CN108865986B

CN108865986B - Mesenchymal stem cell preparation for repairing articular cartilage damage/defect and preparation method and application thereof

Info

Publication number: CN108865986B
Application number: CN201810692499.5A
Authority: CN
Inventors: 张仲文; 刘爱兵; 刘莹
Original assignee: Lin Xiujin
Current assignee: Lin Xiujin
Priority date: 2018-06-29
Filing date: 2018-06-29
Publication date: 2021-11-30
Anticipated expiration: 2038-06-29
Also published as: CN108865986A

Abstract

The invention discloses a mesenchymal stem cell preparation for repairing articular cartilage damage/defect and a preparation method and application thereof. Carrying out indirect co-culture on the mesenchymal stem cells and synoviocytes, and harvesting and freezing the co-cultured mesenchymal stem cells; the cord mesenchymal stem cells obtained after inoculating and co-culturing the synovial cells and the cord mesenchymal stem cells according to the density ratio of 4:1 are more easily differentiated towards the cartilage direction under the same induction condition. According to the invention, the constructed animal knee joint cartilage injury/defect model proves that the umbilical cord mesenchymal stem cells after being co-cultured with synovial cells have obvious effect of promoting articular cartilage injury/defect repair. The invention further provides a preparation prepared by applying the co-cultured mesenchymal stem cells, which can be applied to clinical repair of articular cartilage damage/defect, the preparation is injected into articular cavity to effectively promote repair of articular cartilage damage/defect, and the treatment effect is obviously better than that of a pure mesenchymal stem cell preparation.

Description

Mesenchymal stem cell preparation for repairing articular cartilage damage/defect and preparation method and application thereof

Technical Field

The invention relates to a preparation for repairing articular cartilage damage/defect, in particular to a mesenchymal stem cell preparation for repairing articular cartilage damage/defect and a preparation method thereof.

Background

Articular cartilage is a layer of hyaline cartilage covering the surface of the joint and is composed of chondrocytes and a matrix. The articular cartilage has smooth surface, can reduce friction, protects the joint from being easily worn while bearing the mechanical load of movement, and plays an important role in the movement of the joint. The damage/defect of the articular cartilage is mainly caused by a large amount of exercise for a long time, high load exercise, osteoarthritis of middle-aged and old people and the like. Articular cartilage lacks blood vessels, nerves and lymphatic tissues, so that the self-repair ability is limited, and once damaged, the joint is susceptible to other progressive diseases besides affecting normal joint activities.

Currently, the commonly used articular cartilage damage/defect repair methods mainly include: surgical treatments, such as arthroscopic debridement, micro-fracture, etc.; graft repair, such as osteochondral transplantation; the tissue engineering cartilage comprises cartilage cells transplantation, mesenchymal stem cell transplantation, MACI, transplantation of gel cartilage repair materials loaded with seed cells and the like.

The application of cell transplantation in treating cartilage damage/defect has good application prospect. The major structures within the joint include articular cartilage, meniscus, synovium, and infrapatellar fat pad. The damage repair by directly applying the chondrocytes can obtain better effect, but the chondrocytes are required to be taken from healthy cartilage tissues of donors, so that the trauma to patients is large, and the proliferation capacity of the chondrocytes in vitro is limited and the chondrocytes are easy to dedifferentiate, so that the repair of large-area cartilage damage/defect is difficult. The meniscus is composed primarily of fibrocartilage. Synovial cells are mainly divided into type A and type B, wherein the type B cell is the source of the synovial mesenchymal stem cell, and research shows that the synovial mesenchymal stem cell and the chondrocyte have similar expression profiles and are derived from the same precursor cell, and the synovial mesenchymal stem cell has the potential of differentiating into the chondrocyte in a synovial microenvironment with cartilage damage/defect. In addition, synovial fluid produced by synovial cells is an important component of the environment within the joint cavity, and synovial mesenchymal stem cells are capable of secreting various substances into the synovial fluid. It has been shown that synovial mesenchymal stem cells are capable of forming a cartilage matrix enriched in type II collagen and glycosaminoglycans sulfate. Therefore, synovial cells play an important role in the formation of the environment within the joint cavity.

In recent years, Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) have become seed Cells for various disease model applications due to their simple tissue acquisition, easy cell isolation and culture, and ability to differentiate into various tissue Cells and tissue repair function. Research shows that the application of the MSCs to articular cartilage damage/defect repair has certain curative effect. The direct injection through the joint cavity is a simple and easy operation method, but the MSCs directly injected may cause the number of transplanted cells to be continuously reduced and the cell differentiation function state to be weakened due to reasons such as inadaptation to the microenvironment of the joint cavity, and the cell concentration and quality directly influence the repair effect, so that a large amount of transplanted cells need to be injected for many times to improve the curative effect.

Therefore, there is a high need in clinical practice to reduce the loss rate of MSCs cells and to effectively activate their function of injury repair when MSCs are used for articular cartilage injury/defect repair.

Disclosure of Invention

One of the purposes of the invention is to provide a mesenchymal stem cell preparation which has obvious repairing effect on articular cartilage damage/defect;

the second purpose of the invention is to provide a preparation method of the mesenchymal stem cell preparation applied to clinic;

the invention also aims to apply the mesenchymal stem cell preparation to the repair of articular cartilage damage/defect.

The above object of the present invention is achieved by the following technical solutions:

a process for preparing the mesenchymal stem cells used to repair the damage or defect of articular cartilage includes such steps as indirect coculture between the mesenchymal stem cells and synoviocytes, collecting the cocultured mesenchymal stem cells, and freezing.

Of these, preferred, areThe indirect CO-culture is to inoculate synovial cells to the upper chamber of a Transwell culture plate, inoculate umbilical cord mesenchymal stem cells to the lower chamber of the Transwell culture plate, and place the Transwell culture plate in CO₂Co-culturing in an incubator; wherein the conditions of the culturing comprise: the culture temperature is 37 ℃ and CO₂The concentration of (2) is 5%.

According to the invention, a large number of experiments show that the inoculation ratio of synovial cells and mesenchymal stem cells has great influence on the differentiation of the mesenchymal stem cells to the chondrocytes during co-culture; the invention adopts synovial cells and mesenchymal stem cells according to the following formula (10-1): (1-10) inoculating the inoculation ratios to the upper chamber or the lower chamber of a Transwell culture plate respectively for co-culture, detecting the content of amino glycan and the mRNA expression level of type II collagen after in vitro chondrogenic induction of each group of co-culture cells, and finding the detection result of the content of amino glycan that when the inoculation density ratio of synovial cells to umbilical cord mesenchymal stem cells is 4:1, the content of amino glycan after chondrogenic induction of the co-culture cells is highest and has very significant difference compared with other inoculation density ratios; as a result of detecting the mRNA expression amount of the type II collagen, when the inoculation density ratio of the synovial cells to the umbilical cord mesenchymal stem cells is 4:1, the mRNA expression amount of the type II collagen after chondrogenesis induction of the co-cultured cells is the highest and has a very significant difference compared with other inoculation density ratios.

Preferably, the synovial cells or mesenchymal stem cells are P2 generation cells; wherein, the synovial cells are preferably derived from human healthy synovial tissue, and the mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells.

Wherein, the preparation method of the synoviocytes for co-culture comprises the following steps: obtaining human healthy synovial tissue, separating by enzyme digestion to obtain primary synovial cells (P0), inoculating by passage, culturing to P2 passage, collecting synovial cells, and obtaining synovial cells for co-culture.

The preparation method of the umbilical cord mesenchymal stem cells for co-culture comprises the following steps: obtaining primary human umbilical cord mesenchymal stem cells (P0) by a tissue block adherent culture method, and culturing until P2 generation collecting cells to obtain umbilical cord mesenchymal stem cells for co-culture.

The invention further provides a preparation method of the co-culture mesenchymal stem cell preparation for repairing articular cartilage damage/defect, which comprises the following steps:

(1) recovering the cryopreserved mesenchymal stem cells after the co-culture in a water bath, washing with physiological saline, centrifuging, and discarding the supernatant; (2) resuspending cells by using normal saline as an auxiliary agent, adjusting the cell concentration, and sucking the cells into a syringe for later use.

Wherein, the frozen and co-cultured mesenchymal stem cells are revived by water bath at 37 ℃; the centrifugation in the step (1) is carried out for 5min at 2000 rpm; step (2) adjusting the cell concentration to (1-10) x 10⁶one/mL.

In order to test the repairing effect of the mesenchymal stem cell preparation on articular cartilage damage/defect, the invention constructs an animal knee joint cartilage damage/defect model and uses the mesenchymal stem cell preparation to carry out injection treatment, the general observation of a specimen shows that the repairing effect of the cartilage defect of a high-dose co-culture experimental group is obviously superior to that of a blank control group, a mesenchymal stem cell control group and a low-dose co-culture experimental group, the color and hardness of the repaired cartilage are basically the same as those of surrounding cartilage, and the surface is smooth; the experimental result shows that the umbilical cord mesenchymal stem cells after being co-cultured with the synovial cells have obvious effect of repairing the articular cartilage damage/defect.

The mesenchymal stem cell preparation after co-culture can effectively promote the repair of articular cartilage damage/defect through articular cavity injection, and the treatment effect of the mesenchymal stem cell preparation is obviously better than that of a pure mesenchymal stem cell preparation. The mesenchymal stem cell preparation provided by the invention can be applied to allogeneic or xenogeneic animal cell transplantation and repair of articular cartilage damage/defect.

Drawings

FIG. 1 shows the results of measuring the aminoglycan content of cocultured cells after in vitro chondrogenic differentiation;

FIG. 2 shows the result of detecting the expression level of type II collagen mRNA after in vitro chondrogenic differentiation of co-cultured cells;

FIG. 3 is a comparison of the gross observations before and after the experiment in the blank control group;

fig. 4 comparison graphs of general observations before and after experiments of mesenchymal stem cell control group;

FIG. 5 is a comparison of the general observations before and after the experiment in the low-dose co-culture experimental group;

FIG. 6 is a comparison graph of the general observation before and after the experiment in the high-dose co-culture experimental group;

FIG. 7 Wakitani score results for cartilage surface damage repair.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Preparative example 1 isolation and culture of synoviocytes for Co-culture

1) Screening patients without immune diseases such as rheumatism, rheumatoid disease, osteoarthritis and the like, collecting healthy synovial tissue at the knee joint of a patient with damaged meniscus or cruciate ligament or a synovial donor through arthroscopic surgery, and storing the tissue in PBS buffer containing 1% double antibody.

2) Synovium tissue was aseptically placed in a petri dish and minced, washed 3 times with PBS buffer, and collected into centrifuge tubes.

3) Adding 4% collagenase type I with the volume 5 times that of the tissue, placing the mixture in a constant temperature shaking table, and digesting the mixture for 2 to 3 hours at 37 ℃ and 150 rpm.

4) After the tissue mass was completely digested, the cell suspension was filtered through a 70 μm cell strainer, and the filtrate was collected and centrifuged at 1500rpm for 5 min.

5) The supernatant was discarded, and the cells at the bottom of the centrifuge tube were resuspended in DMEM/F12 medium, centrifuged at 1500rpm for 5min, and washed twice.

6) After the last wash, the cells were resuspended in complete medium to obtain primary synoviocytes (passage P0).

7) Adjusting the cell density to 1.5-2.5 × 10⁵seed/mL, at 25cm²CulturingInoculating 5mL of the suspension into each bottle, and placing at 37 deg.C and 5% CO₂The culture was carried out for P1 generation in the incubator.

8) And after about 48 hours, replacing the liquid to remove the non-adherent cells, and then replacing the liquid every 2 to 3 days until the cell fusion rate reaches more than 80 percent for passage.

9) Subculturing: discarding the original culture medium, adding 2mL of 0.25% trypsin into each bottle, digesting for 3min, adding an equal volume of complete culture medium to stop digestion after the cells become round and float, blowing the cells into single cells, transferring into a centrifuge tube, and centrifuging at 1500rpm for 5 min. The supernatant was discarded, the cell pellet was collected, and the cells were washed 1 time with DMEM/F12 medium and centrifuged. Then, the collected cells were resuspended in the complete medium at 5000 cells/cm²Inoculating to 75cm²The culture flask was cultured for P2 generation.

10) Collecting P2 synovial cells, and freezing for use.

Preparative example 2 isolation and culture of umbilical cord mesenchymal stem cells for Co-culture

1) 1 root of umbilical cord of the term fetus was collected aseptically, and the tissue was stored in PBS buffer containing 1% double antibody.

2) Taking out umbilical cord tissue into a culture dish, shearing off two ends of the umbilical cord tissue under aseptic condition, discarding the umbilical cord tissue 0.5cm respectively, shearing the rest tissues to the length of 1.5cm-2cm, and extruding the umbilical cord section to clean residual blood in the blood vessel.

3) Removing umbilical vein in aseptic dissecting tray, peeling off Wharton jelly, transferring collected Wharton jelly into centrifuge tube, and cutting to 2-5mm³Size.

4) The tissue pieces were collected, washed once with DMEM/F12 medium, and then the tissue pieces were washed at 3-5 pieces/cm²Uniformly distributing in culture flask, adding complete culture medium to cover all tissue blocks, placing at 37 deg.C and 5% CO₂Primary culture was performed in an incubator (passage P0).

5) Culturing for 12-15 days, when more cells climb out of the tissue block, flushing the tissue block, adding a new complete culture medium to continue culturing, and carrying out passage when the cell fusion rate reaches more than 80%.

6) Subculturing: the procedure was the same as synovial cell subculture.

7) Transferring the cells to P2 generation, collecting umbilical cord mesenchymal stem cells, and freezing for later use.

The umbilical cord tissue is easy to obtain, the cell separation culture technology is mature, and a large number of cells can be obtained by one-time culture, so that the umbilical cord tissue has obvious advantages as seed cells.

Example 1 Co-culture of synoviocytes and umbilical cord mesenchymal Stem cells

1) The frozen and preserved P2 generation synovial cells and P2 generation umbilical cord mesenchymal stem cells are respectively revived in water bath at 37 ℃, washed 1 time by using culture medium respectively, centrifuged at 2000rpm for 5min, and then the cells are respectively resuspended by using the complete culture medium.

2) The Transwell plate (pore size 0.4um) was seeded at a density of 5000 cells/cm 2, and the synovial cells were seeded in the upper chamber (area about 4.5 cm)²) Umbilical cord mesenchymal stem cells were seeded into the lower chamber (area about 9.5 cm)²) The resulting mixture was divided into 11 test groups at different inoculation density ratios shown in Table 1, and the test groups were placed at 37 ℃ and 5% CO₂Co-culturing in an incubator.

TABLE 1 different seeding Density ratios of synoviocytes to umbilical cord mesenchymal Stem cells

Group of	Synovial cell, inoculation density ratio of umbilical cord mesenchymal stem cell
		1	10:1
2	8:1
		3	6:1
4	4:1
		5	2:1
6	1:1
		7	1:2
8	1:4
		9	1:6
10	1:8
		11	1:10

3) And (5) co-culturing for 5-7 days, and respectively harvesting the lower-chamber umbilical cord mesenchymal stem cells when the fusion rate of the lower-chamber umbilical cord mesenchymal stem cells reaches more than 80%.

4) And (3) freezing and storing the harvested umbilical cord mesenchymal stem cells after 11 groups of cocultures for later use.

EXAMPLE 2 preparation of the formulations

The 11 groups of cocultured umbilical cord mesenchymal stem cells frozen in example 1 were treated as follows: resuscitating in 37 deg.C water bath, washing with physiological saline for 1 time, centrifuging at 2000rpm for 5min, and discarding the supernatant.

1) Resuspending cells with normal saline as adjuvant, and adjusting cell concentration to 1 × 10⁶and/mL, 1mL each, is sucked into the syringe for use.

2) Resuspending cells with normal saline as adjuvant, and adjusting cell concentration to 10×10⁶and/mL, 1mL each, is sucked into the syringe for use.

Test example 1 coculture cell in vitro chondrogenic induced differentiation test

The mesenchymal stem cells between 11 groups co-cultured in example 1 were inoculated in six-well plates, respectively, and the cell density was adjusted to 2.5X 10⁴one/mL, 2mL per well. And (3) when the cells adhere to the wall and are fused by more than 60%, removing the original culture medium, replacing the original culture medium with a cartilage induction culture medium (a DMEM/F12 culture medium containing TGF-beta 1, dexamethasone, ascorbic acid, sodium pyruvate, insulin, transferrin, selenious acid, bovine serum albumin and linoleic acid), changing the culture medium for 1 time every 2-3 days, and carrying out induction culture for 21 days.

Glycosaminoglycan (GAG) content detection

DMMB (1, 9 dimethyl methylene blue) method: the cells of each group were collected, and the cell pellet was digested with papain (pH6.8) at 60 ℃ for 16 hours. Centrifuging, collecting supernatant, adding DMMB dye solution, mixing, performing color comparison at 525nm, and measuring absorbance value. Chondroitin sulfate is used as a standard curve (0-100 ug/mL), and GAG content in each group of cells is calculated according to the standard curve.

Detection of type II collagen mRNA expression level

Real-time quantitative PCR: and (3) respectively carrying out total RNA extraction and reverse transcription on each group of cells according to the instruction of a related kit to obtain cDNA, carrying out qRT-PCR reaction, and measuring the mRNA expression level of type II collagen in each group of cells.

After each group of co-cultured cells are induced into cartilage, the detection result of the content of the amino glycan is analyzed: when the inoculation density ratio of the synovial cells to the umbilical cord mesenchymal stem cells is 4:1, the content of the amino glycan of the co-cultured cells after chondrogenesis induction is the highest, and the inoculation density ratio is very different from other inoculation density ratios.

After the group of co-cultured cells are induced into cartilage, the detection result of the mRNA expression quantity of type II collagen is analyzed: when the inoculation density ratio of the synovial cells to the umbilical cord mesenchymal stem cells is 4:1, the expression amount of type II collagen mRNA after chondrogenesis induction of the co-cultured cells is the highest, and the co-cultured cells have very significant difference compared with other inoculation density ratios.

The above results indicate that when the seeding density ratio of synovial cells to umbilical cord mesenchymal stem cells is 4:1, umbilical cord mesenchymal stem cells after co-culture are more easily differentiated into cartilage under the same induction conditions.

Experimental example 2 animal knee joint cartilage injury/defect model and application test for repair

Establishing an animal model: 16 adult white rabbits were selected to make a knee joint cartilage injury/defect model. Anesthetizing animals by injecting medical chloral hydrate (2mL/kg body weight) through ear edge vein, selecting the inner side incision of knee joint of both legs, exposing the knee joint, manufacturing 1 defect with the diameter of about 3mm and the depth of about 1-1.5mm on the middle lower part of the femoral medial condyle by using a grinding drill, and suturing the wound.

16 molded white rabbits were randomly divided into 4 groups:

1) blank control group, saline was injected.

2) Mesenchymal stem cell control group, injected with 10 × 10⁶Common umbilical cord mesenchymal stem cells.

3) Low dose Co-culture group, co-cultured umbilical cord mesenchymal stem cells (concentration 1X 10) prepared in step (1) of example 2 were injected⁶/mL)。

4) High dose Co-culture experiment group, co-cultured umbilical cord mesenchymal stem cells (concentration 10X 10) prepared in step (2) of example 2 were injected⁶/mL)。

The animals were injected 1 week after molding. The blank control group is injected with 1mL of physiological saline in the double knee joint cavities respectively; injecting 1mL of common umbilical cord mesenchymal stem cells into the double knee joint cavity by the mesenchymal stem cell control group; the experimental group respectively injects 1mL of co-cultured umbilical cord mesenchymal stem cells with each dose into the double knee joint cavities; the treatment is completed by injecting 1 time every two weeks and 5 times continuously. The experimental animals were sacrificed two weeks after the last 1 injection treatment, gross and histopathological observations were performed on the articular cartilage surface, and the articular cartilage surface damage repair degree was scored according to the Wakitani scoring standard. The results are shown in Table 2.

TABLE 2 articular cartilage surface damage repair degree score results

After cell transplantation, the gross observation of the specimen shows that the cartilage defect repair effect of the high-dose co-culture experimental group is obviously superior to that of a blank control group, a mesenchymal stem cell control group and a low-dose co-culture experimental group, the color and hardness of the repaired cartilage are basically the same as those of surrounding cartilage, and the surface is smooth. The Wakitani score shows that the cartilage repair degree of each cell treatment group is obviously superior to that of a blank control group, and compared with the Wakitani integral of the blank control group, the experimental group and the mesenchymal stem cell control group have significant difference; in addition, under the condition of the same dosage, the cartilage repair degree of the high-dosage co-culture experimental group is obviously superior to that of the mesenchymal stem cell control group and is also superior to that of the low-dosage co-culture experimental group, and the experimental result shows that the umbilical cord mesenchymal stem cells after co-culture with the synovial cells have obvious effect of promoting the articular cartilage damage/defect repair (fig. 3-7).

Claims

1. The application of the mesenchymal stem cell preparation in preparing a medicine for repairing articular cartilage damage or defect caused by movement, wherein the preparation method of the mesenchymal stem cell preparation for repairing articular cartilage damage/defect comprises the following steps: performing indirect co-culture on the mesenchymal stem cells and synoviocytes, harvesting the co-cultured mesenchymal stem cells, and freezing to obtain the mesenchymal stem cells;

the indirect CO-culture is to inoculate synovial cells to an upper chamber of a Transwell culture plate, inoculate umbilical cord mesenchymal stem cells to a lower chamber of the Transwell culture plate, and place the Transwell culture plate after inoculation of the cells in CO₂Co-culturing in an incubator; the synovial cells are derived from human healthy synovial tissues;

inoculating the synovial cells and the umbilical cord mesenchymal stem cells respectively according to an inoculation ratio of 4: 1;

the synovial cells are P2 generation synovial cells; the mesenchymal stem cell is a P2 generation umbilical cord mesenchymal stem cell; the preparation method of the P2 generation synovial cell comprises the following steps: obtaining healthy synovial tissue of a human, separating by using an enzyme digestion method to obtain P0 generation synovial cells, inoculating by passage, and culturing to P2 generation collecting cells to obtain synovial cells for co-culture; the preparation method of the P2 generation umbilical cord mesenchymal stem cells for co-culture comprises the following steps: obtaining human umbilical cord mesenchymal stem cells of P0 generation by a tissue block adherent culture method, and culturing until collecting cells of P2 generation, so as to obtain umbilical cord mesenchymal stem cells for co-culture;

recovering the cryopreserved mesenchymal stem cells after the co-culture in a water bath, washing with physiological saline, centrifuging, and discarding the supernatant; resuspending cells with normal saline as adjuvant, and adjusting cell concentration to 10 × 10⁶Adjusting the cell concentration, and sucking the cell into an injector for later use; and the water bath recovery is to recover the cryopreserved mesenchymal stem cells after the co-culture through a water bath at 37 ℃, and the centrifugation is carried out at 2000rpm for 5 min.