Mesenchymal stem cell composite preparation for treating cartilage injury

Abstract

The invention discloses a mesenchymal stem cell compound preparation for treating cartilage injury, and belongs to the technical field of cell biology. The composite preparation consists of human umbilical cord mesenchymal stem cells (hUC-MSC) subjected to specific pretreatment and a pharmaceutical carrier. The pretreatment method sequentially comprises the following steps: carrying out low-oxygen pretreatment on the hUC-MSC; then, carrying out pretreatment on a ferroptosis inhibitor Ferrostatin-1 under a low-oxygen condition; and finally, carrying out icariin pretreatment under a normal oxygen condition. The unique triple pretreatment strategy generates a remarkable synergistic effect, and can greatly improve the survival rate and the function maintenance capability of the hUC-MSC in the cartilage injury inflammatory microenvironment. Experimental results prove that the preparation can significantly inhibit collagen degradation and promote cartilage matrix regeneration, and the treatment efficiency of stem cell transplantation and the repair effect of cartilage injury are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, and in particular relates to a mesenchymal stem cell complex preparation for treating cartilage damage. Background Technology

[0002] Cartilage injury is a common and challenging clinical condition. Mature cartilage tissue, lacking direct blood supply, nerve innervation, and lymphatic drainage, has extremely limited self-repair capabilities. When full-thickness cartilage injury occurs, the body often only produces fibrocartilage with poor biomechanical properties for repair, failing to restore the original structure and function of hyaline cartilage. This leads to progressive joint dysfunction and even secondary complications such as osteoarthritis. Currently, commonly used first-line treatments include microfracture surgery, autologous or allogeneic cartilage transplantation, autologous chondrocyte implantation, and matrix-induced autologous chondrocyte transplantation. While these methods can partially alleviate symptoms in the short term, they still have many limitations, such as donor site tissue damage, immune rejection, post-repair cartilage hypertrophy, insufficient long-term durability of newly formed tissue, and late-stage degeneration, severely impacting long-term efficacy.

[0003] In recent years, mesenchymal stem cells (MSCs) have been regarded as one of the most promising seed cells in the field of cartilage regeneration due to their multi-lineage differentiation potential, immunomodulatory properties, and strong paracrine function. Among them, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have become one of the preferred cell types for clinical research and therapeutic exploration at home and abroad due to their convenient acquisition, wide availability, strong expansion capacity, low immunogenicity, and fewer ethical controversies.

[0004] However, direct transplantation of untreated hUC-MSCs after routine in vitro culture still faces challenges such as low cell survival and limited actual repair effects. To improve treatment outcomes, multiple injections are often required clinically, which not only increases treatment complexity but may also introduce additional medical risks. Therefore, improving the survival and functional maintenance capabilities of transplanted hUC-MSCs has become a critical issue urgently needing to be addressed in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a mesenchymal stem cell complex preparation for treating cartilage damage.

[0006] To achieve the above objectives, the present invention provides the following technical solution: First, the present invention provides a mesenchymal stem cell composite preparation for treating cartilage injury, the mesenchymal stem cell composite preparation being composed of pretreated mesenchymal stem cells and a pharmaceutically acceptable carrier; The pretreated mesenchymal stem cells were obtained by the following pretreatment method: (1) Seed mesenchymal stem cells into a cell culture dish and culture them until the mesenchymal stem cells are completely adhered to the dish; (2) The mesenchymal stem cells that have adhered to the wall are subjected to hypoxia pretreatment; (3) The mesenchymal stem cells were subjected to ferroptosis inhibition pretreatment under the hypoxic pretreatment environment; (4) After restoring normoxic conditions, the mesenchymal stem cells were pretreated with icariin to obtain pretreated mesenchymal stem cells.

[0007] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0008] Preferably, the preprocessing method comprises the following steps: (a) Human umbilical cord mesenchymal stem cells were seeded into a culture dish and cultured until the cells adhered to the dish. (b) Place the petri dish in a low-oxygen environment of 2% O2 and incubate for 48 hours; (c) While maintaining a low oxygen condition of 2% O2, replace the medium with a final concentration of 0.5 μmol / LFerrostatin-1 and continue culturing for 24 hours; (d) After restoring normoxic conditions, the culture medium was replaced with one containing 5 μmol / L icariin and cultured for another 24 hours to obtain pretreated mesenchymal stem cells.

[0009] Preferably, in the mesenchymal stem cell complex preparation, the number of pretreated mesenchymal stem cells is ≥5×10⁻⁶. 5 indivual.

[0010] Secondly, this invention provides a pretreatment method to improve the survival ability of mesenchymal stem cells in an inflammatory environment and / or the cartilage damage repair effect, the pretreatment method comprising the following steps: 1) Human umbilical cord mesenchymal stem cells were seeded into a culture dish and cultured until the cells adhered to the dish. 2) Place the petri dish in a low-oxygen environment and incubate for 48 hours; 3) While maintaining low oxygen conditions, replace the medium with one containing Ferrostatin-1 and continue culturing for 24 hours; 4) After restoring normoxic conditions, replace the culture medium with one containing icariin and continue culturing for 24 hours to obtain pretreated mesenchymal stem cells.

[0011] Preferably, in step 2), the low-oxygen environment is a 2% O2 environment.

[0012] Preferably, in step 3), the hypoxic condition is 2% O2, and the concentration of Ferrostatin-1 in the culture medium containing Ferrostatin-1 is 0.5 μmol / L.

[0013] Preferably, in step 4), the concentration of icariin in the culture medium containing icariin is 5 μmol / L.

[0014] Furthermore, the present invention provides the application of pretreated mesenchymal stem cells obtained by the above-described pretreatment method in the preparation of a drug for treating cartilage damage.

[0015] The beneficial effects of this invention are as follows: This invention provides a method for treating human umbilical cord mesenchymal stem cells (hUC-MSCs) in a specific sequence of pretreatment with hypoxia, ferroptosis inhibitor (Ferrostatin-1), and icariin, resulting in a mesenchymal stem cell composite preparation with significant advantages. The cell survival rate of hUC-MSCs treated by this method is significantly improved in the inflammatory microenvironment. Simultaneously, the specific triple pretreatment strategy produces a significant synergistic effect, which is significantly superior to any single treatment or simple pairwise combinations. Therefore, the cell composite preparation of this invention improves the therapeutic effect on cartilage damage while reducing the number of treatments, and lowers treatment costs and operational complexity. Attached Figure Description

[0016] Figure 1 This is a bar chart comparing the survival rates of human umbilical cord mesenchymal stem cells before and after pretreatment in an in vitro inflammation model. Figure 2 Bar chart showing the detection results of CTX-II, a marker of type II collagen degradation, in the serum of rats in each treatment group; Figure 3 A bar chart showing the detection results of glycosaminoglycan (GAG) content in the knee joint lavage fluid of rats in each treatment group. Detailed Implementation

[0017] The following details the specific implementation methods of the present invention. It should be noted that the implementation methods described are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. Any non-essential modifications and substitutions made in accordance with the present invention should be within the scope of protection of the present invention.

[0018] Example 1 A pretreatment method to improve stem cell survival and repair efficacy 1 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Place the petri dish in a three-gas incubator with 2% O2, 5% CO2 and balanced N2 for 48 hours of low oxygen culture; (3) While maintaining 2% O2, replace with α-MEM medium containing a final concentration of 0.5 μmol / L Ferrostatin-1 and continue culturing for 24 hours; (4) Restore normoxic conditions, add α-MEM medium containing 5 μmol / L icariin, and continue culturing for 24 hours; (5) After the treatment, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0019] Example 2 A pretreatment method to improve stem cell survival and repair efficacy 2 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were processed at a rate of 5 × 10⁻⁶. 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Place the petri dish in a three-gas incubator with 2% O2, 5% CO2 and balanced N2 for 72 hours under low oxygen conditions; (3) Restore normoxic conditions, replace with new α-MEM medium, and continue culturing for 24 hours; (4) After the treatment, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0020] Example 3 A pretreatment method to improve stem cell survival and repair efficacy 3 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) After culturing under normal oxygen conditions for 48 hours, the medium was replaced with α-MEM medium containing a final concentration of 0.5 μmol / L Ferrostatin-1, and cultured for another 24 hours. (3) Continue to culture under normal aerobic conditions for 24 hours. After the treatment is completed, wash twice with PBS, digest with trypsin and collect the pretreated hUC-MSCs.

[0021] Example 4 A pretreatment method to improve stem cell survival and repair efficacy 4 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×104 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) After culturing the cells under normoxic conditions for 72 hours, add α-MEM medium containing 5 μmol / L icariin and continue culturing for 24 hours; (3) After the treatment was completed, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0022] Example 5 A pretreatment method to improve stem cell survival and repair efficacy 5 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Place the petri dish in a three-gas incubator with 2% O2, 5% CO2 and balanced N2 for 48 hours of low oxygen culture; (3) While maintaining 2% O2, replace with α-MEM medium containing a final concentration of 0.5 μmol / L Ferrostatin-1 and continue culturing for 24 hours; (4) After restoring normoxic conditions and continuing to culture for 24 hours, the cells were washed twice with PBS, digested with trypsin, and collected to obtain pretreated hUC-MSCs.

[0023] Example 6 A pretreatment method to improve stem cell survival and repair efficacy 6 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Place the petri dish in a three-gas incubator with 2% O2, 5% CO2 and balanced N2 for 72 hours of low oxygen culture; (3) Restore normal oxygen, add α-MEM medium containing 5 μmol / L icariin, and continue culturing for 24 hours; (4) After the treatment, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0024] Example 7 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) After 48 hours of normoxic culture, the cells were replaced with α-MEM medium containing a final concentration of 0.5 μmol / L Ferrostatin-1 and cultured for another 24 hours. (3) After the culture is completed, add α-MEM medium containing 5 μmol / L icariin and continue to culture for 24 hours; (4) After the treatment, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0025] Example 8 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Add α-MEM medium containing 5 μmol / L icariin and culture under normal aerobic conditions for 24 hours; (3) Cultured under low oxygen conditions for 48 hours in a three-gas incubator containing 2% O2, 5% CO2, and balanced N2; (4) While maintaining 2% O2, replace with α-MEM medium containing a final concentration of 0.5 μmol / L Ferrostatin-1 and continue culturing for 24 hours; (5) After the treatment, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0026] Example 9 (1) P4 generation human umbilical cord mesenchymal stem cells (hUC-MSC) were prepared at a rate of 1×10 4 cells / cm 2 The amount of the seed is inoculated into a culture dish and cultured until the cells adhere to the dish. (2) Place in an incubator and incubate under normal aerobic conditions for 96 hours; (3) After the treatment was completed, the cells were washed twice with PBS, digested with trypsin and collected to obtain pretreated hUC-MSCs.

[0027] Example 10 To detect the tolerance of cells treated according to the present invention to inflammation. 1. Cell preparation The pretreated umbilical cord mesenchymal stem cells obtained in Examples 1-9 were processed at a ratio of 5 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates (Corning 3599) with 100 μL of complete culture medium (α-MEM + 10% FBS) per well and incubated at 37°C, 21% O2, and 5% CO2 for 24 hours to adhere to the plates.

[0028] 2. Prepare the working solution for inflammatory factors The 100 μg / mL recombinant human IL-1β (PeproTech 200-01B) stock solution was diluted to 20 ng / mL using serum-free α-MEM; The 100 μg / mL recombinant human TNF-α (PeproTech 300-01A) stock solution was diluted to 40 ng / mL using serum-free α-MEM; The diluted solutions were mixed in a 1:1 ratio to obtain a working solution of inflammatory factors containing IL-1β 10 ng / mL + TNF-α 20 ng / mL.

[0029] 3. Inflammatory stimulation After 24 hours of adhesion, the supernatant was discarded, and the cells were washed once with PBS. 100 μL of inflammatory factor working solution was added to each well, and a non-irritating control well (serum-free α-MEM) was set up. The cells were then incubated at 37°C, 21% O2, and 5% CO2 for 72 hours.

[0030] 4. CCK-8 assay for survival rate After 72 h, the supernatant containing inflammatory factors was carefully aspirated, and the sample was washed twice with PBS. 100 μL of CCK-8 working solution (CCK-8 : serum-free α-MEM = 1: 9) was added to each well. The sample was incubated at 37℃ in the dark for 1.5 h. The OD value was measured at 450 nm using a microplate reader, and the survival rate was calculated. Six replicates were set up for each group.

[0031] Table 1. Inflammation tolerance of cells with different pretreatments As can be seen from the results in Table 1, the cell survival rate of hUC-MSCs in Example 9 without pretreatment was only 21.7%, indicating that prolonged inflammatory treatment significantly reduces the cell survival rate of umbilical cord mesenchymal stem cells.

[0032] Further analysis showed that the cell viability of Examples 2, 3, and 4 was significantly improved compared to Example 9 without pretreatment, indicating that pretreatment with hypoxia, Ferrostatin-1, or icariin can effectively improve the cell viability of hUC-MSCs.

[0033] The results from Examples 5-6 show that when hypoxia, Ferrostatin-1, or icariin are pretreated in pairs, the overall effect is essentially the sum of the effects of the two methods. The results from Examples 1 and 8 show that pretreatment with hypoxia, Ferrostatin-1, or icariin in a specific order, resulting in cells of Example 1, significantly improved their tolerance to inflammatory environments compared to simply combining these two methods. This indicates that the pretreatment method described in Example 1 synergistically enhances the tolerance of hUC-MSCs to inflammatory environments, allowing them to effectively treat cartilage damage. However, Example 8, which uses icariin first, followed by hypoxia and Ferrostatin-1 treatment, does not exhibit a significant synergistic effect.

[0034] Example 11 1. Animal selection Male SD rats aged 8-12 weeks were selected, fed a standard laboratory diet, and given free access to water, with a 12-hour light-dark cycle. They were acclimatized for one week before the experiment.

[0035] 2. Anesthesia After weighing the rats, the anesthetic dose of sodium pentobarbital was calculated at 30 mg / kg and administered intraperitoneally. After anesthesia, the rats were placed in a supine position on a constant temperature operating table (37℃), with the right hind limb knee joint elevated and the knee joint flexed at 45°.

[0036] 3. Injection Shave the hair around the right posterior knee joint in a 1 cm × 1 cm area, disinfect with iodine-soaked cotton balls 3 times, and remove iodine once with 75% ethanol. The needle insertion point is located at the anterior medial aspect of the lower border of the patella, forming a 30° angle with the direction of the intercondylar fossa, to a depth of approximately 3-4 mm. 50 μL of sodium iodoacetate solution (containing 0.5 mg of the drug) is slowly injected. After slowly withdrawing the needle, apply pressure for 30 seconds to stop the bleeding. After the procedure, administer a subcutaneous injection of 50,000 U of penicillin for 3 consecutive days to prevent infection.

[0037] 4. Enhanced injection Repeat the above steps, injecting 50 μL of sodium iodoacetate solution again on day 4 and day 7, respectively.

[0038] 5. Model Validation On the 28th day after surgery, rats that passed the Von Frey test (the mechanical pain threshold test of the foot) were used for subsequent experiments (as the first day of treatment).

[0039] Stem cell therapy groups (6 animals per group): Normal control group: Normal male SD rats that were not subjected to modeling treatment, fed normally, and without any injection treatment; Model group: Male SD rats, fed normally, injected with 50 μL PBS on day 1, day 8, day 15 and day 22; Cell treatment group 1: On day 1, hUC-MSCs treated in Example 9 were injected once, and on day 15, the cell preparation was injected again. The dosage of the cell preparation was 50 μL per dose, and the cell quantity was 5 × 10⁶ cells. 5 Cells, prepared with PBS; 50 μL of PBS was injected on days 8 and 22; Cell treatment group 2: On day 1, hUC-MSCs treated in Example 2 were injected once, and on day 15, the cell preparation was injected again. The dosage of the cell preparation was 50 μL per dose, and the cell quantity was 5 × 10⁶ cells. 5 Cells, prepared with PBS; 50 μL of PBS was injected on days 8 and 22; Cell treatment group 3: On day 1, hUC-MSCs treated in Example 3 were injected once, and on day 15, the cell preparation was injected again. The dosage of the cell preparation was 50 μL per dose, and the cell quantity was 5 × 10⁶ cells. 5 Cells, prepared with PBS; 50 μL of PBS was injected on days 8 and 22; Cell treatment group 4: On day 1, hUC-MSCs treated in Example 4 were injected once, and on day 15, the cell preparation was injected again. The dosage of the cell preparation was 50 μL per dose, and the cell quantity was 5 × 10⁶ cells. 5 Cells, prepared with PBS; 50 μL of PBS was injected on days 8 and 22; Cell treatment group 5: On day 1, hUC-MSCs treated in Example 1 were injected once, and on day 15, the cell preparation was injected again. The dosage of the cell preparation was 50 μL per dose, and the cell quantity was 5 × 10⁶ cells. 5 Cells, prepared with PBS; 50 μL of PBS was injected on days 8 and 22; Positive cell treatment group: hUC-MSCs treated in Example 1 were injected once on the first day, and then injected again on the 8th, 15th and 22nd days respectively; Four rats were used in each group, and the entire treatment cycle lasted 28 days.

[0040] 6. Detect the differences in serum CTX-II (type II collagen C-terminal cross-linked peptide) in rats under different treatments. After the treatment cycle was completed, the rats were anesthetized with 3% sodium pentobarbital and 5 mL of blood was collected from their hearts. After standing for 30 minutes, centrifuge at 3000g for 15 minutes at 4℃ and collect the supernatant serum. The levels of CTX-II in the serum of each treatment group were detected using the Rat CTX-II ELISA Kit.

[0041] The results are shown in Table 2 and Figure 2 middle.

[0042] Table 2. CTX-II levels in rats under different treatment groups From Table 2 and Figure 2 The serum CTX-II level detection results showed that the CTX-II level in the model group was significantly higher than that in the normal control group, indicating that the MIA modeling osteogenic induction of the present invention induced type II collagen degradation.

[0043] Compared to the model group, the cell treatment group 1 had a poorer therapeutic effect due to the lower amount of injected cells, indicating that ordinary hUC-MSCs had poor survival in the inflammatory microenvironment and a poor ability to inhibit cartilage matrix degradation.

[0044] The CTX-II levels in cell treatment groups 2-4 decreased by 19.92%-24.24% compared to the model group, and further decreased by 7.42%-17.12% compared to cell treatment group 1. This indicates that pretreatment with hypoxia, Ferrostatin-1, or icariin can enhance the ability of hUC-MSCs to inhibit type II collagen degradation to some extent, but the improvement is limited.

[0045] Further observation revealed that the CTX-II level in cell treatment group 5 was significantly lower than that in the model group, and superior to that in the positive cell group. In terms of enhancement effect, cell treatment group 5 showed a significantly better enhancement effect than cell treatment group 1 than simply adding cell treatment groups 2, 3, and 4 together. These results demonstrate that hUC-MSCs treated using the method of this invention can significantly improve long-term survival and functional maintenance in an inflammatory environment, thereby achieving better therapeutic effects on cartilage damage while reducing the injection volume.

[0046] 7. Detect the differences in GAG (glycosaminoglycan) levels in rats from different treatment groups. After sacrificing the rats, the right knee joint of the rats was exposed, and a 30G insulin needle was used to insert into the joint cavity from the outside of the patellar ligament. 100 μL of PBS containing 0.1% protease inhibitor was injected, and the fluid was aspirated back and forth 10 times to recover the irrigation fluid. Centrifuge the rinsing solution at 4°C and 12000g for 10 minutes to remove cell debris, and add 40μL of supernatant to a 96-well cell culture plate. Quickly add 200 μL DMMB staining solution, mix immediately, and then measure the absorbance at A525 (main wavelength) and A590 (background) using an ELISA reader. Calculate ΔA = A525 – A590. A standard curve was plotted using chondroitin sulfate standards, and the GAG ​​concentration was calculated from the standard curve.

[0047] The results are displayed in Figure 3 And in Table 3.

[0048] Table 3. Differences in GAG levels in rats from different treatment groups. From Table 3 and Figure 3 The results of GAG level detection show that the GAG ​​in the joint lavage fluid of the model group was significantly lower than that of the control group, indicating that the MIA modeling of the present invention led to severe loss of cartilage matrix.

[0049] Compared to the model group, the GAG ​​level in cell treatment group 1 increased by only 21.47%. Although this was better than the effect of CTX-II, it was still effective, indicating that hUC-MSCs have limited therapeutic effect on cartilage damage when the cell injection volume is insufficient.

[0050] Compared to cell treatment group 1, cell treatment groups 2, 3 and 4 showed a further increase in GAG levels, but the effect was still not ideal.

[0051] The effects of cell treatment group 5 were not only significantly better than those of cell treatment groups 2-4, but also, due to the expected effect of combining Examples 2-4 (52.87% based on the Bliss independent model), it indicates that the combined pretreatment strategy produced a significant synergistic effect. Furthermore, the GAG ​​level in cell treatment group 5 was also significantly better than that in the positive cell treatment group. This result is similar to the CTX-II inhibition trend in Table 2, further demonstrating that hUC-MSCs pretreated using the method of this invention can achieve better cartilage damage repair in an inflammatory environment.

Claims

1. A mesenchymal stem cell complex preparation for treating cartilage injury, characterized by, The mesenchymal stem cell complex preparation is composed of pretreated mesenchymal stem cells and a pharmaceutically acceptable carrier. The pretreated mesenchymal stem cells are obtained by a pretreatment method as follows: (1) inoculating mesenchymal stem cells into a cell culture dish and culturing until the mesenchymal stem cells are completely adherent; (2) performing hypoxic pretreatment on the adherent mesenchymal stem cells; (3) performing ferroptosis inhibition pretreatment on the mesenchymal stem cells under the hypoxic pretreatment environment; (4) after restoring normoxic conditions, performing icariin pretreatment on the mesenchymal stem cells to obtain pretreated mesenchymal stem cells.

2. The mesenchymal stem cell complex formulation according to claim 1, characterized by, The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

3. The mesenchymal stem cell complex formulation according to claim 2, wherein The specific steps of the pretreatment method are as follows: (a) inoculating human umbilical cord mesenchymal stem cells into a culture dish and culturing until the cells are adherent; (b) placing the culture dish in a hypoxic environment of 2% O2 for 48 hours; (c) under the continued hypoxic conditions of 2% O2, replacing the culture medium with one containing a final concentration of 0.5 μmol / L Ferrostatin-1 and continuing to culture for 24 hours; (d) after restoring normoxic conditions, replacing the culture medium with one containing 5 μmol / L icariin and continuing to culture for 24 hours to obtain the pretreated mesenchymal stem cells.

4. The mesenchymal stem cell complex formulation according to claim 2, wherein The amount of the pretreated mesenchymal stem cells in the mesenchymal stem cell complex preparation is ≥5×10 5 cells.

5. A pretreatment method for improving the survival ability of mesenchymal stem cells in an inflammatory environment and / or the effect of cartilage damage repair, characterized by, The pretreatment method comprises the following steps: 1) inoculating human umbilical cord mesenchymal stem cells into a culture dish and culturing until the cells are adherent; 2) placing the culture dish in a hypoxic environment for 48 hours; 3) under the continued hypoxic conditions, replacing the culture medium with one containing Ferrostatin-1 and continuing to culture for 24 hours; 4) after restoring normoxic conditions, replacing the culture medium with one containing icariin and continuing to culture for 24 hours to obtain pretreated mesenchymal stem cells.

6. The pre-treatment method according to claim 5, characterized in that, In step (2), the hypoxic environment is a 2% O2 environment.

7. The pre-treatment method according to claim 6, characterized in that, In step (3), the hypoxic conditions are 2% O2 conditions, and in the culture medium containing Ferrostatin-1, the concentration of Ferrostatin-1 is 0.5 μmol / L.

8. The pre-treatment method according to claim 7, characterized in that, In step (4), in the culture medium containing icariin, the concentration of icariin is 5 μmol / L.

9. Use of pretreated mesenchymal stem cells obtained by the pretreatment method of any one of claims 5-8 in the preparation of a medicament for treating cartilage injury.

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