to the tympanic membrane perforation site to repair the perforation.

[0019] The tympanic membrane perforation mentioned above includes chronic tympanic membrane perforation or acute tympanic membrane perforation.

[0020] Beneficial effects: This application represents the first domestic and international attempt to utilize in vitro cryopreservation technology to preserve fat grafts for tympanic membrane repair and transplantation, reducing cell metabolism and apoptosis during the waiting period before transplantation. By adding L-alanyl-L-glutamine, a parenteral nutrient, to the cryopreservation solution, the study promotes the synthesis of glutathione (GSH) from fat cells, regulates ROS mitochondrial oxidative stress, mitigates cryopreservation damage to the fat cell, and improves the quality of fat cell cryopreservation. Compared to current interventions during and after tympanic membrane transplantation, this application goes beyond simply changing surgical repair methods and finding artificial substitutes for autologous grafts. It not only focuses on improving the pre-transplant quality of autologous grafts but also, based on this, utilizes a classic formula containing collagen, an important repair-promoting agent, to form a functional biomaterial with supporting and angiogenesis-promoting effects. This creates a "pre-transplantation-post-transplantation dual-stage regulation" linkage model, closely addressing the practical difficulties of prolonged tympanic membrane repair leading to fat cell damage and reduced repair success rates. This research explores a novel approach to improving tympanic membrane repair, with profound clinical significance. Furthermore, due to the unique nature of packing and bandaging after tympanic membrane repair, it's impossible to routinely change dressings like for open wounds. Repeated removal and repacking of the external auditory canal packing material can alter the position of the graft, leading to repair failure and thus missing the opportunity for post-repair medication. The use of medicated collagen offers limitless possibilities for post-tympanic membrane repair medication, especially topical Chinese medicine preparations. It paves a new path for improving clinical efficacy and lays a methodological foundation for the inheritance and development of traditional Chinese medicine. Existing research often focuses on the mechanism of action of drugs on specific cells or cell types. However, in the body, the effects of drugs often target multiple cell types, especially in wound repair. When applying medication locally, the tissues surrounding the wound respond first and most directly to the drug's stimulation. Fat pad tympanic membrane repair centers on adipose-derived stem cells and vascular endothelial cells. This project focuses on studying the changes in the responses of these two cell types in a "cold preservation-transplantation" model, more comprehensively and accurately reflecting the role of fat pads in tympanic membrane repair. It also better interprets the "one-to-many" drug action characteristics of traditional Chinese medicine.

[0021] 1. The use of Shengji Yuhong collagen for external auditory canal packing in patients after tympanic membrane repair promotes angiogenesis and epithelial formation. This provides clinical evidence demonstrating the unique advantages of topical Chinese medicine preparations in the postoperative treatment of tympanic membrane perforation, increasing surgical benefits, improving postoperative recovery, and preventing postoperative complications. This application uniquely utilizes drug-containing collagen sustained-release technology in wound repair after tympanic membrane repair, using inflammatory factors (IL-1β, IL-6, IL-8, TNF-α) as a link. The role of inflammatory factors in repair is two-sided; excessively high levels can cause infection and affect repair, while excessively low levels can slow down the repair process and lead to transplantation failure. The "pre- and post-transplant dual-stage regulation" model (high inflammatory factors before transplantation, low inflammatory factors after transplantation) is the key technology and concept of this study, involving the phased dynamic regulation of inflammatory factors, providing a new research approach for regulating physiological changes in cells.

[0022] 2. Establish a fat pad "cold preservation-transplantation" model (EVFP cell model and EVFP rat model) to elucidate the possibility of the above hypothesis and reveal its potential regulatory mechanism. This application aims to provide a theoretical basis and clinical guidance for "providing cold preservation protection for ex vivo fat pads and improving the cold preservation quality of donor fat pads, increasing the utilization rate of repair materials, forming a "pre-transplantation-post-transplantation dual-stage regulation" model, promoting the healing of fat pads and tympanic membrane remnants, and shortening the healing time." In existing studies, some researchers have attempted to improve the performance of grafts or add additional functions by dipping or rapidly soaking transplanted materials before transplantation. This application focuses on the protection and performance improvement of grafts throughout the pre-transplantation and post-transplantation stages. The pre-transplantation cold preservation model provides a new approach and research platform for graft performance improvement research, and also provides a good way to evaluate cell function and explore related mechanisms under different cold preservation conditions at the cellular and molecular level.

[0023] 3. This study referenced advanced animal models of chronic tympanic membrane perforation in existing research to create a tympanic membrane perforation model. Compared with the fresh wounds formed by physical drilling commonly used in previous studies, this model more effectively avoids interference from the self-healing nature of tympanic membrane perforation on experimental results. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 The diagram shows a cell cryopreservation culture apparatus, where 57.5 represents the oxygen content measured by an oxygen analyzer in the culture apparatus.

[0026] Figure 2 This is a schematic diagram of the EVFP cell model.

[0027] Figure 3This chart shows the growth of adipocytes under different cold storage times and simulated transplantation times without the addition of L-alanyl-L-glutamine in the cold storage solution. * represents... P <0.001.

[0028] Figure 4 The growth of adipocytes under different cold storage times and simulated transplantation times when different concentrations (0-16 mM) of L-alanyl-L-glutamine were added to the cold storage solution. A represents CIT 0 h, B represents CIT 1 h, C represents CIT 2 h, and D represents CIT 4 h. * indicates... P <0.001.

[0029] Figure 5 The effect of adding L-alanyl-L-glutamine to the cryopreservation solution on inflammatory factors and the angiogenesis marker VEGF in human primary adipose stem cells.

[0030] Figure 6 The effect of adding L-alanyl-L-glutamine to the cryopreservation solution on inflammatory factors in human primary adipose microvascular endothelial cells.

[0031] Figure 7 A schematic diagram illustrating the process of investigating the dosage of Shengji Yuhong Collagen.

[0032] Figure 8 The image shows the cell morphology of EVFPs observed under a 4x microscope at a simulation time of 12 h. A represents human primary adipose stem cells, and B represents human primary adipose microvascular endothelial cells.

[0033] Figure 9 The image shows cell activity, where A represents primary human adipose stem cells and B represents primary human adipose microvascular endothelial cells.

[0034] Figure 10 This is a schematic diagram of the rat EVFP model.

[0035] Figure 11 The images show the culture process of rats in different groups. "Initial" represents the initial state immediately before modeling. D7, D14, D21, and D28 represent tympanic membrane images at different time points. Among them, A is the blank group, B is the perforation model group, C is the NS group, D is the STEEN group, E is the STEEN+8mM Glu group, F is the STEEN+8mM Glu+BSO group, G is the STEEN+8mM Glu+SJYH group, and H is the STEEN+8mM Glu+SJYH+VEGF group.

[0036] Figure 12 This is a schematic diagram of the treatment of the STEEN+8mM Glu+SJYH group.

[0037] Figure 13 The inflammatory factors in rats from different groups after transplantation. Detailed Implementation

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0039] The two types of adipocytes used in the following examples are as follows: the human primary adipose stem cells (hADSCs) were purchased from Nanjing Meditech Biochemical Co., Ltd., and the human primary adipose microvascular endothelial cells (hAMECs) were purchased from Nanjing Invegene Biotechnology Center.

[0040] In the following examples, unless otherwise specified, the PBS referred to is 1×, pH 7.4 PBS buffer.

[0041] In the following examples, the cryopreservation solution was prepared by adding L-alanyl-L-glutamine to STEEN solution. Specifically, the formulation of the cryopreservation solution (500 mL) is as follows: 2.5 g sodium chloride, 175 mg potassium chloride, 110 mg calcium chloride dihydrate, 121 mg magnesium chloride hexahydrate, 82 mg sodium dihydrogen phosphate monohydrate, 53 mg sodium bicarbonate, 1 g glucose, 2.5 g dextran-40, 35 g human serum albumin, and 0-16 mM L-alanyl-L-glutamine, with PBS as the solvent.

[0042] Example 1: Cellular Experiment Validation of L-Alanyl-L-Glutamine Dosage Concentration hADSCs and hAMECs cells were cultured separately as simulated grafts, and cold preservation conditions were tested separately. The culture methods were as follows: cells were routinely cultured in a carbon dioxide incubator (37 ℃, 5% CO2) using the initial culture medium. The initial culture medium was a commonly used cell culture medium, specifically mesenchymal stem cell culture medium MSCM (containing matching serum and antibiotics, purchased from Sciencell, catalog number 7501) or DMEM + 10% v / v FBS medium.

[0043] The culture medium for both hADSCs and hAMECs cells obtained above was replaced with cryopreservation medium to preserve the hADSCs and hAMECs cells, respectively. Then, the culture dishes were placed... Figure 1 In the sealed apparatus shown, pure oxygen was introduced, and the oxygen meter reading was observed. When the oxygen content stabilized at 50.0%, oxygen supply was stopped, and the entire apparatus was placed in a 4 ℃ environment for routine cold storage.

[0044] In this embodiment, the cold preservation time (also known as cold ischemia time, CIT, set to 1, 2 or 4 h) of two types of adipocytes and the concentration of L-alanyl-L-glutamine added to the cold preservation solution (set to 0, 2, 4, 8 or 16 mM) were screened and optimized.

[0045] After cold preservation as described above, the cold preservation solution was replaced with the initial culture medium, and the culture conditions were changed to the conventional 37℃, 5% CO2. This simulated the transition of the graft from a cold-preserved state to a state where blood supply was restored after transplantation into the human body. This was a simulated EVFP transplantation, with the post-transplantation time set to 4, 12, or 24 hours, i.e., the EVFP time was 4, 12, or 24 hours. Figure 2 As shown. Cell viability (cell proliferation, apoptosis) was detected at different time points and the EVFP endpoint after different treatments in two cell line models. Cell morphology at each experimental observation point was observed under an inverted optical microscope (OLYMPUS CK40 inverted optical microscope), and OD was measured. 450 By quantifying specific cell growth, comparing the control group and experimental groups with different concentrations, suitable cold preservation models and L-alanyl-L-glutamine concentrations for two cell types, hADSCs and hAMECs, were comprehensively screened. This application establishes a novel in vitro fat graft cold preservation cell model EVFP for the first time both domestically and internationally. It uses human adipose-derived stem cells (hADSCs) and human adipose microvascular endothelial cells (hAMECs) to simulate the entire clinical process of "graft fat graft acquisition - cold preservation - tympanic membrane transplantation," serving as an indispensable and important model for cell experiments, providing in vitro experimental model support and data support for subsequent animal experiments.

[0046] When L-alanyl-L-glutamine was not added to the cold storage solution (0 mM), the results were as follows: Figure 3 As shown, cold storage for 1–4 hours can significantly induce rewarming damage in cells. However, the addition of L-alanyl-L-glutamine can effectively reverse the rewarming damage induced by CIT, exhibiting a dose-dependent effect of 0–8 mM. Figure 4 As shown, the results were significant at 4 h of CIT, but excessively high doses (16 mM) could potentially reduce efficacy. However, L-alanyl-L-glutamine did not exert a protective effect when cells were undamaged (0 h of CIT). Figure 4 In the A), even transient dose injury (EVFP 4 h) occurred after administration.

[0047] In summary, the optimal concentration of L-alanyl-L-glutamine in the cryopreservation solution was determined to be 8 mM, the optimal cryopreservation time (CIT) was 4 h, and the optimal time to implantation (EVFP) was 12 h, based on the above screening results.

[0048] Example 2: Cellular experiments to verify the mechanism of action of L-alanyl-L-glutamine Based on the model and drug concentration obtained from Example 1, hADSCs and hAMECs cells were grouped separately under their appropriate model and drug concentration: (1) Control group (0 mM): routine cold storage, the concentration of L-alanyl-L-glutamine in the cold storage solution was 0 mM; (2) Experimental group (8 mM): Conventional cold storage, the concentration of L-alanyl-L-glutamine in the cold storage solution was 8 mM; (3) GSH inhibition group: routine cold storage, the concentration of L-alanyl-L-glutamine in the cold storage solution was 8 mM, and 1 mmol / L GSH synthesis inhibitor BSO (butyrosine sulfoxide) was also added to the cold storage solution.

[0049] The conventional cold preservation method is as described in Example 1, in which cells are immersed in cold preservation solution, placed in a sealed incubator with a vent valve and oxygenated to a concentration of 50% (±2%), and the entire container is placed in a 4°C refrigerator with a cold preservation time of 4 h.

[0050] After cold storage for 4 hours, ROS, cell viability, GSH, and inflammatory factors were measured in each of the above groups. The specific detection methods are as follows: ROS detection: CellROX® Oxidative Stress Reagents were used to label ROS products in the endpoint cells of EVFP in each group and then quantitatively analyzed using a fluorescent microplate reader. Cell viability assay: MTT assay was used to detect cell proliferation and apoptosis; GSH assay: EVFP endpoint cell lysis products were collected from each group, and the expression level of glutathione GSH was detected using the Glutathione Fluorometric Assay Kit (GSH, GSSG and Total). Inflammatory factor detection: The levels of major inflammatory factors such as IL-8, IL-6, IL-1β and TNF-α and vascular endothelial growth factor (VEGF) in the cell culture supernatant of EVFP endpoint cells in each group were detected by ELISA kit.

[0051] Results of human primary adipose-derived stem cells (hADSCs) are as follows Figure 5 As shown, human primary adipose microvascular endothelial cells (hAMECs) are as follows: Figure 6As shown, the results indicate that in the cold preservation model of human hADSCs and hAMECs, the addition of L-alanyl-L-glutamine to STEEN solution resulted in increased IL-6, increased IL-8, and increased TNF-α, while IL-1β remained unchanged or decreased. This has clear positive significance and is highly consistent with the core objective of protecting the fat pad in tympanic membrane transplantation in this invention. On the one hand, IL-6 and IL-8, as important pro-inflammatory factors, can activate the early inflammatory response of the body by moderately increasing their levels, recruiting immune cells to clear apoptotic cells and metabolic waste generated during cold preservation, laying the foundation for subsequent cell repair. At the same time, IL-8 can specifically promote the proliferation and migration of hAMECs, accelerate angiogenesis, help restore blood supply to the fat pad after cold preservation, and improve the survival quality of the graft. Moderately increasing TNF-α can activate the cell stress defense mechanism, promote the secretion of growth factors by hADSCs, enhance the anti-apoptotic ability of cells, and reduce cell damage during cold preservation and hot perfusion. On the other hand, maintaining or decreasing IL-1β can prevent tissue damage caused by excessive inflammatory response and avoid cytokine storms leading to increased apoptosis and impaired function of hADSCs and hAMECs, thus achieving a balance between "moderate pro-inflammatory and avoiding excessive inflammation." In summary, the change in this cytokine expression is an important manifestation of L-alanyl-L-glutamine regulating cellular oxidative stress and mitigating cold preservation damage. It not only ensures the activity and function of graft cells but also provides favorable conditions for the subsequent repair process after tympanic membrane transplantation, which is highly consistent with the core research objective of this invention to improve the quality of fat pad cold preservation.

[0052] Example 3: Investigation of the concentration and mechanism of regenerating red collagen This example investigated the effects of Regenerist collagen on hADSCs and hAMECs under a "cold preservation-transplantation" model and screened the appropriate concentration. Both cell types were cultured under the cold preservation model conditions selected in Example 1 (CIT 4 h, L-alanyl-L-glutamine concentration in the cold preservation solution 8 mM, EVFP simulation time set to 12 h to facilitate observation of the effects of Regenerist collagen on the two cell types). The cold preservation and EVFP simulation methods were the same as in Example 1, except that during EVFP transplantation simulation, three concentration gradients of Regenerist collagen (S: 0 (Normal control group), 50 μg / mL, 100 μg / mL, and 200 μg / mL) were added to the culture medium (experimental design diagram shown in Figure 1). Figure 7As shown in the figure, a control group containing only an equal volume of collagen (without Shengji Yuhong solution) was used. Cell viability (cell proliferation, apoptosis), expression of VEGF and inflammatory factors in cells and cell culture supernatant were detected at different time points after transplantation under cold preservation model conditions. The control group and experimental groups with different concentrations were compared to investigate the changes in hADSCs and hAMECs of the two cell lines under the action of Shengji Yuhong collagen. Appropriate animal observation time points and Shengji Yuhong collagen concentrations were determined. The results are as follows: Figure 8 and Figure 9 As shown in the figure. The results indicate that Regenerist collagen can alleviate cell rewarming damage, with 100 μg / mL Regenerist collagen showing the best effect in alleviating the damage.

[0053] This embodiment also explored the mechanism by which Shengji Yuhong collagen may promote angiogenesis by increasing IL-8 and VEGF expression, and the investigation was conducted through the following grouping: (1) Control group: routine simulated transplantation culture, the culture medium at the time of transplantation was the initial culture medium, which did not contain regenerating red collagen; (2) Experimental group: conventional simulated transplantation culture, the culture medium at the time of transplantation was the initial culture medium containing the target concentration of Regenerating Red Collagen; (3) IL-8 inhibition group: routine simulated transplantation culture, the culture medium at the time of transplantation was the initial culture medium containing the target concentration of regenerating collagen and 15 μmol / L IL-8 inhibitor Reparixin; (4) VEGF inhibition group: routine simulated transplantation culture, the culture medium at the time of transplantation was the initial culture medium containing the target concentration of dermal collagen and 5 μg / mL bevacizumab (a common VEGF inhibitor).

[0054] The conventional simulated transplantation culture described herein is consistent with the conventional culture method and simulated transplantation method for the two types of cells described in Example 1.

[0055] After culture, inflammatory factors and VEGF were detected: the levels of IL-8 and VEGF in the cell culture supernatant of each EVFP endpoint were detected by ELISA kit.

[0056] The regenerating collagen described in this embodiment was prepared as follows: The regenerating collagen composition consisted of 6 parts Angelica sinensis, 6 parts Glycyrrhiza uralensis, 6 parts Angelica dahurica, 6 parts Lithospermum erythrorhizon, 2.4 parts Dragon's Blood, 6 parts Terminalia chebula, and 6 parts Hibiscus syriacus. The preparation process was as follows: The prescribed amounts of Angelica sinensis, Glycyrrhiza uralensis, Terminalia chebula, and Hibiscus syriacus were mixed and extracted twice by reflux with 70% v / v ethanol. Each extraction was performed with 8 times the volume of ethanol, and the mixture was refluxed for 1 hour. The filtrates were combined, and the ethanol was recovered until no alcohol odor remained, yielding the first extract for later use. Separately, take the prescribed amounts of Angelica dahurica and Lithospermum erythrorhizon, mix them well, pulverize them into coarse powder, pass them through a 20-mesh sieve, and use 8 times the volume of 70% v / v ethanol as solvent. After soaking for 8 hours, perform percolation extraction at a rate of 0.5 mL / (min / kg). Collect the percolate, combine it, add the prescribed amount of Dragon's Blood to the percolate, stir well to dissolve, then add the first extract, stir well to dissolve, filter, and add 70 v / v% ethanol aqueous solution to make up to 100 mL. When the total weight of the prescription does not exceed 64 g, the total volume should be 100 mL to obtain the Shengji Yuhong medicinal solution. Load 12.5 mL of the prepared Shengji Yuhong medicinal solution into a rectangular collagen sponge (bovine Achilles tendon type I collagen, purchased from Wuxi Bedi Biotechnology Co., Ltd., batch number 20143642302) with dimensions of 50 mm in length and width and 1 mm in height, freeze-dry at -50℃ for 24 h, package it individually, and sterilize it with ethylene oxide to obtain the Shengji Yuhong collagen sponge drug. The collagen pore size is between 15 and 25 μm, with a porosity of 98%.

[0057] Example 4: Animal Model Research Forty-two male SD rats weighing 480-520g were used in this embodiment and prepared in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, 1996 Revision).

[0058] The rat chronic unilateral tympanic membrane perforation model is identical to that in Chinese patent CN121337817A. Specifically, it was constructed as follows: Before induction anesthesia, ketamine (5 mg / kg) was administered intramuscularly to the rats, followed by induction anesthesia in the induction chamber using 4% isoflurane / oxygen (1.8 L / min). During maintenance anesthesia, 2.5% isoflurane / oxygen (1 L / min) was inhaled through a face mask. Both ears of the rats were examined using a pen-type fiber optic microscope. The side with the cleaner ear, less curvature, fewer ear deformities, and less curvature of the malleus handle was selected for unilateral testing. The tympanic membrane perforation model was established, and subsequent tympanic membrane observation was performed under a pen-type fiber optic microscope. First, an acute tympanic membrane perforation model was constructed using the following method: A rat earwax hook (the length of the earwax hook was 136 mm, the length of the needle head was 50 mm, and the rest was the handle; the diameter of the needle head was 1.4 mm, and the length of the hook was 0.3 mm) was used to perform a tympanic membrane puncture (TMP) in the posterior quadrant of the tympanic membrane. The tympanic membrane is punctured to a depth equivalent to one-third of the visible length of the malleus handle, ensuring that the resulting perforation occupies approximately 60% of the pars tensa region of the posterior quadrant of the tympanic membrane. The posterior quadrant of the rat tympanic membrane is determined as follows: the malleus handle divides the tympanic membrane into anterior and posterior quadrants; the portion facing the head and face belongs to the anterior quadrant, and the portion facing the back of the head belongs to the posterior quadrant. During the puncture, under a pen-type fiber optic microscope, the field of view should be perpendicular to the perforation area to facilitate vertical needle insertion, control of the puncture depth, and observation of the distance between the needle insertion point and surrounding tissues. The operator can clearly see the malleus handle and distinguish between the anterior and posterior quadrants of the tympanic membrane, and can also clearly see the perforation site after the earwax hook has entered. The "posterior" refers to the area behind the tympanic membrane, i.e., within the middle ear cavity. The puncture depth is one-third of the length of the malleus handle (the length of the malleus handle is visible under the microscope), resulting in rats with acute tympanic membrane perforation.

[0059] Under a pen-type fiber optic microscope, a drug-impregnated gelatin sponge was inserted into the perforation of rats with acute tympanic membrane perforation. Specifically, a dexamethasone (DEX) solution was prepared with sterile PBS to a final concentration of 10 mg / mL. A dry gelatin sponge (1 mm × 1 mm × 1 mm; two type A sponges) was soaked in the 10 mg / mL DEX solution and then inserted into the perforation to fix the initial position of the gelatin sponge. After 24 hours and 48 hours, new gelatin sponges soaked in DEX solution were used to replace the sponge. After 72 hours, the gelatin sponge was removed, resulting in a rat model of chronic unilateral tympanic membrane perforation. Care was taken to avoid the malleus handle and the external auditory canal wall when inserting the drug-impregnated gelatin sponge.

[0060] The graft material used in this application is a fat graft. In the rat model, the fat graft was obtained as follows: The left groin area of ​​the rat from which the fat graft (obtained from the same organism) was shaved, and an incision of approximately 2 cm was made. A fat graft of at least twice the perforation size (approximately 0.5 g) was taken, and the incision was sutured with 5.0 absorbable sutures. Figure 10 As shown.

[0061] The rats in this embodiment were grouped as follows (N=6 per group, total N=42): (1) Blank group: Normally fed under standard laboratory conditions, without modeling or medication. Figure 11 (A in the middle) (2) Perforation Model: Establishing a chronic unilateral tympanic membrane perforation model ( Figure 11 (B in the middle) (3) NS group (Control): A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region, soaked in physiological saline (NS) for 4 h, and then transplanted to the unilateral tympanic membrane perforation site of the experimental mice. Figure 11 (C in the middle) (4) STEEN group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region, soaked in a cold preservation solution without L-alanyl-L-glutamine for 4 h, and then transplanted to the tympanic membrane perforation sites of the left and right ears of experimental mice. Figure 11 (D in the middle) (5) STEEN+8mM Glu group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region and soaked in a cryopreservation solution containing the target concentration (8 mM) L-alanyl-L-glutamine for 4 h. Then, they were transplanted to the unilateral tympanic membrane perforation site of the experimental mice. Figure 11 (E in the middle) (6) STEEN+8mM Glu+BSO group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region and soaked in a cold preservation solution containing the target concentration (8 mM) L-alanyl-L-glutamine and 1 mmol / L GSH synthesis inhibitor BSO for 4 h. Then, they were transplanted to the unilateral tympanic membrane perforation site of the experimental mice. Figure 11 (F in the middle) (7) STEEN+8mM Glu+SJYH group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region and soaked in a cryopreservation solution containing the target concentration (8 mM) L-alanyl-L-glutamine for 4 h. The grafts were then transplanted to the unilateral tympanic membrane perforation site of the experimental mice. After transplantation, the unilateral ear was supported and packed with 100 μg / mL Shengji Yuhong collagen sponge. Figure 11 (G in the text). The filling method of the Shengji Yuhong Collagen Sponge described here is as follows: Figure 12 As shown, the size of the Shengji Yuhong collagen sponge is related to the size of the tympanic membrane perforation. It is generally large enough to cover and repair the wound, but not wider than the external auditory canal. (8) STEEN+8mM Glu+SJYH+VEGF group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region and soaked in a cold preservation solution containing the target concentration (8 mM) L-alanyl-L-glutamine and 100 ng / mL VEGF for 4 h. The grafts were then transplanted to the unilateral tympanic membrane perforation site of the experimental mice. After transplantation, the unilateral ear was supported and packed with 100 μg / mL Shengji Yuhong collagen sponge. Figure 11 (H in the middle) (9) 8 mM Glu group: A chronic unilateral tympanic membrane perforation model was established. Fat grafts were obtained from the left inguinal region, soaked in 8 mM L-alanyl-L-glutamine solution for cold preservation for 4 h, and then transplanted to the unilateral tympanic membrane perforation site of the experimental mice.

[0062] Day 1 of observation was defined as the transplantation of fat pads. Following surgery, the experimental rats were given a 21-day healing period. Water and food (feed pellets) were readily available under standard laboratory conditions. Graft growth was assessed using a micro-electronic otoscope on days 3, 5, 7, 10, 14, 21, and 28 after modeling. This example assessed wound healing in the above-grouped rats. Figure 11 As shown, compared with the perforation model group (B) and the NS group (C), the STEEN group (D) containing STEEN solution showed improved healing speed. The effect was further enhanced by combining STEEN with L-alanyl-L-glutamine in the STEEN+8mM Glu group (E). However, the addition of BSO (glutathione synthase inhibitor) (STEEN+8mM Glu+BSO group F) resulted in delayed healing at time points D14 and D21. The most effective regimen was the combination with regenerating collagen (STEEN+8mM Glu+SJYH group G), which showed better epithelial coverage, tissue regeneration, and angiogenesis. The additional addition of VEGF to this regimen (STEEN+8mM Glu+SJYH+VEGF group H) may have shown a further synergistic effect at specific time points (such as D21), suggesting the crucial role of angiogenesis in later healing. All images were obtained through direct visualization under tympanoscopy.

[0063] In this embodiment, on day 21 after modeling, animals were intraperitoneally injected with a lethal dose of sodium pentobarbital, followed by decapitation. After decapitation, the tympanic membrane of one side was removed and directly observed. The tympanic membrane was dissected under a microscope, and the surrounding bony ring and 1-2 mm of the external auditory canal were removed as experimental samples. The protein content and nucleic acid expression levels of GSH, ROS, IL-6, IL-8, IL-1β, TNF-α, and VEGF in the samples were detected. Some results are shown below. Figure 13 As shown, the results indicated that the levels of various inflammatory factors (including IL-1β, IL-6, IL-8 and TNF-α) in the STEEN+8mM Glu group were significantly reduced after graft transplantation.

[0064] This invention provides a concept and method for the application of a cryopreservation solution and its combined traditional Chinese medicine compound biological scaffold drug delivery system in tympanic membrane repair. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A cryopreservation solution, characterized in that, The cold preservation solution comprises a base solution and L-alanyl-L-glutamine; wherein the base solution comprises STEEN solution.

2. The cryopreservation solution according to claim 1, characterized in that, The concentration of L-alanyl-L-glutamine in the cold preservation solution is 4~16 mM.

3. The application of the cryopreservation solution according to claim 1 or 2 in the cryopreservation of fat grafts, preferably, the fat graft is an adipocyte and / or adipose tissue.

4. The application according to claim 3, characterized in that, The applications include any one or more combinations of the following (1) to (4): (1) Improve the activity of fat grafts; (2) Regulate the levels of inflammatory factors and / or vascular endothelial growth factor; (3) Adjusting the content of reactive oxygen species; (4) Increase glutathione content.

5. The application according to claim 3, characterized in that, The cold storage is performed for 1 to 4 hours at a temperature of 2 to 8 °C and with an oxygen content of 48% to 52%.

6. The use of the cryopreservation solution according to claim 1 or 2 in the cryopreservation of fat grafts in tympanic membrane perforation repair materials.

7. The application according to claim 6, characterized in that, The tympanic membrane perforation repair materials include fat grafts and traditional Chinese medicine compound biological scaffold drug delivery systems.

8. The application according to claim 7, characterized in that, The aforementioned traditional Chinese medicine compound biological scaffold drug delivery system is Shengji Yuhong collagen sponge.

9. The application according to claim 8, characterized in that, The aforementioned Shengji Yuhong collagen sponge is prepared by immersing a collagen sponge in a Shengji Yuhong drug solution with a concentration of 50~200 μg / mL.

10. The application according to claim 6, characterized in that, After the fat graft was placed in a cryopreservation solution for cryopreservation, it was transplanted to the tympanic membrane perforation site to repair the perforation.

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