Method for rapidly evaluating repairing function of cartilage cells on cartilage injury at low cost

Abstract

The invention relates to the technical field of cell preparations, in particular to a method for rapidly evaluating the repairing function of cartilage cells on cartilage injury at low cost, which comprises the following steps: constructing a cartilage injury model by adopting pig knee and femoral joint cartilage tissues, inoculating cell spheres formed by the to-be-evaluated cartilage cells to an injury part of the cartilage injury model, and evaluating the cartilage injury part of the cartilage injury model. And transplanting the cartilage cells into an experimental animal body, and evaluating the repair function of the cartilage cells according to the repair condition of the cartilage injury model. The method provides a systematic scheme for evaluating the repairing function of the cartilage cells, has the advantages of simplicity in operation, short time consumption, low cost, high stability and the like, and provides an effective method for evaluating the functions of the cartilage cells and screening and evaluating the functions of cells such as induced pluripotent stem cells with cartilage injury repairing potential; the method has a good application prospect in development of cell preparations for cartilage injury repair.

Description

Technical Field

[0001] The present invention relates to the technical field of cell preparations, and in particular to a method for rapidly evaluating the repairing function of chondrocytes on cartilage damage at low cost. Background Art

[0002] Cartilage tissue is an important connective tissue in the human body with important physiological functions. For example, articular cartilage can reduce friction during joint movement, buffer mechanical pressure, and make joint movement more flexible and stable. Articular cartilage damage is one of the main causes of joint pain and dysfunction. Because the self-repair ability of cartilage tissue is extremely limited, cartilage tissue engineering and cell therapy (such as chondrocyte transplantation) have become important research directions for repairing cartilage damage. In these studies, accurate and efficient evaluation of the repair function of implanted chondrocytes is crucial, which is directly related to the evaluation of repair effects, standardization of cell products, and optimization of treatment plans. Existing evaluation methods generally have problems such as long time consumption, complex operation, high cost, and poor reproducibility. Therefore, it is necessary to develop a set of systematic methods to evaluate the repair function of chondrocytes on cartilage damage. Summary of the Invention

[0003] The present invention provides a method for evaluating the repair function of chondrocytes on cartilage damage.

[0004] Specifically, the present invention provides the following technical solutions.

[0005] The present invention provides a method for evaluating the repair function of chondrocytes on cartilage damage, the method comprising: constructing a cartilage damage model using pig knee and femoral joint cartilage tissue, inoculating cell spheres formed by chondrocytes to be evaluated into the damaged site of the cartilage damage model, and then transplanting the cell spheres into experimental animals, and evaluating the repair function of the chondrocytes based on the repair status of the cartilage damage model.

[0006] In the process of constructing a cartilage injury model suitable for evaluating the repair function of chondrocytes, the present invention screened the animal and part sources of the cartilage tissue used to construct the injury model. It was verified that, compared with other animals, cartilage tissue from pigs, especially knee-femoral joint cartilage tissue, was more stable during the model construction process, with smaller group and batch differences, more accurate repair function evaluation results, and better repeatability; and the thickness of the cartilage layer of the pig knee-femoral joint is 2 mm, which is basically the same as the human cartilage layer. The model constructed can be a standard full-thickness cartilage injury, which is more conducive to reflecting the repair function of chondrocytes.

[0007] Preferably, the cartilage tissue is osteochondral tissue of the distal femur of a pig's knee-femoral joint, wherein the osteochondral tissue specifically retains a height of 2 mm in the subchondral bone.

[0008] Preferably, the cartilage tissue is circular or approximately circular, with a diameter of 5-8 mm, more preferably 6-7 mm. The cartilage tissue is obtained from animal samples.

[0009] In the above method, the cartilage injury model is constructed by punching holes in the cartilage tissue, and the diameter of the holes is 2-4 mm.

[0010] Preferably, a hole is punched in the center of the cartilage tissue to obtain a hollow cartilage tissue, which is a cartilage injury model.

[0011] In the above method, the inoculation comprises: placing the cell spheres in holes punched in cartilage tissue and fixing them with biological glue.

[0012] The bioglue fixes the cell spheroids within the pores of the cartilage tissue through adhesion. The bioglue can be natural bioglue (such as gelatin, chitosan, alginate, fibrin, etc.), synthetic bioglue (such as polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), etc.), genetically engineered bioglue (such as genetically engineered polypeptide-based bioglue), or other bioglue.

[0013] In some embodiments of the invention, the bioglue comprises fibrinogen.

[0014] In some embodiments of the present invention, the preparation method of the fibrin adhesive comprises: using a freeze-dried fibrinogen dissolving solution to dissolve freeze-dried fibrinogen, using a freeze-dried thrombin dissolving solution to dissolve thrombin, and using a double injection stand to mix equal amounts of fibrinogen and thrombin.

[0015] In some embodiments of the present invention, chondrocyte spheres are placed in the hollow of the hollow cartilage tissue, and then biological glue is added dropwise, and transplanted after solidification.

[0016] In the above method, the experimental animal is a mouse, preferably an immunodeficient mouse.

[0017] In the above method, the method for evaluating the repair function of chondrocytes comprises one or more methods selected from the group consisting of visual observation, tissue staining, immunohistochemistry, ICRS cartilage repair scoring, O'Driscoll scoring system, or modified O'Driscoll scoring system.

[0018] Preferably, the tissue staining includes one or more selected from HE, Safranin O, and EVG staining.

[0019] Wherein, the tissue staining further comprises the step of semi-quantitatively analyzing the specifically stained tissue area.

[0020] Preferably, the proteins targeted by the immunohistochemistry include one or more proteins selected from Collagen I, Collagen II, and S-100.

[0021] Wherein, the immunohistochemistry further comprises a step of semi-quantitatively analyzing the positive area.

[0022] Preferably, the evaluation of the repair function of chondrocytes is performed at least 2 days after transplantation.

[0023] In the above method, the cell sphere can be a chondrocyte sphere obtained by culturing chondrocytes from any source.

[0024] In some embodiments of the present invention, the chondrocytes are chondrocytes differentiated from induced pluripotent stem cells (iPSCs).

[0025] The method for producing chondrocyte spheres from induced pluripotent stem cells can employ culture and differentiation methods commonly used in the field of pluripotent stem cells. For example, induced pluripotent stem cells can be induced to form mesenchymal stem cells, which are then induced to differentiate into chondrocyte spheres. The culture medium and induction factors used in the induction and differentiation process can be conventional culture medium and induction factors in the art, or they can be optimized based on the characteristics and properties of a specific induced pluripotent stem cell.

[0026] Preferably, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0027] In some embodiments of the present invention, the chondrocyte spheres are obtained by culturing and differentiating human induced pluripotent stem cells (hiPSCs). The evaluation method of the present invention has no particular limitation on iPSCs and the method and process for preparing chondrocyte spheres by inducing differentiation from iPSCs.

[0028] In the present invention, the repair function includes but is not limited to the degree of integration of chondrocytes and cartilage tissue, hyaline cartilage formation, cartilage tissue structure repair, promotion of cartilage tissue regeneration, degenerative changes in cartilage tissue cells, subchondral bone reconstruction, and the combination of cartilage repair and subchondral bone reconstruction.

[0029] The above evaluation method can be used to evaluate the cartilage repair function of chondrocytes. If the chondrocytes are differentiated from stem cells such as induced pluripotent stem cells, it can also be used to evaluate the function and potential of the stem cells for repairing cartilage damage.

[0030] Based on this, the present invention also provides the use of the above-mentioned method for evaluating the repair function of chondrocytes on cartilage damage in screening cells or cell lines with the function of repairing cartilage damage.

[0031] The beneficial effects of the present invention include at least: the present invention provides a method for evaluating the repair function of chondrocytes on cartilage damage, which provides a systematic solution for evaluating the repair function of chondrocytes. By transplanting the chondrocyte spheres to be evaluated and the pig knee and femoral joint cartilage tissue into the body of an experimental animal, the cartilage damage repair function such as the degree of integration of the chondrocyte spheres and the cartilage tissue is evaluated. This method can be used to screen cell lines with the function of repairing cartilage tissue, has the advantages of simple operation, short time consumption, low cost, and high stability, and provides an effective method for the functional evaluation of chondrocytes and the screening and functional evaluation of cells such as induced pluripotent stem cells with cartilage damage repair potential, and has good application prospects in the development of cell preparations for cartilage damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Schematic diagram of cartilage tissue sampling and drilling specifications in the experimental examples of the present invention.

[0034] Figure 2 The results of the section staining (HE, Safranin O Fast Green, slow stripping and rapid sectioning results) of the transplanted grafts of the control group 20 days after transplantation were evaluated using the method of Example 1 in the experimental example of the present invention.

[0035] Figure 3 The results of the section staining (HE, Safranin O Fast Green, slow stripping and rapid sectioning results) of the grafts of the experimental group (i.e., the filling group) 20 days after transplantation in the evaluation method of Example 1 in the experimental example of the present invention are shown. The scale bar is 1000 μm.

[0036] Figure 4 1 is a comparison between the rabbit cartilage sampling area (left) and the pig cartilage sampling area (right) in the experimental example of the present invention.

[0037] Figure 5 These are the results of section staining of the graft 20 days after transplantation, evaluated using the method of Comparative Example 1 in the experimental example of the present invention. The upper image is HE staining, and the lower image is Safranin O Fast Green staining, both of which are the results of slow and rapid sectioning. The scale bar is 1000 μm.

[0038] Figure 6 Comparison between the sheep cartilage sampling area (left) and the pig cartilage sampling area (right) in the experimental example of the present invention. DETAILED DESCRIPTION

[0039] The full names and definitions of the abbreviations involved in the present invention are shown in Table 1.

[0040] Table 1

[0041] The experimental materials used in the specific embodiments of the present invention are as follows: 1. Test substance information Name: Human iPSC-derived chondrocyte spheres Appearance: translucent ball; Carrier: distal femoral cartilage tissue from porcine knee joint; Storage conditions: 2~8℃.

[0042] 2. Experimental Animals (1) Animal information Strain: B-NDG mice; Level: SPF level; Gender: female; Age: 6 weeks old.

[0043] (2) Breeding environment Rearing environment: temperature 20~26℃, humidity 40%~70%, light intensity 12 hours alternating between light and dark; Cage dimensions: IVC, 398 × 188 × 202 mm; Stocking density: ≤5 per cage; Feed name: experimental mouse maintenance feed; Drinking water: high temperature and high pressure sterilized drinking water.

[0044] 3. Main instruments, reagents and consumables The information of main instruments, reagents and consumables is shown in Table 2.

[0045] Table 2

[0046] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0047] Example 1 This embodiment provides a method for evaluating the cartilage damage repair function of chondrocytes, which comprises the following steps: 1. Graft preparation (1) Preparation of pig distal femoral cartilage tissue carrier One day in advance, fresh porcine knee-femoral joints were obtained. Uniformly sized osteochondral tissue was removed from the distal femur using an electric drill (leaving 2 mm of subchondral bone in height and 6 mm in diameter). A uniform hole was punched in the center using a 2 mm or 4 mm punch to obtain hollow cartilage tissue, which served as the porcine distal femoral cartilage tissue carrier (i.e., cartilage injury model). The hollow cartilage tissue was immersed in gentamicin injection and stored at 2–8°C until use.

[0048] (2) Graft preparation The hollow cartilage tissue obtained in step (1) is placed on a culture dish, the chondrocyte spheres to be evaluated are placed in the hollow part, fibrin adhesive is added dropwise, and after it solidifies, it is used for subcutaneous transplantation in mice.

[0049] 2. Transplantation After the animals were received, the experiment began after 3 to 7 days of adaptive feeding. The transplantation process was as follows: (1) Place the mouse in a gas anesthesia induction box and anesthetize with isoflurane; (2) After the mouse loses consciousness, remove it and shave the left / right ventral side; (3) Fix the animal on the operating board, connect the mask, and use isoflurane to maintain anesthesia; (4) Apply iodine to the left and right sides for disinfection; (5) Make a small incision on the left / right ventral side, use hemostatic forceps to bluntly separate the skin and muscle tissue, and insert the graft from step 1 above from the incision to the left / right rib side (1-2 cm away from the incision); (6) Align the open skin and seal the wound with animal tissue glue; (7) Place the animal on a heating pad to rewarm it, and return it to the cage after it regains consciousness.

[0050] 3. Sample collection Samples were collected 20 days after transplantation, mice were euthanized using CO2, and the grafts were removed, immersed in 10% formalin neutral fixative, and stored at room temperature until examination.

[0051] 4. Observation indicators (1) General observation The basic conditions of the animals were observed three times a week after transplantation, including appearance and hair, behavioral activities, mental state and diet.

[0052] (2) Weight The animals were weighed before and after transplantation.

[0053] (3) Histopathology The grafts were stained with HE, Safranin O, and EVG, and the area of ​​specifically stained tissue was semi-quantified.

[0054] (4) Immunohistochemistry The grafts were subjected to immunohistochemistry for Collagen Ⅰ, Collagen Ⅱ, and S-100, and the positive areas were semi-quantified.

[0055] 5. Data processing The experimental data were expressed as mean ± standard deviation (mean ± SD) and statistically analyzed using Graphpad Prism 9 software. The homogeneity of variance of the data in each group was first tested, and then the corresponding test method in One-way ANOVA was used to check whether the differences between the groups were statistically significant. If the homogeneity of variance was not met, the Brown-Forsythe and Welch ANOVA Test was used; if the homogeneity of variance was met, the Ordinary ANOVA Test was used. The test results of each group were compared with the model control group. P <0.05 indicated that the difference compared with the control group was statistically significant.

[0056] The International Cartilage Repair Society (ICRS) score or the modified O'Driscoll score was used to evaluate the cartilage repair function.

[0057] Comparative Example 1 This comparative example provides a method for evaluating the cartilage damage repair function of chondrocytes, which differs from the method in Example 1 only in that the distal femoral cartilage tissue of the pig knee-femoral joint is replaced by the distal femoral cartilage tissue of the rabbit knee-femoral joint.

[0058] Comparative Example 2 This comparative example provides a method for evaluating the cartilage damage repair function of chondrocytes, which differs from the method in Example 1 only in that the distal femoral cartilage tissue of the pig knee-femoral joint is replaced with the distal femoral cartilage tissue of the sheep knee-femoral joint.

[0059] Experimental example The cartilage damage repair function of human iPSC-derived chondrocyte spheres was evaluated using the method provided in Example 1. The experimental grouping is shown in Table 3. Figure 1 shown.

[0060] Table 3 Experimental group information Note: NA stands for not vaccinated.

[0061] After 20 days of transplantation, the grafts on both sides were removed, tissue sections were prepared and stained with HE and Safranin O Fast Green. The results of the control group and the experimental group were shown in Figure 2. Figure 2 and Figure 3 The results showed that compared with the model group, the experimental group could completely fill the damaged area after filling with chondrocyte spheres, and spontaneous fusion between chondrocyte spheres and with the surrounding cartilage tissue could be observed, proving that this method can quickly reflect the repair effect of human iPSC-derived chondrocyte spheres on cartilage damage.

[0062] Furthermore, the cartilage damage repair status of the model control group and the experimental group in Table 3 was scored using the modified O'Driscoll scoring method, and the results are shown in Table 4. The results show that the evaluation method of Example 1 of the present invention can be used to evaluate the cartilage damage repair function of chondrocyte spheres.

[0063] Table 4 Cartilage damage repair score table Note: The model injury group is the model control group, the chondrocyte filling group is the experimental group, L and R represent the left and right subcutaneous grafts, respectively, and 20 represents 20 days after transplantation.

[0064] Using the method provided in Comparative Example 1, we evaluated the cartilage repair capacity of human iPSC-derived chondrocyte spheroids. The evaluation revealed that rabbit-derived cartilage tissue exhibited significantly greater self-healing capacity than the porcine cartilage tissue used in Example 1, potentially impacting the stability of the cartilage injury model. The rabbit-derived cartilage tissue model in Comparative Example 1 exhibited significantly greater intragroup variability and poorer model stability.

[0065] Among them, the comparison between rabbit cartilage sampling area and pig cartilage sampling area is as follows Figure 4 After 20 days of transplantation, the grafts containing cartilage carrier + fibrin + human iPSC-derived chondrocyte spheres from the distal femur of rabbits were removed, tissue sections were prepared, and HE and Safranin O fast green staining were performed. The results are shown in Figure 2. Figure 5 The results showed that subchondral bone damage occurred in the rabbit cartilage sampling area (the rabbit cartilage layer is only 0.5 mm thick), but it is difficult to create full-thickness cartilage damage, which will have an unpredictable impact on the stability of the model.

[0066] The method provided in Comparative Example 2 was used to evaluate the cartilage damage repair function of human iPSC-derived chondrocyte spheres. During the evaluation process, it was found that compared with pig knee and femoral joint cartilage, the self-healing ability of sheep knee and femoral joint cartilage was significantly stronger, and the individual differences were large, which had a greater impact on the stability of the evaluation results. In comparison, the self-healing ability of pig knee and femoral joint cartilage was worse, and the inter-group and batch differences in cartilage damage modeling were small, and the evaluation results were more stable. Among them, the sheep cartilage sampling area was compared with the pig cartilage sampling area. Figure 6 As shown in the figure: the area of ​​sheep knee joint is smaller than that of pig joint, the actual use cost is high, and there are large individual differences in sheep knee joints, which has a great impact on experimental data analysis. Using pig knee joints can not only reduce costs, but also reduce the impact of individual differences on the stability of evaluation results.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for evaluating the repair function of chondrocytes on cartilage damage, characterized in that: The method comprises: constructing a cartilage injury model using pig knee-femoral joint cartilage tissue, inoculating cell spheres formed by chondrocytes to be evaluated into the damaged site of the cartilage injury model, and then transplanting them into experimental animals, and evaluating the repair function of the chondrocytes based on the repair status of the cartilage injury model.

2. The method according to claim 1, characterized in that The cartilage tissue is the osteochondral tissue of the distal femur of the pig knee-femoral joint.

3. The method according to claim 1 or 2, characterized in that The cartilage tissue is circular or approximately circular, and has a diameter of 5-8 mm.

4. The method according to any one of claims 1 to 3, characterized in that The cartilage injury model is constructed by punching holes in the cartilage tissue, and the diameter of the holes is 2-4 mm.

5. The method according to claim 4, characterized in that The punching of the cartilage tissue is to punch a hole in the center of the cartilage tissue to obtain a hollow cartilage tissue.

6. The method according to any one of claims 1 to 5, characterized in that The inoculation comprises: placing the cell spheres in holes punched in cartilage tissue and fixing them with biological glue.

7. The method according to any one of claims 1 to 6, characterized in that The method for evaluating the repair function of chondrocytes comprises one or more methods selected from the group consisting of macroscopic observation, tissue staining, immunohistochemistry, ICRS cartilage repair scoring method, O'Driscoll scoring method, and modified O'Driscoll scoring method.

8. The method according to claim 7, characterized in that: The tissue staining includes one or more selected from HE, Safranin O, and EVG staining; And / or, the proteins targeted by the immunohistochemistry include one or more proteins selected from Collagen I, Collagen II, and S-100.

9. The method according to claim 7 or 8, characterized in that: The repair function of chondrocytes was evaluated at least 2 days after transplantation.

10. Use of the method according to any one of claims 1 to 9 in screening cells or cell lines capable of repairing cartilage damage.

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