An olfactory mucosal mesenchymal stem cell preparation, its preparation method, and its application in degenerative osteoarthritis.

Abstract

This application provides an olfactory mucosal mesenchymal stem cell preparation, its preparation method, and its application in degenerative osteoarthritis, relating to the field of mesenchymal stem cell technology. The invention involves preparing a cell suspension from passaged olfactory mucosal mesenchymal stem cells, adding it to a plasma-treated hydrogel scaffold, and culturing it with culture medium until the olfactory mucosal mesenchymal stem cell preparation is obtained. The invention aims to provide an olfactory mucosal mesenchymal stem cell preparation pre-cultured on a hydrogel scaffold obtained through plasma jet treatment and its application in osteoarthritis, thereby improving the functional stability, anti-inflammatory capacity, and cartilage-protective potential of the cells in the inflammatory microenvironment of osteoarthritis. This preparation is intended to address the problem that existing osteoarthritis treatments, such as conventional drugs or injections, primarily focus on symptom relief and are insufficient to sustainably improve the intra-articular inflammatory microenvironment and cartilage degeneration.

Description

Technical Field

[0001] This invention relates to the field of mesenchymal stem cell technology, and particularly to an olfactory mucosal mesenchymal stem cell preparation, its preparation method, and its application in degenerative osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease characterized by cartilage degeneration, synovitis, subchondral bone changes, and joint dysfunction. Current clinical treatments primarily include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular corticosteroids, sodium hyaluronate, platelet-rich plasma (PRP), physical therapy, and end-stage artificial joint replacement. While these treatments offer some relief for pain and short-term improvement in joint function, most are symptomatic and lack the ability to sustainably reverse cartilage degeneration or improve the intra-articular inflammatory microenvironment in the long term. Therefore, developing novel treatment regimens that combine anti-inflammatory, cartilage-protective, and joint function-improving effects is a crucial direction in the treatment of osteoarthritis.

[0003] In recent years, mesenchymal stem cells (MSCs) have been increasingly applied in research on osteoarthritis and cartilage repair due to their multi-lineage differentiation potential, immunomodulatory capabilities, and paracrine repair effects. Commonly used cell sources in current techniques include bone marrow MSCs, adipose-derived MSCs, synovial MSCs, and umbilical cord MSCs. The typical technical approach involves isolating MSCs from the corresponding tissues, expanding and culturing them in vitro to prepare a cell suspension, and then administering the solution via intra-articular injection. The aim is to regulate local joint inflammation through the secretion of cytokines, growth factors, and extracellular vesicles, promoting extracellular matrix synthesis in chondrocytes and delaying the progression of osteoarthritis. Related studies suggest that mesenchymal stem cell therapy has certain application potential in osteoarthritis treatment, but its efficacy is influenced by various factors such as cell source, culture conditions, cell viability, dosage, and the inflammatory microenvironment of the joint cavity.

[0004] The influencing factors are as follows: 1. Current mesenchymal stem cell therapy mostly uses cells derived from bone marrow, adipose tissue, umbilical cord, or synovium, which has certain limitations in cell source and biological properties. Bone marrow-derived mesenchymal stem cell collection is relatively invasive, and cell quantity and activity may be affected by donor age, underlying diseases, and collection site; although adipose-derived mesenchymal stem cells can be obtained in larger quantities, the cell population exhibits significant heterogeneity; while allogeneic cells are easy to prepare and store, issues such as immunocompatibility, batch stability, and long-term safety still need to be considered. These factors may lead to individual differences and insufficient stability in the efficacy of cell therapy. 2. Osteoarthritis involves multiple pathological changes in the joint cavity, including elevated inflammatory factors, synovial hyperplasia, cartilage matrix degradation, and abnormal local microenvironment. After injection of ordinary cell suspension into the joint cavity, the cells need to function in an inflammatory and mechanical stress environment, and their survival, retention, and paracrine effects may all be affected. If the cells themselves lack anti-inflammatory and repair capabilities, it is difficult to simultaneously improve multiple pathological aspects such as synovial inflammation, cartilage degeneration, and motor dysfunction. 3. Current technologies lack comprehensive evaluation of the three aspects of "anti-inflammation, cartilage protection, and recovery of motor function." Treatment of osteoarthritis requires not only reducing local inflammation but also improving cartilage structure and joint function.

[0005] Olfactory Mucosa / Nasal Mucosa-derived mesenchymal stem cells (OM-MSCs) are a type of mesenchymal stem cell with unique advantages. Compared to traditional sources such as bone marrow and adipose tissue, olfactory mucosa / nasal mucosa tissue harvesting is relatively minimally invasive, and the cells possess certain proliferative capacity, multi-lineage differentiation potential, and tissue repair potential. When used as autologous cells, they also offer advantages in immunocompatibility. Existing research has demonstrated that olfactory mucosa-derived mesenchymal stem cells possess the relevant biological characteristics of mesenchymal stem cells and are considered a cell source with potential applications in regenerative medicine.

[0006] Current cell therapy technologies for osteoarthritis mainly focus on mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, or synovium, while research on the application of olfactory mucosa / nasal mucosa mesenchymal stem cells in the treatment of osteoarthritis is relatively limited.

[0007] Therefore, although existing technologies have disclosed various treatment options for osteoarthritis using mesenchymal stem cells, the issues of formulation, functional enhancement, and local joint cavity intervention of olfactory mucosa / nasal mucosa mesenchymal stem cells in osteoarthritis have not yet been fully resolved. Summary of the Invention

[0008] Therefore, the purpose of this invention is to propose an olfactory mucosal mesenchymal stem cell preparation, its preparation method, and its application in degenerative osteoarthritis. Specifically, it aims to provide an olfactory mucosal mesenchymal stem cell preparation pre-cultured on a hydrogel scaffold obtained through plasma jet treatment and its application in osteoarthritis. This aims to improve the functional stability, anti-inflammatory capacity, and cartilage-protective potential of the cells in the inflammatory microenvironment of osteoarthritis, and to prepare it as a cell preparation for osteoarthritis intervention. This addresses the problem that existing osteoarthritis treatments, such as conventional drugs or injections, primarily focus on symptom relief and are unable to sustainably improve the intra-articular inflammatory microenvironment and cartilage degeneration.

[0009] This invention further aims to address the problems of limited cell sources, relatively large invasiveness in harvesting, and the susceptibility of cell activity and therapeutic efficacy to donor conditions in existing mesenchymal stem cell therapies. By using olfactory mucosa / nasal mucosa-derived mesenchymal stem cells as the cell source, and leveraging their relatively minimally invasive harvesting, good proliferation capacity, and strong tissue repair potential, this invention provides a new source of cell preparations for the local treatment of osteoarthritis.

[0010] This invention also aims to address the shortcomings in the anti-inflammatory capacity, paracrine function, and chondrogenic protective effects of mesenchymal stem cells (MSCs) under conventional culture conditions. Due to the differences between the conventional in vitro culture environment and the in vivo tissue microenvironment, MSCs, when injected into the joint cavity and directly exposed to the inflammatory microenvironment, typically experience limitations in survival, functional maintenance, and local regulatory effects. This invention pre-cultures olfactory / nasal mucosa MSCs to enhance their functional stability, anti-inflammatory effects, and chondrogenic protective potential within the osteoarthritis microenvironment, enabling them to exert rapid anti-inflammatory responses and regulate the cartilage microenvironment, thereby improving the overall therapeutic effect of osteoarthritis.

[0011] This invention also aims to provide a cell preparation application scheme suitable for intra-articular local administration, enabling pre-cultured olfactory mucosa / nasal mucosa mesenchymal stem cells to act on the lesion site of osteoarthritis through intra-articular injection, thereby rapidly reducing local inflammatory response, improving synovial inflammation, promoting the expression of cartilage matrix synthesis-related markers, and continuously improving osteoarthritis-related cartilage structure damage and decreased motor function.

[0012] The technical solution of this invention is implemented as follows: A method for preparing an olfactory mucosal mesenchymal stem cell preparation includes the following steps: After passaged olfactory mucosal mesenchymal stem cells were prepared into a cell suspension, they were added to a plasma-treated hydrogel scaffold, culture medium was added and cultured until the target olfactory mucosal mesenchymal stem cells were harvested, and the target olfactory mucosal mesenchymal stem cells were prepared into a cell suspension to obtain the olfactory mucosal mesenchymal stem cell preparation. The method for preparing the plasma-treated hydrogel scaffold includes: treating sodium alginate solution with plasma, then adding calcium chloride solution to form hydrogel beads, followed by freeze-drying to obtain the plasma-treated hydrogel scaffold.

[0013] The present invention preferably uses olfactory mucosa / nasal mucosa mesenchymal stem cells, but mesenchymal stem cells derived from nasal septum mucosa, nasal turbinate mucosa or other nasal mucosal tissues may also be used.

[0014] The cells can be autologous or allogeneic, as long as they are identified as conforming to the mesenchymal stem cell phenotype and have the potential for in vitro expansion, immunomodulation, and tissue repair, they can be used in this invention.

[0015] This invention preferably uses the tissue block adhesion method to obtain primary cells, but enzymatic digestion or a combination of tissue block adhesion and enzymatic digestion can also be used. The enzymes used may include collagenase, trypsin, dispersase, or hyaluronidase, as long as they can yield olfactory mucosa / nasal mucosa mesenchymal stem cells that meet the requirements.

[0016] The cells can be P2 to P6 generation cells, preferably early to mid-stage passaged cells, to ensure cell proliferation capacity, phenotypic stability and biological function.

[0017] Cell expansion media can include DMEM / F12, low-glucose DMEM, α-MEM, or other media suitable for mesenchymal stem cell culture. Fetal bovine serum (FBS), human platelet lysate (hPL), serum-free supplements, or animal-free culture supplements can be added to the culture system.

[0018] Furthermore, the conditions for plasma processing are as follows: helium and oxygen are used as carrier gases.

[0019] Furthermore, the helium flow rate is 0.8~1.2 L / min, and the oxygen flow rate is 0.1~0.3 L / min; Furthermore, the plasma treatment temperature is 20~30℃.

[0020] Furthermore, each 80-120 mL sodium alginate solution is subjected to plasma treatment for 10-50 min.

[0021] Furthermore, the sodium alginate solution is an aqueous solution of sodium alginate with a concentration of 2.5% w / v to 4.5% w / v; the calcium chloride solution is an aqueous solution of calcium chloride with a concentration of 0.05% w / v to 0.3% w / v.

[0022] Furthermore, the target olfactory mucosal mesenchymal stem cells were harvested after culturing in culture medium for 10-50 hours. The culture period of 10-50 h also includes shaking culture, with a shaking rate of 100-200 rpm; Before culturing, the cell concentration is 1~5×10⁻⁶. 6 The amount of the hydrogel scaffold used is 180~220 μL, and the amount of the culture medium used is 350~450 μL.

[0023] The present invention provides an olfactory mucosal mesenchymal stem cell preparation prepared by the above-mentioned method.

[0024] The formulation can be formed by combining cells with biocompatible materials such as hyaluronic acid, hydrogel, collagen gel, fibrin gel, or thermosensitive hydrogel.

[0025] The present invention prepares pretreated cells into cell suspensions, which can also be resuspended in PBS, physiological saline, sodium hyaluronate solution or other pharmaceutically acceptable injectable carriers.

[0026] Cells can also be loaded into biocompatible materials such as hyaluronic acid hydrogel, thermosensitive hydrogel, collagen gel, and fibrin gel to increase the retention time of cells in the joint cavity.

[0027] On one hand, this invention provides the application of the olfactory mucosal mesenchymal stem cell preparation described above in the preparation of a drug for treating degenerative osteoarthritis. This is achieved by intra-articular injection of 1×102 5 ~1×10 7 A dose of 1 cell / 100 μL is used to treat osteoarthritis, and the dosage can be adjusted according to the size of the joint.

[0028] Furthermore, the olfactory mucosal mesenchymal stem cell preparation improves at least one of the following aspects: 1) Reduce synovial inflammation; 2) Repair cartilage tissue; 3) Improves motor dysfunction caused by osteoarthritis; 4) Reduce inflammation within the joints.

[0029] On the other hand, the present invention provides the application of the olfactory mucosal mesenchymal stem cell preparation as described above in the preparation of a drug that inhibits the level of inflammatory factors in chondrocytes.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes olfactory mucosa / nasal mucosa mesenchymal stem cells as the cell source, providing a novel seed cell option for cell therapy of osteoarthritis. Olfactory mucosa tissue harvesting is relatively minimally invasive, and the cells possess good in vitro expansion capacity, chondrogenic differentiation potential, and immunomodulatory capabilities. When used as autologous cells, it also helps reduce the risk of immune rejection. Therefore, compared to traditional mesenchymal stem cells from bone marrow, adipose tissue, and other sources, this invention offers advantages in terms of cell source, including a relatively gentle harvesting method and better cell application suitability.

[0031] Secondly, this invention introduces a pre-culture step of hydrogel scaffold obtained through plasma jet treatment during cell preparation, enabling olfactory mucosal mesenchymal stem cells to undergo adaptive culture under controlled oxidative / nitrosation stress conditions. This invention constructs a three-dimensional scaffold using sodium alginate and calcium chloride, combined with controlled O2 / He carrier gas plasma jet treatment, to build a hydrogel scaffold primarily containing reactive oxygen species. The inflammatory signaling molecules present in ROS / RNS during liquid culture can induce an adaptive anti-inflammatory pretreatment response in OM-MSCs, allowing them to regulate cell functional status before drug administration. This enhances cell viability, stemness maintenance, paracrine activity, and tissue repair-related functions, resulting in more pronounced anti-inflammatory and cartilage-protective effects of OM-MSCs in osteoarthritis models.

[0032] Third, the pre-cultured olfactory mucosal mesenchymal stem cell preparation obtained in this invention can be used via intra-articular injection. Intra-articular injection allows the cells to act directly on the affected joint in osteoarthritis, facilitating contact between the cells and the inflammatory microenvironment of the synovium, cartilage, and synovial fluid, thereby exerting local immunomodulatory and tissue protective effects. Compared with systemic administration, this local administration method is more suitable for diseases like osteoarthritis, which are primarily characterized by local joint lesions.

[0033] Fourth, this invention helps improve the intra-articular inflammatory microenvironment in osteoarthritis. Increased levels of inflammatory factors such as IL-1β and TNF-α are often observed in the joint cavity of osteoarthritis patients, further promoting synovial inflammation and cartilage matrix degradation. This invention enhances the anti-inflammatory regulatory capacity of OM-MSCs through pre-culture treatment. Cell experiments showed a faster decrease in TNF-α levels, and animal experiments showed a decrease in IL-1β and TNF-α levels in synovial fluid, indicating that this cell preparation has an inhibitory effect on the local inflammatory response in osteoarthritis.

[0034] Fifth, this invention helps promote the restoration of cartilage matrix homeostasis and the protection of cartilage structure. OM-MSCs pre-cultured on hydrogel scaffolds obtained through plasma jet treatment, when applied to the joints of osteoarthritis, can increase the expression of cartilage anabolic metabolism markers, such as Sox9 and type II collagen (Col II), and improve cartilage surface integrity, collagen arrangement, and tissue structure. These effects help alleviate cartilage degeneration caused by chondrocyte phenotypic damage and extracellular matrix degradation during osteoarthritis.

[0035] Sixth, this invention not only targets single inflammatory markers but also exerts a comprehensive improving effect from multiple levels, including synovial inflammation, cartilage tissue structure, molecular markers, and motor function. Previous animal experiments showed that the OM-MSCs group pre-cultured on hydrogel scaffolds obtained through plasma jet treatment showed improvements over the model group in terms of inflammatory factors, HE / Masson staining, Sox9 / ColII immunohistochemistry, rotarod behavior, and MRI imaging, with overall results superior to the conventionally scaffold-cultured OM-MSCs group. This indicates that this pre-culture treatment step can enhance the comprehensive regulatory capacity of OM-MSCs in the complex pathological environment of osteoarthritis.

[0036] Seventh, the invention's preparation process is relatively clear, mainly including steps such as tissue sampling, primary cell extraction, in vitro expansion, cell identification, pre-culture treatment, and cell suspension preparation. Among these, the pre-culture treatment conditions, cell identification indicators, and administration methods can all be standardized and controlled, facilitating subsequent quality control, dosage optimization, and preclinical translational research. Attached Figure Description

[0037] Figure 1 These figures show surgical diagrams, intra-articular injection diagrams, and changes in IL-1β and TNF-α levels in the synovial fluid of rats in the sham-operated group and various experimental groups during the DMM modeling process of this invention. In the figures, A represents the surgical procedure during DMM modeling, where sham represents the sham-operated group (joint exposure without ligament transection) and OA represents the experimental group (meniscus and tibial ligament transection). B represents the injection of phosphate-buffered saline (PBS) and olfactory mucosal mesenchymal stem cells into rats in each experimental group. C represents the changes in IL-1β levels in the synovial fluid of rats in the sham-operated group and various experimental groups. D represents the changes in TNF-α levels in the synovial fluid of rats in the sham-operated group and various experimental groups. Figure 2 The figures show the results of the rotarod experiment on rats in the sham-operated group and various experimental groups of this invention; wherein, Figure 2 In the graph, A represents a comparison of the total distance traveled by each group of mice in the rotarod experiment. Figure 2B in the figure is a comparison of the escape latency of mice in each group during the rotarod experiment; * in the figure represents significant differences (**, p <0.05; ***, p <0.01,****, p <0.001), compared with the sham surgery group, the data are mean ± SD, n=8; Figure 3 The graph shows the synovial inflammation status of rats in the sham-operated group and each experimental group; Figure 4 The images show the results of HE and Masson staining; in the images, A is the HE staining result of bone and joint tissue sections, and B is the Masson staining result of bone and joint tissue sections. Figure 5 The image shows the results of immunofluorescence detection of extracellular matrix degradation markers related to Aggrecan (proteoglycan, ACAN) and Col II (type II collagen); Figure 6 MRI images of the knee joints of rats in the sham-operated group and each experimental group; Note: The sham procedure shown in the image represents the sham surgery group, where only the joint is exposed without severing the ligaments. OA+PBS group: PBS was injected into the joint cavity; OA+nOM-MSCs refers to the OA+nOM-MSCs group: olfactory mucosal mesenchymal stem cells obtained by intra-articular injection of comparative example 1; OA+hOM-MSCs refers to the OA+hOM-MSCs group: olfactory mucosal mesenchymal stem cells obtained in Example 1 were injected into the joint cavity. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0041] A coaxial DBD plasma jet device (quartz tube outer diameter 6 mm / inner diameter 4 mm) was used, with a frequency of 20 kHz and a discharge power of 10~15 W.

[0042] Example 1 Step 1: Cell Acquisition, Culture, and Identification Methods Human olfactory mucosal tissue samples were collected and washed with phosphate-buffered saline under aseptic conditions to remove blood, secretions, and obvious non-target tissue. The tissue was cut into small tissue blocks of approximately 0.5–2 mm³ and seeded into cell culture flasks or dishes for primary culture using the tissue block adherence method.

[0043] After the tissue blocks adhered to the culture medium, culture was initiated using DMEM / F12 basal medium containing 8% FBS and 2.5% hPL. The cell culture conditions were 37 ℃ and 5% CO2. Once the cells had migrated out from around the tissue blocks and reached a suitable degree of confluence, they were digested and passaged.

[0044] The expanded cells were then identified. Immunofluorescence assay showed that the cells expressed STRO-1 and Nestin; flow cytometry analysis showed that the cells were positive for CD44, CD73, CD90, CD105, CD133, and CD146, and negative for CD34 and CD45. After identification confirming that the cells conformed to the mesenchymal stem cell phenotype, the cells were further expanded and cultured.

[0045] Step 2: Plasma-treated hydrogel scaffolds for culturing OM-MSCs Olfactory mucosal mesenchymal stem cells expanded to passage P3 were seeded into culture containers. After the cells reached 80%–90% confluence, they were collected, washed with sterile phosphate-buffered saline (PBS) to remove residual culture medium, centrifuged, and resuspended to prepare a cell suspension with a cell count of 5 × 10⁶ cells / mL. 6 indivual.

[0046] Sodium alginate powder was added to deionized water at 60°C while stirring to prepare a 3.2% w / v sodium alginate solution. Calcium chloride was dissolved in deionized water to prepare a 0.17% w / v calcium chloride solution.

[0047] 100 mL of sodium alginate solution was placed in a flask and treated with a non-thermal plasma jet at room temperature and atmospheric pressure. Helium (1 L / min) and oxygen (0.2 L / min) were used as carrier gases. The distance between the plasma jet and the medium was 10 mm. The treatment lasted for 30 min to obtain a plasma-treated sodium alginate solution.

[0048] A calcium chloride solution was continuously added dropwise to a plasma-treated sodium alginate solution to achieve cross-linking and solidification. The hydrogel beads were then filtered out and freeze-dried to obtain a gel scaffold rich in active particles.

[0049] Take the UV-sterilized gel scaffold, add it to DMEM / F12 basal medium for immersion and rehydration, then add 200 μL of scaffold to the culture wells, and add 5×10⁻⁶ ppm of the medium. 6The target cells were obtained by mixing 400 μL of DMEM / F12 medium with a pipette and incubating at 37 °C, 5% CO2, and 100 rpm for 24 h. The cells were then denoted as hOM-MSCs.

[0050] Comparative Example 1 Olfactory mucosal mesenchymal stem cells expanded to passage P3 were seeded into culture containers. After the cells reached 80%–90% confluence, they were collected, washed with sterile phosphate-buffered saline (PBS) to remove residual culture medium, centrifuged, and resuspended to prepare a cell suspension with a cell count of 5 × 10⁶ cells / mL. 6 indivual.

[0051] Sodium alginate powder was added to deionized water at 60°C while stirring to prepare a 3.2% w / v sodium alginate solution. Calcium chloride was dissolved in deionized water to prepare a 0.17% w / v calcium chloride solution.

[0052] Calcium chloride solution was continuously added dropwise to sodium alginate solution to achieve cross-linking and solidification. The hydrogel beads were then filtered out and freeze-dried to obtain a gel scaffold.

[0053] Take the UV-sterilized gel scaffold, add it to DMEM / F12 basal medium for immersion and rehydration, then add 200 μL of scaffold to the culture wells, and add 5×10⁻⁶ ppm of the medium. 6 One cell and 400 μL of DMEM / F12 medium were mixed by pipetting and cultured in a shaker at 37 ℃, 5% CO2, and 100 rpm for 24 h to obtain the target cells, which were designated as OM-MSCs.

[0054] Test Example 1 - In vitro therapeutic effect of olfactory mucosal mesenchymal stem cells Step 1: Obtaining chondrocytes Newborn SD rats were euthanized by dislocation. Under aseptic conditions, the skin was cut open to expose the femoral head. Cartilage with a thickness of about 0.1 to 0.2 mm was cut off, washed with PBS containing 1% penicillin and antibiotics, chopped, and digested in a 37°C water bath for 6 hours with 0.1% type II collagenase added.

[0055] Centrifuge at 1500 rpm for 3 min, discard the digestion solution, add culture medium to wash the cells, and resuspend to prepare a cell suspension with a cell count of 5 × 10⁶ cells / mL. 5 Add complete chondrocyte culture medium (DMEM / F12 basal medium containing 10% FBS + 1% penicillin-streptomycin) to each cell, and culture at 37°C with 5% CO2. Change the medium every 2 days. Passage the cells after they reach 80%–90% confluence.

[0056] P2 generation chondrocytes were used for experiments.

[0057] A cell model of osteoarthritis was established by culturing P2 generation chondrocytes (OA-ACs) with 10 ng / mL IL-1β in the culture medium for 24 h.

[0058] OM-MSCs and OA-ACs were co-cultured indirectly in Transwell chambers. After 48 h of culture, OM-MSCs were removed, and OA-ACs were cultured for another 24 h after changing the medium. The supernatant was collected, and the TNF-α secretion level was detected using an ELISA kit.

[0059] The results are shown in Table 1 below.

[0060] Table 1. OM-MSCs inhibit TNF-α levels in OA chondrocytes

[0061] As shown in Table 1, OM-MSCs cultured on hydrogel scaffolds obtained through non-thermal plasma jet treatment can rapidly exert their paracrine effects to improve the inflammatory level of OA-ACs and increase the TNF-α secretion level of chondrocytes. This indicates that OM-MSCs cultured on hydrogel scaffolds obtained through non-thermal plasma jet treatment can simulate the in vivo inflammatory microenvironment and induce OM-MSCs to produce a certain adaptive anti-inflammatory pretreatment response, enabling OM-MSCs to rapidly alleviate the inflammatory effects of OA-ACs upon contact.

[0062] Example 2 The difference between this embodiment and Embodiment 1 lies in the adjustment of the culture time and conditions of the olfactory mucosal mesenchymal stem cells, as detailed below: Olfactory mucosal mesenchymal stem cells expanded to generation P3 in Example 1 were seeded in a culture vessel. After the cells grew to 80%–90% confluence, the cells were collected, washed with sterile phosphate-buffered saline to remove residual culture medium, and resuspended after centrifugation to prepare a cell suspension with a cell count of 5 × 10⁶ cells / mL. 6 indivual.

[0063] Sodium alginate powder was added to deionized water at 60°C while stirring to prepare a 3.2% w / v sodium alginate solution. Calcium chloride was dissolved in deionized water to prepare a 0.17% w / v calcium chloride solution.

[0064] 100 mL of sodium alginate solution was placed in a flask and treated with a non-thermal plasma jet at room temperature and atmospheric pressure. Helium (1 L / min) and oxygen (0.2 L / min) were used as carrier gases. The distance between the plasma jet and the medium was 10 mm. The treatment lasted for 30 min to obtain a plasma-treated sodium alginate solution.

[0065] A calcium chloride solution was continuously added dropwise to a plasma-treated sodium alginate solution to achieve cross-linking and solidification. The hydrogel beads were then filtered out and freeze-dried to obtain a gel scaffold rich in active particles.

[0066] 1) Take the UV-sterilized gel scaffold, add it to DMEM / F12 basal medium for immersion and rehydration, then add 200 μL of scaffold to the culture wells, and add 5×10⁻⁶ mol / L of basal medium. 6 One cell and 400 μL of DMEM / F12 medium were mixed by pipetting and cultured in a shaker at 37 ℃, 5% CO2, and 100 rpm for 12 h to obtain the target cells, which were designated as 12h-OM-MSCs.

[0067] 2) Take the UV-sterilized gel scaffold, add it to DMEM / F12 basal medium for immersion and rehydration, then add 200 μL of scaffold to the culture wells, and add 5×10⁻⁶ ppm of the medium. 6 400 μL of DMEM / F12 medium and 100 μL of cells were mixed by pipetting and cultured in a shaker at 37 °C, 5% CO2, and 100 rpm for 48 h to obtain the target cells, which were designated as 48h-OM-MSCs.

[0068] 3) Take the UV-sterilized gel scaffold, add it to DMEM / F12 basal medium for immersion and rehydration, then add 200 μL of scaffold to the culture wells, and add 5×10⁻⁶ ppm of the medium. 6 One cell and 400 μL of DMEM / F12 medium were mixed by pipetting and cultured at 37 ℃ and 5% CO2 for 24 h to obtain the target cells, which were denoted as 24h-OM-MSCs.

[0069] Test Example 2 The OM-MSCs obtained in Example 2 were tested using the detection method of Test Example 1, and the following results were obtained: Table 2. Inhibition of TNF-α levels in OA chondrocytes by different cultured OM-MSCs

[0070] As shown in Table 2, the improvement of OA-ACs inflammation level by OM-MSCs obtained by culturing on hydrogel scaffolds treated with non-thermal plasma jet decreased with the extension of culture time. The effect of omitting the shaking culture in the culture stage also decreased. However, the obtained OM-MSCs still have good anti-inflammatory effects overall.

[0071] Application Example 1 - Application of olfactory mucosal mesenchymal stem cells in an osteoarthritis model Four-week-old male SD rats were used, and the DMM model is a classic surgical model for studying the progression of osteoarthritis (OA). In this study, four-week-old male rats weighing 200-250g were selected, and the surgery was performed under anesthesia. First, a small incision was made on the medial side of the knee joint to expose the joint cavity and identify the medial meniscus-tibial ligament (MMTL). This ligament was cut, thereby disrupting meniscal stability and inducing joint degeneration. After surgery, the wound was sutured, and antibiotics were administered to prevent infection. After recovery, the rats underwent running training for four weeks to simulate conditions leading to articular cartilage damage. The control group underwent joint exposure without ligament transection. After modeling, the animals were divided into a sham surgery group, an OA+PBS group, an OA+nOM-MSCs group, and an OA+hOM-MSCs group.

[0072] The OA+PBS group received intra-articular injection of PBS; Olfactory mucosal mesenchymal stem cells obtained in Comparative Example 1 were injected intra-articularly into the OA+nOM-MSCs group; Olfactory mucosal mesenchymal stem cells obtained in Example 1 were injected into the joint cavity of the OA+hOM-MSCs group.

[0073] The injection volume of olfactory mucosal mesenchymal stem cells is 100 μL per injection, with a cell count of 5 × 10⁶ cells. 6 indivual.

[0074] The efficacy of the drug was evaluated in each group of animals after administration. The evaluation included: ELISA to detect IL-1β and TNF-α levels in synovial fluid; rotarod test to evaluate motor coordination and recovery of motor function; HE and Masson staining to evaluate synovial inflammation and cartilage structural changes; immunofluorescence detection of Aggrecan and Col II-related extracellular matrix degradation indicators; and MRI to observe joint structure, cartilage surface, synovial thickness, and joint effusion.

[0075] The experimental results are as follows: Figure 1 Figure 1 shows that after DMM modeling, the levels of IL-1β and TNF-α in the synovial fluid of the OA+PBS group increased; after intervention with OM-MSCs, the levels of inflammatory factors decreased, with a more significant decrease in the pre-cultured OM-MSCs group, suggesting that pre-culture treatment can enhance the inhibitory effect of OM-MSCs on intra-articular inflammatory response in osteoarthritis.

[0076] Figure 2The results showed that the rotarod latency and total distance traveled by rats in the OA+PBS group decreased; after intervention with conventional scaffold cultured OM-MSCs and pre-cultured OM-MSCs, the above behavioral indicators were improved, with the pre-cultured OM-MSCs group showing more significant improvement, suggesting that this cell preparation can improve osteoarthritis-related motor dysfunction.

[0077] Figure 3 The results showed that the OA+PBS group had significant synovial thickening, congestion, and inflammatory changes; after intervention with pre-cultured OM-MSCs, synovial hyperplasia and inflammation were reduced, suggesting that this cell preparation has an ameliorative effect on synovial inflammation in osteoarthritis.

[0078] Figure 4 The results showed that the OA+PBS group exhibited cartilage surface damage, cartilage layer thinning, and disordered cell arrangement, accompanied by synovial inflammation and abnormal collagen fiber arrangement. After intervention with pre-cultured OM-MSCs, the integrity of the cartilage surface, chondrocyte arrangement, and collagen fiber structure were all improved, suggesting that the preparation has cartilage protection and tissue repair effects.

[0079] Figure 5 The results showed that the expression of Aggrecan and Col II chondrogenic metabolic markers was reduced in the cartilage tissue of the OA+PBS group; after intervention with OM-MSCs, the expression of the above-mentioned metabolic markers increased, and the expression of the pre-cultured OM-MSCs group recovered more significantly, suggesting that pre-culture treatment helps to enhance the promoting effect of OM-MSCs on chondrocyte phenotype maintenance and extracellular matrix synthesis.

[0080] Figure 6 The imaging findings showed that the knee joint in the OA+PBS group had changes in joint space, irregular cartilage surface, synovial thickening, and joint effusion. After intervention with pre-cultured OM-MSCs, joint structure, cartilage signal, and synovial inflammation-related manifestations were all improved, suggesting that this cell preparation can improve joint structural damage related to osteoarthritis.

[0081] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing an olfactory mucosal mesenchymal stem cell preparation, characterized in that, Includes the following steps: After passaged olfactory mucosal mesenchymal stem cells were prepared into a cell suspension, they were added to a plasma-treated hydrogel scaffold, culture medium was added and cultured until the target olfactory mucosal mesenchymal stem cells were harvested, and the target olfactory mucosal mesenchymal stem cells were prepared into a cell suspension to obtain the olfactory mucosal mesenchymal stem cell preparation. The method for preparing the plasma-treated hydrogel scaffold includes: treating sodium alginate solution with plasma, then adding calcium chloride solution to form hydrogel beads, followed by freeze-drying to obtain the plasma-treated hydrogel scaffold.

2. The method for preparing the olfactory mucosal mesenchymal stem cell preparation according to claim 1, characterized in that, The conditions for plasma processing are: using helium and oxygen as carrier gases.

3. The method for preparing the olfactory mucosal mesenchymal stem cell preparation according to claim 2, characterized in that, The helium flow rate is 0.8~1.2 L / min, and the oxygen flow rate is 0.1~0.3 L / min; The plasma treatment temperature is 20~30℃.

4. The method for preparing the olfactory mucosal mesenchymal stem cell preparation according to claim 1, characterized in that, Each 80-120 mL sodium alginate solution is subjected to plasma treatment for 10-50 min.

5. The method for preparing the olfactory mucosal mesenchymal stem cell preparation according to claim 1, characterized in that, The sodium alginate solution is an aqueous solution of sodium alginate with a concentration of 2.5% w / v to 4.5% w / v; the calcium chloride solution is an aqueous solution of calcium chloride with a concentration of 0.05% w / v to 0.3% w / v.

6. The method for preparing the olfactory mucosal mesenchymal stem cell preparation according to claim 1, characterized in that, Add culture medium and culture for 10-50 h to harvest target olfactory mucosal mesenchymal stem cells; The culture period of 10-50 h also includes shaking culture, with a shaking rate of 100-200 rpm; Before culturing, the cell concentration is 1~5×10⁻⁶. 6 The amount of the hydrogel scaffold used is 180~220 μL, and the amount of the culture medium used is 350~450 μL.

7. An olfactory mucosal mesenchymal stem cell preparation prepared by the method of any one of claims 1 to 6.

8. The use of the olfactory mucosal mesenchymal stem cell preparation as described in claim 7 in the preparation of a drug for treating degenerative osteoarthritis.

9. The application according to claim 8, characterized in that, The aforementioned olfactory mucosal mesenchymal stem cell preparation improves at least one of the following aspects: 1) Reduce synovial inflammation; 2) Repair cartilage tissue; 3) Improves motor dysfunction caused by osteoarthritis; 4) Reduce inflammation within the joints.

10. The use of the olfactory mucosal mesenchymal stem cell preparation as described in claim 7 in the preparation of a drug that inhibits the level of inflammatory factors in chondrocytes.

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