CN117165518A - Method for separating chondroprogenitor cells and application of separated chondroprogenitor cells - Google Patents

Abstract

The application discloses a method for separating cartilage progenitor cells in the field of medical preparations and application of the separated cartilage progenitor cells. The technical problem underlying the present application is how to simply and/or rapidly isolate chondrocyte progenitors having the capacity to inhibit the formation of osteoclasts and/or bone resorption. In order to solve the technical problem, the present application provides a method for isolating chondroprogenitor cells, the method comprising the steps of: and (3) performing enzymolysis on the isolated articular hyaline cartilage to obtain an enzymolysis product, and culturing the enzymolysis product by utilizing the migration characteristic of the chondrocyte progenitor cells to obtain the chondrocyte progenitor cells. The application can be used for treating and/or preventing and/or alleviating and/or improving osteoarthritis.

Description

Method for separating chondroprogenitor cells and application of separated chondroprogenitor cells

Technical Field

The application belongs to a method for separating cartilage progenitor cells in the field of medical preparations and application of the separated cartilage progenitor cells.

Background

Osteoarthritis (OA) is a common chronic degenerative disease and is most commonly seen with knee Osteoarthritis, mainly manifested as knee pain and limited movement. The joint pathology caused by OA includes injury to cartilage, subchondral bone, leading to joint inflammation. Its incidence and progression is associated with a variety of risk factors. The treatment of OA is now still dominated by symptomatic and step treatments. The latest version of the guidelines for osteoarthritis treatment issued by the chinese medical society in 2018 mentions: existing treatment modes of osteoarthritis include rehabilitation training, medicines and surgery, but the treatment mode of osteoarthritis before the operation has limited effect, and no method can reverse the pathological process of OA. Surgical interventions (including osteotomies or joint replacements) can only be performed in the late OA stage. Therefore, new therapeutic approaches are urgently needed to interfere with the progression of the bone joint and to improve the quality of life of the patient.

Disclosure of Invention

The technical problem to be solved by the application is how to obtain cartilage progenitor cells without utilizing the dedifferentiation of cartilage cells. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

Experiments prove that the cartilage progenitor cells have strong immunoregulation and osteogenic cartilage differentiation effects and can be used as seed cells for repairing bone cartilage diseases; the human cartilage progenitor cells which are subjected to primary separation culture of the joints are injected through joint cavities, so that the effect of relieving osteoarthritis is achieved, abnormal bone remodeling of osteoarthritis patients can be improved, and the curative effect is improved.

In order to solve the technical problems, the application provides the following technical scheme:

the present application provides a method for isolating chondroprogenitor cells, comprising the steps of: enzymatic digestion of isolated articular hyaline cartilage gives a cartilage tissue mass which is cultured using the migration characteristics of chondroprogenitors, without dedifferentiation of the chondrocytes.

Further, the above method does not include dedifferentiation of chondrocytes.

Further, the culturing in the above method includes a step of culturing with a medium in which the adherent cells are cultured.

The culture medium for culturing adherent cells may contain 10% by volume of FBS (fetal bovine serum).

The culture medium for culturing the adherent cells may be a liquid medium (complete medium) obtained by adding FBS, penicillin and streptomycin to an alpha-MEM medium, the volume percentage of FBS in the culture medium for culturing the adherent cells may be 10%, the penicillin content may be 100 units/ml, and the streptomycin content may be 100ng/ml.

Further, the articular hyaline cartilage in the above method may be derived from a human.

Further, the enzymatic digestion in the above method may be performed with collagenase.

The collagenase may be a type ii collagenase.

The enzymolysis digestion can be carried out in an enzymolysis reaction system with the content of type II collagenase of 125CDU/mL for 2 hours at 37 ℃.

Further, the chondroprogenitors in the above methods have the ability to inhibit the formation of osteoclasts and/or bone resorption by secretion.

The application also provides the application of the chondrocyte progenitor cells in preparing products for treating and/or preventing and/or relieving and/or improving the chondrogenic diseases, wherein the chondrocyte progenitor cells are prepared according to the method.

The product may be a medicament.

Further, the cartilage disorder in the above application includes at least one of degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, scapulohumeral periarthritis, hyperosteogeny, rheumatoid arthritis, and rheumatoid arthritis.

The application also provides a medicine for treating and/or preventing and/or relieving and/or improving osteoarthritis, which consists of a pharmaceutical adjuvant and chondrocyte progenitor cells, wherein the chondrocyte progenitor cells are prepared according to the method.

Further, the dosage form of the medicament can be injection.

The application discloses a method for separating human cartilage progenitor cells with immunoregulation capability and application thereof in osteoarthritis, wherein the surface layer of articular cartilage is rich in the cartilage progenitor cells, which are important seed cells for promoting tissue regeneration. The isolated chondrocyte progenitor cells have strong immunoregulation capacity and can inhibit the formation of osteoclasts. The application uses the rat osteoarthritis model to prove that the cartilage progenitor cells can inhibit the formation of osteoclasts, and can effectively treat osteoarthritis.

Drawings

FIG. 1 is a graph showing the growth of chondroprogenitors of generation P0-P5 and a graph showing the doubling time of the population of generation P1-P5 according to example 1 of the present application.

Fig. 2 is a morphology of chondroprogenitors according to example 1 of the present application.

FIG. 3 is a flow chart of the chondrocyte progenitor cell according to example 1 of the present application.

FIG. 4 is a graph showing the identification of the three-lineage differentiation ability of chondrocyte progenitor cells according to example 1 of the present application. The crystal violet staining results, ALP staining results, toluidine Blue (tolucidine Blue) staining results and Oil Red O (Oil Red O) staining results are shown in order from left to right.

FIG. 5 is a chart showing the karyotype analysis of chondroprogenitor cells according to example 1 of the present application.

Fig. 6 is a color chart of the chondrocyte progenitor cells provided in example 1 of the present application in vivo as a tumor Ma Songran.

FIG. 7 is a chart showing RNA sequencing enrichment analysis of chondrocyte progenitor cells according to example 2 of the present application.

Fig. 8 is a graph showing the ability of chondroprogenitors provided in example 2 of the present application to regulate osteoclast formation. The upper panel of the first column from left to right shows the staining results of the monocyte group (monocyte cultured alone), and the lower panel of the first column shows the staining results of the monocyte and chondrocyte progenitor direct co-culture group; the upper panel of the second column shows the staining results of the Transwell monocyte group (monocytes cultured alone), and the lower panel of the second column shows the staining results of the Transwell monocyte and chondroprogenitor indirect co-culture group; the upper panel of the third column shows the staining results of the monocyte bone resorption group (monocyte cultured alone), and the lower panel of the third column shows the staining results of the monocyte and chondroprogenitor co-cultured bone resorption group.

FIG. 9 is an evaluation chart of the chondrocyte progenitor cells of the present application for treating rats by OAmicro-CT provided in example 3. The upper graph of the first column from left to right is a 3D reconstruction graph of the false operation group micro CT, the middle graph of the first column is a coronal graph of the false operation group micro CT, and the lower graph of the first column is a 3D reconstruction graph of the false operation group micro CT sagittal plane; the upper graph of the second column is a model group micro CT 3D reconstruction graph, the middle graph of the second column is a model group micro CT coronal plane graph, and the lower graph of the second column is a model group micro CT sagittal plane 3D reconstruction graph; the upper graph of the third column is a micro CT 3D reconstruction graph of the treatment group, the middle graph of the third column is a coronal graph of the treatment group, and the lower graph of the third column is a sagittal 3D reconstruction graph of the treatment group.

FIG. 10 is a chart showing the evaluation of the OA histochemical staining of rats treated with chondrocyte progenitor cells according to example 3 of the present application.

Wherein, the upper graph of the first column from left to right is a solid green dyeing result graph of the artificial operation group safranine O, the middle graph of the first column is a blue dyeing result graph of the artificial operation group toluidine, the lower graph of the first column is a two-type collagen dyeing result graph of the artificial operation group, and the arrow represents a damage part result graph; the upper graph of the second column is a model group safranin O solid green dyeing result graph, the middle graph of the second column is a model group toluidine blue dyeing result graph, and the lower graph of the second column is a model group type II collagen dyeing result graph; the upper panel of the third column shows the treatment group safranin O solid green staining results, the middle panel of the third column shows the treatment group toluidine blue staining results, and the lower panel of the third column shows the model group type II collagen staining results.

FIG. 11 is a statistical result of the evaluation of OA histochemical staining of rats treated with chondrocyte progenitor cells according to example 3 of the present application. ", P <0.001.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 culture of chondroprogenitors

1. Acquisition of joint platform tissue

Discarded human knee tibial plateau tissues (patient signed informed consent) were collected from the hospital.

2. Isolation and culture of chondroprogenitors

The following operations were all performed under aseptic conditions.

1. Transferring the collected human knee joint tibia plateau tissue into a sterile collection bottle, and preserving at 2-8 ℃. The joint platform tissue was washed in an ultra clean bench with pre-chilled 1% double antibody (penicillin/streptomycin) PBS phosphate buffer (penicillin 100 units/ml, streptomycin 100 ng/ml) for 5 minutes.

2. The method comprises the steps of separating articular hyaline cartilage of a middle area of a human knee joint tibia platform relative to a normal side by adopting a sterile scalpel, firstly holding the scalpel by hand to take lamellar cartilage during separation, taking a bearing surface of the center of a joint surface layer, avoiding edges, (the joint surface layer is hyaline cartilage, is rich in stem cell groups and has more fibrocartilage at the joint edges), obtaining articular hyaline cartilage, and placing the hyaline cartilage in a six-hole plate.

3. Cutting the articular hyaline cartilage with an ophthalmic scissors to obtain cut cartilage.

4. The sheared cartilage was transferred to a 10mL centrifuge tube, washed three times with 1% double-resistant PBS and the supernatant removed.

Preheating a 0.1% type II collagenase (Sigma, product number V900892) solution at 5.37 ℃, adding a 0.1% type II collagenase solution with a volume which is 2 times that of the tissue into the centrifuge tube in the step 4 to obtain an enzymolysis reaction system, enabling the content of the type II collagenase in the enzymolysis digestion reaction system to be 125CDU/mL, and placing the mixture in a shaking table at 80rpm at 37 ℃ for digestion for 2 hours after vortex mixing to obtain digested cartilage tissue.

6. Centrifuging the cartilage tissue subjected to enzymolysis digestion in the step 5, sucking out collagenase, adding 5mL PBS containing 1% of double antibodies into the centrifuge tube for resuspension, centrifuging at 500rpm for 3min, discarding the supernatant, and taking the precipitate to obtain the cleaned cartilage tissue block (namely the zymolyte).

7. Culture and passage of chondroprogenitors

Culture medium for culturing adherent cells: liquid medium (also called complete medium) obtained by adding FBS, penicillin and streptomycin to alpha-MEM medium (Gibco, cat# C12571500 BT). In the culture medium for culturing the adherent cells, the volume percentage of FBS is 10 percent, the penicillin content is 100 units/ml, and the streptomycin content is 100 ng/ml).

(1) Transferring the cartilage tissue block washed in the step 6 to 25cm by forceps ² And (3) adding about 5mL of culture medium for culturing the adherent cells into the culture flask, transferring the culture flask into the culture box for culturing for 3 days, and keeping the culture flask motionless, wherein the cells are the P0 generation chondroprogenitor cells (P0 for short).

(2) Removing culture medium from culture flask when P0 generation cartilage progenitor cells grow to fusion rate of 80% -90%, adding 0.25% EDTA-pancreatin (Servicebio, cat# G4004-100 ML) for digestion, adding 2 times volume of the complete culture medium for stopping digestion when cells become round, collecting, centrifuging, re-suspending with proper amount of complete culture medium, and adjusting cell density to 5×10 ⁵ The cells/flask were inoculated, and the flask was placed in a 5% carbon dioxide incubator at 37℃and incubated with 10% FBS in an alpha-MEM medium (liquid medium obtained by adding FBS to the alpha-MEM medium, the volume content of FBS in the liquid medium being 10%) for every 3 days to obtain P1-generation chondroprogenitors (abbreviated as P1).

(3) Subculturing the P1-generation chondroprogenitors to the P3-generation, designated as P3-generation chondroprogenitors (abbreviated as P3) according to the method shown in step (2). The P4-generation chondroprogenitors (abbreviated as P4), P5-generation chondroprogenitors (abbreviated as P5) and P8-generation chondroprogenitors (abbreviated as P8) were cultured and harvested as described above. The graph of P0-P5 generation chondroprogenitor cell growth is shown in the left panel of FIG. 1, and the population doubling time (population doubling time, PDT) of P1-P5 generation chondroprogenitor cells is shown in the right panel of FIG. 1. (PDT=t× [ lg 2/(lgNt-lgN 0)]) Nt is the final number of cells cultured at time t, t is the initial number of cells cultured at 2X 10 on day 5 (P1), day 6.5 (P2), day 9 (P3), day 12 (P4) and day 16 (P5) ⁵ Number of cells inoculated per well.

8. Identifying the cells in step 1:

(1) Morphological identification

The P0, P4 and P8 generation chondroprogenitors were observed and photographed, respectively, using an optical microscope. As a result, the chondrocyte progenitor cells were in the form of a spindle and were grown as fibroblasts, as shown in FIG. 2.

(2) Flow authentication

In flow tubes, the P3-generation chondrocyte progenitor concentration was adjusted to 5X 10 ⁵ The final volume per tube was 100. Mu.L. CD45 (Biolegend, cat 304016), CD31 (Biolegend, cat 303118), CD235A (Biolegend, cat 349112) and CD29 are added to each flow tubeBody (manufacturer eBioscience, cat No. 12-0299-41), CD44 flow antibody (manufacturer Invitrogen, cat No. 17-0441-81), CD90 flow antibody (manufacturer Biolegend, cat No. 328108), CD105 flow antibody (manufacturer Invitrogen, cat No. 1930337) and PDPN flow antibody (manufacturer Biolegend, cat No. 337022) 5 μl were incubated at 4 ℃ for 30min in the absence of light, and after three pbs washes, were detected using a flow cytometer. As a result, as shown in FIG. 3, the isolated P3-generation chondroprogenitors did not express the surface markers of hematopoietic cells, endothelial cells, and erythrocytes, such as CD45, CD31, and CD235A, but expressed the markers of chondroprogenitors, CD29, CD44, CD90, CD105, and PDPN (podoplanin).

(3) Self-renewal and trilinear differentiation ability identification

A. Self-renewal capacity of chondroprogenitors

Plating the P3 generation cartilage progenitor cells at 500 cells/hole, adding 2mL of 10% FBS alpha-MEM culture medium, and standing for the first three days; then the complete culture medium is replaced every 3 days; after 10 days, the mixture was fixed and stained with 0.1% crystal violet solution.

The crystal violet staining results are shown in the first left panel of fig. 4, with purple staining indicating cell colony formation.

B. Induction of differentiation of chondroprogenitors into osteoblasts

Experimental group: p3 generation chondroprogenitor cells were cultured at 5X 10 ³ The cells/well were plated in 48-well plates and replaced with osteoinductive medium every 3 days (manufacturer cyagen, cat. HUXMX-90021). After induction for 10 days, staining was performed using alkaline phosphatase (ALP).

Control group: the osteogenic medium was replaced with 10% FBS in alpha-MEM medium and the other procedures were the same as in the experimental group.

ALP staining results are shown in the second plot from the left in FIG. 4, the presence of red stained cells, indicating the presence of osteoblasts, i.e., P3-generation chondroprogenitors, which can be induced to differentiate into osteoblasts.

C. Induction of differentiation of chondroprogenitors into chondrocytes

Experimental group: p3 generation chondroprogenitor cells were cultured at 5X 10 ³ The cells/wells were seeded in 48-well plates and replaced with cartilage every 3 daysInduction medium (manufacturer cyagen, cat. HUXMX-90041). After 14 days of induction, staining was performed with Toluidine Blue (tolucine Blue).

Control group: the cartilage induction medium was replaced with 10% FBS. Alpha. -MEM medium, and the other procedures were the same as those of the experimental group.

The results of the tolucidine Blue staining are shown in the third panel from the left in fig. 4, with purple staining, indicating the presence of chondroblasts, i.e., the P3-generation chondroprogenitors can be induced to differentiate into chondroblasts.

D. Induction of differentiation of chondroprogenitors into adipocytes

Experimental group: p3 generation chondroprogenitor cells were cultured at 1X 10 ⁴ The cells/wells were plated in 48-well plates and replaced with fat induction medium every 3 days (manufacturer cyagen, cat. HUXMX-90031). After 14 days of induction, staining was performed with Oil Red O (Oil Red O).

Control group: the fat induction medium was replaced with 10% FBS. Alpha. -MEM medium, and the other procedures were the same as in the experimental group.

The results of Oil Red O staining are shown in the fourth panel from the left in fig. 4, with Red staining indicating the presence of adipocytes, i.e., P3-generation chondrocyte progenitors, that can be induced to differentiate into adipocytes.

(4) Nuclear analysis

Prior to harvesting, cells were harvested by centrifugation at 1100rpm for 10 minutes with 20. Mu.L colchicine solution (20. Mu.g/mL) added to the P3-generation cell culture flask and treated for 20 minutes. Adding 0.075M potassium chloride solution (the solute is potassium chloride and the solvent is water), treating in 37 ℃ water bath for 15min, pre-fixing by using 1mL of fresh fixing solution (acetic acid: methanol=1:3), centrifuging, reserving a proper amount of fixing solution according to cell count, dripping 100 mu L of cell suspension on a glass slide, drying in air, and baking in an oven at 90 ℃ for 2h. The baked slide is digested by pancreatin for 30s, FBS is used for stopping digestion, finally, the slide is immersed in Jim Sa staining solution for 2.5min, washed by tap water, dried and sealed.

The slides were scanned using a GSL120 fully automated fluorescence microscope, chromosome morphology was examined using Cytovision software, 20 per sample, chromosome number, morphology and structure were examined for each cell, and abnormal cells seen were classified and counted. The nomenclature of chromosome banding and karyotype results is referred to the International nomenclature System for human cytogenomics (ISCN 2020). The results showed that the isolated chondroprogenitor cell chromosomes were normal (fig. 5).

(5) Subcutaneous nodulation experiment of nude mice

Experimental group: the P3 generation chondroprogenitor cells were digested with trypsin, and the cell concentration was adjusted to 5X 10 using an appropriate amount of Matrix gel ⁶ And each mL. The chondrocyte progenitor cells were subcutaneously injected in 8W nude mice (commercially available from Vetolihua, cat# line code: 401) at an injection rate of 0.2ml. The cell pellet size was observed daily after injection, the cell pellet was sampled after 4 weeks, paraffin embedded after fixation and sections were Masson stained.

Control group: the Matrix gel was used to replace the P3-generation chondroprogenitors, and the other procedures were the same as in the experimental group.

As a result, as shown in FIG. 6, no abnormality was observed in the injection site after 4 weeks, no cell arrangement in a nest or gland shape was observed, and no cell deformation or necrosis was observed.

Example 2 evaluation of immunomodulatory Activity of Soft diaphyseal progenitor cells

1. Chondroprogenitor enrichment pathway analysis

The P3 generation chondrocyte progenitor cells obtained in example 1 were subjected to RNA sequencing, pre-quality control and pretreatment, and GO enrichment analysis was performed on the RNA sequencing data of the chondrocyte progenitor cells, and the results are shown in fig. 7, wherein the pathways related to regulation of development, differentiation and bone resorption of the osteoclast cells are shown in fig. 7, and the number of genes represents the number of genes enriched in the corresponding pathways, so that the pathways related to enrichment of osteoclast cells by the chondrocyte progenitor cells can be seen, and the effect of regulation of osteoclast cells is suggested.

2. Chondroprogenitors can inhibit the formation of osteoclasts by secretion

Human monocytes: for isolation by collecting peripheral blood from healthy volunteers, the isolation method is described in non-patent documents "Li PL, wang YX, zhao ZD, et al clinical-grade human dental pulp stem cells suppressed the activation ofosteoarthritic macrophages and attenuated cartilaginous damage in a rabbit osteoarthritis model. Stem Cell Res Ther.2021;12 (1):260.".

Macrophage colony stimulating factor (M-CSF): purchased from PeproTech under the accession number 30025.

NF- κb activator ligand (RANKL): purchased from PeproTech under the product number 310-01.

Chondrocyte progenitor Co-culture (Co-culture) group: chondrocyte progenitor cells were transformed according to 5X 10 ³ After plating the cells/well on 48-well plates and culturing them in 10% FBS in alpha-MEM for 24 hours, human monocytes were cultured according to 1X 10 ⁵ The cells/wells were inoculated into a culture plate containing chondrocyte progenitors, and macrophage colony-stimulating factor (M-CSF) and NF- κB activator ligand (RANKL) were added simultaneously to give a final content of M-CSF of 20ng/mL and a final content of RANKL of 20ng/mL. Culturing was continued for 9d. The a-MEM medium of 10% fbs was changed every 3 days. Experiments were repeated three times, 3 wells each. How much of the polynuclear mature osteoclasts were observed by tartrate-resistant acid phosphatase (TRAP) staining. Mature osteoclasts can be stained wine red with tartrate-resistant acid phosphatase (TRAP).

Chondroprogenitor cells and monocytes were co-cultured indirectly (Transwell): chondroprogenitors according to 5X 10 ³ The cells were inoculated into 48-well Transwell culture plate cells, and human monocytes were cultured in accordance with 1X 10 ⁵ The cells/well were inoculated into the lower layer of a 48-well transwell plate, and macrophage colony-stimulating factor (M-CSF) and NF- κB activator ligand (RANKL) were added simultaneously to give a final content of M-CSF of 20ng/mL and a final content of RANKL of 20ng/mL. Culturing was continued for 9d. The a-MEM medium of 10% fbs was changed every 3 days. Experiments were repeated three times, 3 wells each.

As a result, as shown in fig. 8, the number of polynuclear mature osteoclasts in the chondrocyte progenitor cell and monocyte Co-culture (Co-culture) group was significantly reduced relative to the chondrocyte progenitor cell and monocyte indirect Co-culture (Transwell) group.

3. Chondrocyte progenitor cells can inhibit bone resorption by secretion

The report group: sterile waste elephant ivory sheets (purchased from IDS, cat No. AE-8050) were subjected to bone resorption tests. When in use, a proper amount of ivory slices are taken and sterilized under an ultraviolet lamp for 30min, and thenThe ivory pieces were then soaked with 1% diabody, 10% FBS in alpha-MEM medium for 15min and then placed at the bottom of the wells of a 48-well plate. Chondroprogenitor cells were plated at 5X 10 per well ³ Density of individual cells was inoculated onto ivory plaque, and after overnight attachment, the isolated monocytes (1X 10) ⁵ And/or wells) and macrophage colony-stimulating factor (M-CSF) and NF- κB activator ligand (RANKL) were added together to give a final content of M-CSF of 20ng/mL and a final content of RANKL of 20ng/mL. The a-MEM medium containing M-CSF and RANKL 10% FBS was changed every 3 days. Cultivation was continued for 21d. The cells adhered to the surface of the ivory piece were removed with a cotton swab, PBS was washed until no visible cells were observed under the mirror, stained with toluidine blue dye for 15min, washed with PBS, and the number of bone resorption pits was counted under a microscope. Experiments were repeated three times, 3 wells each.

Control group: only the isolated monocytes (1X 10) ⁵ Per well), no chondrocyte progenitor cells were added, the remainder being the report group.

As shown in fig. 8, the co-cultured group significantly inhibited the formation of the recesses positive for toluidine blue staining compared with the induced group, and it was found that chondroprogenitors can inhibit the formation of osteoclasts and bone resorption function by secretion.

EXAMPLE 3 establishment of rat anterior cruciate ligament off-joint unstable osteoarthritis model and cell transplantation therapy

1. Preparation of chondrocyte progenitor cell preparation

Collecting the P3 generation chondroprogenitor cells of example 1 in logarithmic phase, centrifuging, collecting cell pellet, and re-suspending the cell pellet with phosphate buffer to adjust cell concentration to 1×10 ⁷ and/mL, obtaining the cartilage progenitor cell preparation.

2. Construction of anterior cruciate ligament separation injury model

(1) 8W rats (vendor Vetong Lihua, strain code: 101) were divided into two groups of a blank control group and a model group, 3 each. The anterior cruciate ligament disruption model building method is as follows:

all rats were kept under standard conditions prior to surgery and were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (sodium pentobarbital as solute and physiological saline as solvent) at a dose of 45mg/kg prior to the experiment. After anesthesia, the cefazolin sodium solution is intramuscular injected (solute is cefazolin sodium, solvent is physiological saline), and the injection dosage is 5mg/100g; fixing the operation table upward, and scraping hair around the knee joint. Sterilizing the knee joint with 5% povidone iodine solution, and performing conventional operation towel; the rat knee joint was incised 1cm in longitudinal lines, and the skin, fascia, muscle were incised layer by layer. Dislocates the patella to the outside, exposing the knee joint; and bending the knee, cutting off the anterior cruciate ligament by using a pointed surgical knife, performing anterior drawer verification, closing the wound layer by layer after the patella is reset, suturing the skin, and raising for one week to obtain the model rat.

Group of sham operations: the incision is made on the knee joint of the rat in a longitudinal line of 1cm, the skin, fascia and muscle are incised layer by layer, the anterior cruciate ligament is not damaged, and the skin is sutured.

(2) Model rats were randomized into 2 groups, model and treatment groups of 3 rats each, 1 week after joint injury. Mice used in the sham group, i.e., mice in the sham group in step (1).

Treatment group: the rat-side knee joint cavity was injected with 100 μl of the chondrocyte progenitor preparation and then fed in animal houses without specific pathogenic bacteria (specific pathogen free, SPF).

Group of sham operations: the rat side knee joint cavity was injected with 100 μl of phosphate buffer.

Model group: the chondrocyte progenitor preparation was replaced with an equal volume of physiological saline and the rest was treated in the same way as the treatment group.

3. Bone imaging structure for detecting micro CT system

(1) The SD rat was euthanized 6 weeks after the joint cavity injection, and the tissue samples were subjected to microCT (Scanco Medical, basersodrf, zurich, switzerland) scanning imaging with the whole knee joint, and the bone imaging structure was examined to evaluate the extent of the lesions of osteoarthritis based on the extent of the formation of hyperosteogeny and the extent of joint destruction.

(2) Bone imaging structure test results (fig. 9): in the model group, the bone trabecular structure of the subchondral bone region was disturbed, and the cartilage stem progenitor cells could improve the osteo-graphic structure of traumatic osteoarthritis after articular cavity injection of cartilage progenitor cells (fig. 9).

4. Histologic pathology and immunohistochemical staining

(1) The SD rat joint cavity was euthanized 6 weeks after injection, knee joint tissue was decalcified in 10% EDTA decalcification solution (neutral phosphate buffer solution as solvent) for 4 weeks, paraffin embedding was then performed, and 4 μm sections were cut.

(2) Tissue morphology changes were observed by safranin O-fast green (SOFG) and toluidine blue staining.

The results showed (fig. 10): compared with the control group, the joint surface of the artificial operation group is uneven, the cartilage layer is thinned, and the cartilage matrix is reduced; compared with the treatment group, the pseudo-operation group has the advantages that after the treatment of the human cartilage progenitor cells, cartilage matrixes are increased, joint surfaces are smooth, and cartilage tissues are protected to a certain extent.

(3) Subchondral bone remodeling was assessed by immunohistochemical staining of collagen type I (COL ii) to analyze subchondral bone COL ii in joint tissue samples. After endogenous peroxidase quenching and blocking of the non-specific binding sites, the sections were subjected to immunohistochemical staining by incubation with anti-COL II (1:100; abcam, ab 34712).

The experimental results (fig. 10) show: compared with the control group, the COL II staining area of subchondral bone of the sham operation group is obviously reduced; the areas of COL II staining of subchondral bone after treatment with human chondroprogenitor cells increased compared to sham and model groups. The positive cell rate of COL II staining was analyzed by Image J, the COL II staining results were processed by GraphPad Prism 8 statistical software, the experimental results were expressed as mean.+ -. Standard deviation, and significant differences were expressed by using One-way ANOVA test, P <0.001. As a result, as shown in FIG. 11, COL II positive cell rates of the treatment group (P < 0.001) and the sham operation group (P < 0.001) were significantly higher than those of the model group.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (9)

1. A method of isolating chondroprogenitors, comprising the steps of: enzymatic digestion of isolated articular hyaline cartilage gives a cartilage tissue mass which is cultured using the migration characteristics of chondroprogenitors, without dedifferentiation of the chondrocytes.

2. The method of claim 1, wherein the culturing comprises the step of culturing with a medium that cultures adherent cells.

3. The method of claim 1, wherein the articular hyaline cartilage is of human origin.

4. A method according to any one of claims 1-3, wherein the enzymatic digestion is performed with collagenase.

5. A method according to any one of claims 1 to 3, wherein said chondroprogenitor cells have the ability to inhibit the formation of osteoclasts and/or bone resorption by secretion.

6. Use of chondroprogenitors prepared according to the method of any one of claims 1-5, for the preparation of a product for the treatment and/or prevention and/or alleviation and/or amelioration of a chondrogenic disease.

7. The use according to claim 6, wherein the cartilage disorder comprises at least one of degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, scapulohumeral periarthritis, hyperosteogeny, rheumatoid arthritis, and rheumatoid arthritis.

8. A medicament for the treatment and/or prevention and/or alleviation and/or amelioration of osteoarthritis, characterized in that the medicament consists of a pharmaceutical adjuvant and chondroprogenitors prepared according to the method of any one of claims 1-5.

9. The medicament according to claim 8, wherein the dosage form of the medicament is an injection.