Use of mesenchymal stem cell spheroids in the preparation of a preparation for treating osteoarthritis

Abstract

The application discloses application of mesenchymal stem cell spheroids in preparation of a preparation for treating osteoarthritis, and relates to the technical field of cell therapy and osteoarthritis treatment, and specifically comprises mesenchymal stem cell spheroids with an average diameter of 120-180 mu m and a solution containing 0.5%-10% human serum albumin. The cells are prepared into 120-180 mu m cell spheroids, and the solution containing specific concentration human serum albumin is used as a carrier, so that the cell survival rate is stabilized at more than 90%, and high-activity delivery is realized; the optimized size ensures smooth injection and long-term retention in the joint cavity, and overcomes the shortcoming that single cells are easily removed; after joint cavity injection, the preparation can synergistically promote bone reconstruction and cartilage repair, and produces clear curative effects such as bone mass increase of more than 30%, cartilage degeneration score reduction of more than 50%, and the like, which are quantifiable and verifiable, so that a stable, long-acting and curative drug preparation and application scheme are provided for osteoarthritis treatment.

Description

Technical Field

[0001] This invention relates to the field of cell therapy and osteoarthritis treatment technology, specifically the application of mesenchymal stem cell spheres in the preparation of preparations for treating osteoarthritis. Background Technology

[0002] Osteoarthritis is a common degenerative joint disease, mainly characterized by damage to articular cartilage, pain, and functional impairment. Traditional treatments are difficult to fundamentally repair damaged cartilage, and their therapeutic effects are limited.

[0003] With the development of regenerative medicine, stem cell preparations have been used to treat osteoarthritis. In prior art document CN117338816A, a technical solution for a stem cell preparation for treating arthritis and its preparation method is disclosed. However, the stem cells used in this solution are in the form of single cells. When using single-cell injection, the stem cells are easily and quickly cleared in the joint cavity, and cannot achieve long-term retention, resulting in unstable treatment effects and short duration, which makes it difficult to meet the needs of clinical treatment. Summary of the Invention

[0004] The purpose of this invention is to provide an application of mesenchymal stem cell spheres in the preparation of agents for treating osteoarthritis, in order to solve the problem in the prior art that single-cell stem cells are easily and rapidly cleared from the joint cavity and cannot achieve long-term retention.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The application of a mesenchymal stem cell sphere in the preparation of a drug for treating osteoarthritis.

[0006] Preferably, the drug is administered via intra-articular injection.

[0007] Furthermore, the volume of the drug preparation injected into the joint cavity each time is 100-300 μL.

[0008] Furthermore, each intra-articular injection of the drug formulation contains 5000±500 mesenchymal stem cell spheres.

[0009] Preferably, the drug is used to improve the trabecular bone structure and / or reduce cartilage degeneration in patients with osteoarthritis.

[0010] A medicament for treating osteoarthritis, comprising mesenchymal stem cell spheres as used in the described application and a pharmaceutically acceptable delivery vehicle.

[0011] Preferably, the delivery carrier is a buffer solution containing 0.5%-10% (w / v) human serum albumin.

[0012] A method for preparing intermediate mesenchymal stem cell spheres as described above includes the following steps: S1 uses human umbilical cord mesenchymal stem cells to prepare a single-cell suspension; S2 involves inoculating the single-cell suspension obtained in S1 into a serum-free culture medium to obtain a cell suspension; S3. The cell suspension obtained in S2 is placed into a container and sealed. S4 involves periodically shaking the sealed container from S3 at varying speeds for 36–48 hours to form cell spheres. S5 collects the cell spheres obtained in S4.

[0013] Preferably, the serum-free culture medium is based on αMEM and consists of the following added components and their final concentrations: vitamin C 0.1-0.2 mM, potassium dihydrogen phosphate 1-2 mM, dexamethasone 0.01-0.05 μM, and human serum albumin 2-5%.

[0014] Preferably, the periodic speed change in S4 is to run at the first speed for a first time, then run at the second speed for a second time, and so on.

[0015] Furthermore, the first rotational speed is lower than the second rotational speed.

[0016] Furthermore, the first rotational speed is 40 rpm, the first time is 3 min, the second rotational speed is 60 rpm, and the second time is 30 s.

[0017] A mesenchymal stem cell sphere, prepared by the aforementioned culture method, has an average diameter of 120μm-180μm and cell viability ≥90%.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes the spherical form of mesenchymal stem cells, which can effectively achieve long-term retention within the joint cavity, significantly overcoming the disadvantage of single-cell stem cells being easily and rapidly cleared from the joint cavity, and prolonging the duration of action of stem cells at the lesion site.

[0019] Meanwhile, mesenchymal stem cell spheres can maintain their own activity and paracrine function, thereby improving the trabecular bone structure and reducing cartilage degeneration in patients with osteoarthritis, enhancing the therapeutic effect and making the efficacy more stable. This solves the technical problems of unstable therapeutic effects and short duration of existing single-cell stem cell preparations, and meets the clinical treatment needs of osteoarthritis. Attached Figure Description

[0020] Figure 1 This is a dynamic tracking imaging image of Example 3.

[0021] Figure 2 This is a bar chart showing the dynamic tracking results of Example 3.

[0022] Figure 3The images show the cell spheroidization after culturing for 0 / 12 / 24 / 48 hours according to this invention.

[0023] Figure 4 This is a diameter distribution diagram of the cell spheroid diameter in Example 1 of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Statistical analysis: Quantitative data are expressed as mean ± standard deviation (mean ± SD). Two-tailed Welcht tests were used for comparisons between the two groups (without assuming homogeneity of variance). A p-value < 0.05 was considered statistically significant. Significance notation: ns indicates p ≥ 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. The mesenchymal stem cells in this application are at least one of human umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, and placental mesenchymal stem cells, preferably human umbilical cord mesenchymal stem cells. Example 1

[0026] A method for the large-scale preparation of mesenchymal stem cell (MSC) spheres includes the following steps: (1) Provide a single-cell suspension of MSCs; (2) Add the single-cell suspension to a closed disposable soft bag container for suspension culture. The soft bag container is a blood bag or cell culture bag that allows gas exchange. (3) The soft bag container is placed on a shaker-type bioreactor, and the single cells are induced to aggregate and form MSC spheres by periodic variable speed shaking; wherein the periodic variable speed shaking speed is set to 20–120 rpm, preferably 30–80 rpm, more preferably 40–60 rpm; specifically, the shaking conditions are set to first shake at 40 rpm for 3 minutes, then shake at 60 rpm for 30 seconds, and the shaking is repeated in the above conditions. (4) After 36-48 hours of cultivation, the MSC spheres formed are collected to obtain MSC sphere products.

[0027] like Figure 3 The images shown are cell spheroidization patterns after 0 / 12 / 24 / 48 hours of culture. It can be seen that the cells form spheroids best after 36-48 hours.

[0028] like Figure 4As shown, the results indicate that the average diameter of the cell spheroids in the example group was 150.6 ± 19.1 μm, of which 85% had a diameter in the range of 120–180 μm.

[0029] The disposable soft bag container is a commercially available disposable PVC blood bag with a volume of 50–500 mL, preferably 100 mL.

[0030] The quality properties of the MSC spheres obtained through the above steps are shown in Table 1.

[0031] Table 1 index Results (mean ± SD) or percentage Average sphere diameter (μm) 150±19.6 Percentage of samples with diameters between 120 and 180 μm (%) 85% Survival rate (%) 90±2.5 Recovery rate before injection (%) 86.2±3.4 Number of balls per dose (per joint) 5000±500 Example 2

[0032] A pharmaceutical formulation containing MSC spheres is provided, comprising mesenchymal stem cell spheres obtained as described in Example 1 and a pharmaceutically acceptable carrier solution, wherein the carrier solution contains human serum albumin (HSA), for intra-articular injection administration.

[0033] The mass-volume fraction of human serum albumin is 0.5%–10%, more preferably 1%–8%, and even more preferably 5%.

[0034] In this embodiment, the injection volume of the drug preparation can be set to 50–500 μL / joint, preferably 100–300 μL / joint, and more preferably 200 μL / joint. Example 3

[0035] To verify the long-term retention of mesenchymal stem cell spheres within the joint cavity, dynamic tracking of the retention time was performed for up to 36 days using DIR near-infrared fluorescence staining imaging.

[0036] Experimental methods Labeling method: Mesenchymal stem cell spheres were pre-labeled using DIR cell membrane fluorescent probes; Administration method: 200 μl of mesenchymal stem cell sphere suspension was injected into the joint cavity of osteoarthritis model animals using a 5 ml syringe; Monitoring time points: 1 day, 8 days, 15 days, 22 days, 29 days, and 36 days post-surgery; Detection method: Small animal in vivo imaging system to collect fluorescence signal intensity in the joint cavity area.

[0037] Experimental results are as follows Figure 1 and Figure 2 As shown, significant specific fluorescence signals were still detected in the joint cavity region in the imaging results on day 29, and 3D-MSCs at 36 days still had significant signals, while 2D-MSCs no longer had obvious signals.

[0038] in conclusion Experimental data confirms that the cell sphere diameter set in this invention prevents it from being discharged through the joint capsule and maintains structural stability in the synovial fluid environment, thereby achieving long-term retention in the joint cavity for up to 28 days, providing crucial time assurance for its continuous cartilage repair function. Example 4

[0039] The drug formulation of this MSC sphere can be used to treat osteoarthritis, and this can be verified through the following experimental steps: ACLT osteoarthritis model establishment, grouping and time axis: Healthy rats were selected, and an anterior cruciate ligament injury / resection (ACLT) surgery was performed on the right knee after anesthesia to establish an osteoarthritis model.

[0040] Animals were randomly assigned to groups and evaluated using a blinded method; drug administration was performed one month after model establishment; animals were sacrificed two months after drug administration for sampling and testing. Grouping and timelines are shown in Table 2.

[0041] Table 2 Animal grouping, sample size and time axis Group n per group Dosage time Sampling and testing time SHAM 10 — same batch as the model group CONTROL 10 One month after modeling, inject an equal volume of HSA. Corresponding time point 2 months after administration 2D single cell 10 One month after modeling 2 months after administration 3D Cell Ball 10 One month after modeling 2 months after administration Specifically, the dosing parameters are as follows: One month after modeling, intra-articular injections were performed on the "2D single-cell group" and the "3D cell sphere group".

[0042] The administration record is as follows: Each animal was injected with 2 × 10^6 cell equivalents, the carrier was 5% HSA, the total volume was 200 μL, and the administration was once. The administration parameters are shown in Table 3.

[0043] Table 3. Dosing formulations and dosing parameters project parameter route of administration Intra-articular injection (right knee) Dosage time One month after modeling Cell dose 2×10^6 cell equivalents / joint Formulation carrier 5% HSA Injection volume 200μL / joint Dosage frequency 1 time To evaluate the above therapeutic results, the following trial was conducted.

[0044] Experimental Case 1: Significant improvement in micro-CT trabecular bone parameters (BV / TV, Tb.N, Tb.Sp, Tb.Th) Two months after drug administration, rats were sacrificed and their knee joints were scanned using micro-CT. BV / TV, Tb.N, Tb.Sp, and Tb.Th were calculated using fixed ROIs. The results are shown in Table 4.

[0045] Compared with the CONTROL group, the 3D cell sphere group showed that BV / TV increased from 0.3864±0.0234 to 0.5260±0.0584 (an increase of 36.1%, or 1.361 times), Tb.N increased from 1.2232±0.0941 to 1.5804±0.1211 (an increase of 29.2%, or 1.292 times), Tb.Sp decreased from 0.4895±0.0598 to 0.3464±0.0489 (a decrease of 29.2%, or 0.708 times), and Tb.Th increased from 0.3100±0.0221 to 0.3757±0.0482 (an increase of 21.2%, or 1.212 times), suggesting that 3D cell spheres have a significant effect on improving bone trabecular structure.

[0046] The 3D cell spheroid group showed improvement compared to the 2D single-cell group. Specifically, the 3D cell spheroid group was superior to the 2D single-cell group in BV / TV (9.6% higher than 2D, P=0.041) and Tb.N (13.5% higher than 2D, P=0.0107), while Tb.Sp showed no significant difference (P=0.301, ns), and Tb.Th was higher than the 2D group (11.9% higher than 2D, P=0.041, *).

[0047] Table 4. Micro-CT trabecular bone parameters (mean ± SD, n = 10) and the magnitude / significance of improvement compared with the CONTROL group. index SHAM CONTROL 2D single cell 3D Cell Ball BV / TV 0.5459±0.0537 0.3864±0.0234 0.4798±0.0272（+24.2%，***） 0.5260±0.0584（+36.1%，***） Tb.N 1.6503±0.1582 1.2232±0.0941 1.3926±0.1672（+13.8%，*） 1.5804±0.1211（+29.2%，***） Tb.Sp 0.3403±0.0428 0.4895±0.0598 0.3879±0.1111（−20.8%，*） 0.3464±0.0489（−29.2%，***） Tb.Th 0.4116±0.0965 0.3100±0.0221 0.3357±0.0294（+8.3%，*） 0.3757±0.0482（+21.2%，**） Experimental Example 2: Significant Improvement in SO Staining Score After fixation, decalcification, embedding, and sectioning of the joint tissue, safranin O-Fixed Green (SO) staining was performed, and cartilage degeneration was scored according to a pre-set semi-quantitative scoring system (the higher the score, the more severe the degeneration). The results are shown in Table 5.

[0048] Compared with the CONTROL group, the SO score of the 3D cell spheroid group decreased from 4.00±0.866 to 1.50±0.50 (a decrease of 62.5%, 0.375-fold, **), suggesting that 3D cell spheroids can significantly alleviate cartilage degeneration; the score of the 2D single-cell group was 2.5857±1.0779, which was significantly different from that of the CONTROL group (P=0.0174). Furthermore, the 3D cell spheroid group was significantly better than the 2D single-cell group (a decrease of 42%, P=0.039, *).

[0049] Table 5 SO staining degeneration score (mean ± SD) and comparison with the CONTROL group Group Effective n Score (mean ± SD) Relative CONTROL change Significance (vs. CONTROL) SHAM 8 1.25±0.4330 −68.8% *** CONTROL 8 4.00±0.8660 — — 2D single cell 7 2.5857±1.0779 −35.4% *（P=0.0174） 3D Cell Ball 8 1.50±0.50 −62.5% ***（P=1.88×10^-5） Experimental Example 3: Significant Improvement in COL2A1 Immunohistochemical Score COL2A1 immunohistochemical staining was performed on articular cartilage sections, and scores were assigned according to a pre-defined semi-quantitative scoring system (higher scores indicate more severe degeneration / higher degree of type II collagen-related abnormalities in cartilage). The results are shown in Table 6.

[0050] Compared with the CONTROL group, the COL2A1 score in the 3D cell spheroid group decreased from 3.1667±0.5528 to 1.1667±0.3727 (a decrease of 63.2%, 0.368-fold, *), and recovered to a level comparable to the SHAM group (ns); the score in the 2D single-cell group was 2.1667±0.4714, a decrease of 31.6% compared with the CONTROL group (**). Furthermore, the 3D cell spheroid group was significantly superior to the 2D single-cell group, with a decrease of 46.2% compared with the 2D single-cell group (P=0.00248, **).

[0051] Table 6. COL2A1 immunohistochemical degeneration score (mean ± SD, n=6) and comparison with the CONTROL group. Group Score (mean ± SD) Relative CONTROL change Significance (vs. CONTROL) SHAM 1.1667±0.3727 −63.2% *** CONTROL 3.1667±0.5528 — — 2D single cell 2.1667±0.4714 −31.6% **（P=0.00735） 3D Cell Ball 1.1667±0.3727 −63.2% ***（P=4.98×10^-5） In summary, MSC cell spheres prepared on a large scale using PVC blood bags with heterogeneous shaking, when injected intra-articularly to treat an ACLT osteoarthritis model, showed statistically significant improvements compared to the untreated group in micro-CT trabecular bone structure (BV / TV, Tb.N, Tb.Sp, Tb.Th), cartilage histology (SO score), and COL2A1 immunohistochemical score. Furthermore, the MSC cell spheres were superior to the 2D single-cell suspension group in key indicators, demonstrating the clear and quantifiable therapeutic efficacy of this invention.

[0052] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Furthermore, the contents not described in detail in this specification are all prior art known to those skilled in the art.

Claims

1. The use of a mesenchymal stem cell sphere in the preparation of a drug for treating osteoarthritis.

2. The application according to claim 1, characterized in that, The drug is administered via intra-articular injection.

3. The application according to claim 2, characterized in that, The volume of the drug preparation injected into the joint cavity each time is 100-300 μL.

4. The application according to claim 3, characterized in that, Each intra-articular injection of the drug formulation contains 5000±500 mesenchymal stem cell spheres.

5. The application according to claim 1, characterized in that, The drug is used to improve the trabecular bone structure and / or reduce cartilage degeneration in patients with osteoarthritis.

6. A medicament for treating osteoarthritis, characterized in that, Includes mesenchymal stem cell spheres and pharmaceutically acceptable delivery carriers used in any of the applications described in claims 1-5.

7. The drug according to claim 6, characterized in that, The delivery carrier is a buffer solution containing 0.5%-10% (w / v) human serum albumin.

8. A method for culturing intermediate mesenchymal stem cell spheres according to any one of claims 1-5, characterized in that, Includes the following steps: S1 uses human umbilical cord mesenchymal stem cells to prepare a single-cell suspension; S2 involves inoculating the single-cell suspension obtained in S1 into a serum-free culture medium to obtain a cell suspension; S3. The cell suspension obtained in S2 is placed into a container and sealed. S4 involves periodically shaking the sealed container from S3 at varying speeds for 36–48 hours to form cell spheres. S5 collects the cell spheres obtained in S4.

9. The cultivation method according to claim 8, characterized in that, The serum-free culture medium is based on αMEM and consists of the following added components and their final concentrations: vitamin C 0.1-0.2 mM, potassium dihydrogen phosphate 1-2 mM, dexamethasone 0.01-0.05 μM, and human serum albumin 2-5%.

10. The cultivation method according to claim 8, characterized in that, The periodic speed change in S4 involves running at the first speed for a first time, then running at the second speed for a second time, and so on.

11. The cultivation method according to claim 10, characterized in that, The first rotational speed is lower than the second rotational speed.

12. The cultivation method according to claim 11, characterized in that, The first rotational speed is 40 rpm, the first time is 3 min, the second rotational speed is 60 rpm, and the second time is 30 s.

13. A mesenchymal stem cell sphere, characterized in that, Prepared by the culture method described in any one of claims 8-12, the cells have an average diameter of 120 μm-180 μm and a cell viability of ≥90%.

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