A method for promoting chondrocyte growth

Abstract

The present invention discloses a method for promoting chondrocyte growth, comprising the following steps: (1) constructing BMP-2 gene recombinant MSC (MSCdBMP2) cells to express BMP-2 protein on the surface of MSCdBMP2 cells; (2) preparing exosomes (MSCdBMP2Exo) derived from MSCdBMP2; (3) stimulating MSCdBMP2 cells and MSCdBMP2Exo to significantly promote chondrocyte growth. The present invention clones the BMP-2 gene into MSC cells through genetic recombination technology to prepare new BMP-2 gene recombinant MSC (MSCdBMP2) cells; at the same time, the exosomes (MSCdBMP2Exo) prepared from MSCdBMP2 cells have a biological function similar to that of MSCdBMP2 cells in promoting chondrocyte growth.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for promoting the growth of chondrocytes. Background Art

[0002] Cartilage damage is a common disease. Destruction of articular cartilage is the primary pathological change in osteoarthritis. Cartilage damage caused by trauma and other factors can ultimately lead to osteoarthritis. Therefore, cartilage repair has become a major issue in orthopedics. Although cartilage can repair itself after damage, the process is lengthy and the results are often unsatisfactory. The first reason why cartilage is difficult to repair is that cartilage has an active metabolism but lacks a blood vessel supply, so its repair capacity is limited. The second reason is that there is a lack of undifferentiated cells at the site of injury. Chondrocytes are highly differentiated cells and cannot migrate and grow into the damaged cartilage after injury. The third reason is that with aging, the ability of chondrocytes to divide and produce cellular matrix decreases.

[0003] The application of cell and tissue engineering methods to repair cartilage is a research direction that scientists are currently working hard on. The main methods of artificial intervention in cartilage repair include: (1) drilling holes in the subchondral bone to repair cartilage damage, (2) cartilage transplantation, (3) periosteum and perichondrium transplantation, (4) chondrocyte transplantation, (5) undifferentiated MSC transplantation, etc. There are many studies on the use of transplantation to repair cartilage, and there is great hope; however, there are also many influencing factors, mainly including: (1) A sufficient amount of repair cells is required, usually 10 6 The volume of the transplanted cells is on the order of 100 ml / ml, ranging from 0.1 ml to several ml depending on the size of the injury. (2) A suitable carrier is required. The ideal carrier should be tissue compatible, absorbable, easy to repeat, and have affinity with the transplanted cells. Current carriers include synthetic materials such as polylactic acid (PLA) and polyglycolic acid (GPA); and biological components such as type II collagen.

[0004] MSC transplantation for the treatment of cartilage damage and arthritis has been extensively studied and is generally considered effective. However, the primary effect of MSCs is their paracrine function, which promotes collagen and glycosaminoglycan synthesis, thereby alleviating symptoms. In reality, cartilage repair is not significant. To date, no other treatment, including MSCs, can significantly promote cartilage repair.

[0005] Introduction to human bone morphogenetic protein-2 (BMP-2). Bone morphogenetic proteins (BMPs), also known as bone morphogenetic proteins, are a group of highly conserved functional proteins with similar structures and belong to the TGF-β family. BMPs can stimulate DNA synthesis and cell replication, thereby promoting the directional differentiation of MSCs into osteoblasts. They are also the main factors that induce bone and cartilage formation in the body. They are expressed during limb growth, endochondral ossification, early fractures, and cartilage repair, playing an important role in the embryonic development and regenerative repair of bones. Today, the BMP family has 43 members, which are widely present in tissues such as embryos, blood cells, kidneys, and spleens of pigs, cattle, sheep, rabbits, mice, and humans, and have a high degree of homology between different species. BMPs are not only involved in bone regulation but also play a role in the development of fat, kidneys, liver, bones, and the nervous system.

[0006] After secretion, BMPs bind to the extracellular matrix and soluble antagonists and interact with various protein receptors on the cell membrane surface. Not only do they possess unique structures and physicochemical properties that differ from other bone growth factors, but different members of the BMP family also differ in their gene localization and osteogenesis mechanisms. BMPs primarily act through two signaling pathways: the Smad pathway and the p38-MAPK pathway. These signaling pathways are regulated at four levels: extracellular antagonists, membrane receptors, the cytoplasmic microenvironment, and transcription. Therapeutic approaches, BMPs not only mediate osteogenesis independently but also in combination with other bone growth factors, accelerating disease recovery.

[0007] BMP-2 is synthesized in vivo as a precursor. The signal peptide and propeptide are removed by proteolytic cleavage, resulting in a mature peptide consisting of 114 amino acid residues. The mature peptide correctly folds its conserved structure through seven pairs of disulfide bonds; only homo- or heterodimers of the mature peptide are biologically active. Therefore, the production of recombinant BMP-2 protein is challenging. Another common drawback of BMPs is their short half-life and susceptibility to degradation in the body. Consequently, sustained-release technologies are often used for local administration to delay degradation. Consequently, despite the known potential of BMPs to promote chondrocyte growth, no viable products have yet been developed. Summary of the Invention

[0008] The main purpose of the present invention is to provide a method for promoting the growth of chondrocytes. Based on the research on MSC and BMP-2, two of the most promising new means for cartilage growth, we found that both of them have advantages, but at the same time they also have serious defects. Attempts have been made to use the two together, and although the results showed improvement, they were not significant; the main problems have been introduced above. On the one hand, BMP-2 has many disulfide bonds, and eukaryotic expression not only has a low amount but also a complicated subsequent purification process, and it is difficult to renature when expressed in the prokaryotic cell; on the other hand, its half-life is too short and it cannot fully play its role. The present invention seeks solutions to the two main problems in the process of using MSC and BMP-2 together. The breakthrough point is not to use the traditional eukaryotic or prokaryotic expression followed by purification and renaturation, and then use sustained-release technology to extend its half-life, but to clone the BMP-2 gene into MSC cells through genetic recombination technology to prepare a brand-new BMP-2 gene-recombinant MSC (MSCdBMP2) cell.

[0009] To achieve the above object, the present invention provides a method for promoting chondrocyte growth, comprising the following steps:

[0010] S101: Preparation of hUC-MSC cells;

[0011] S102: The BMP-2 coding gene was cloned into a eukaryotic expression vector pDisplay with a transmembrane domain structure to form the BMP-2 cell membrane surface display plasmid pdBMP2;

[0012] S103: Transfecting pdBMP2 into MSC cells to generate MSCdBMP2 cells; MSCdBMP2 cells can be used directly or used to prepare MSCdBMP2 Exo;

[0013] S104: Use MSCdBMP2 cells and MSCdBMP2Exo to stimulate and promote chondrocyte growth.

[0014] Preferably, in said S102, the BMP-2 protein consists of 396 amino acids, and its amino acid sequence is:

[0015] "MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLL

[0016] SMFGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEES

[0017] LEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNSSFHHRINIYEI

[0018] IKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEE

[0019] KQGVSKRHVRISRSLHQDEHSWSQIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKS

[0020] SCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPPFLADHLNSTNHAIVQTLVNSVN

[0021] SKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR"

[0022] Preferably, in S102, the gene DNA of the BMP-2 protein consists of 1191 bases, and its coding sequence is the 1st to 1191st bases from the 5' end, and its nucleotide sequence is:

[0023] atggtggccgggacccgctgtcttctagcgttgctgcttccccaggtcctcctgggcggcgcggctg

[0024] gcctcgttccggagctgggccgcaggaagttcgcggcggcgtcgtcgggccgcccctcatcccagcc

[0025] ctctgacgaggtcctgagcgagttcgagttgcggctgctcagcatgttcggcctgaaacagagaccc

[0026] acccccagcagggacgccgtggtgcccccctacatgctagacctgtatcgcaggcactcaggtcagc

[0027] cgggctcacccgccccagaccaccggttggagagggcagccagccgagccaacactgtgcgcagctt

[0028] ccaccatgaagaatctttggaagaactaccagaaacgagtgggaaaacaacccggagattcttcttt

[0029] aatttaagttctatccccacggaggagtttatcacctcagcagagcttcaggttttccgagaacaga

[0030] tgcaagatgctttaggaaacaatagcagtttccatcaccgaattaatatttatgaaatcataaaacc

[0031] tgcaacagccaactcgaaattccccgtgaccagacttttggacaccaggttggtgaatcagaatgca

[0032] agcaggtgggaaagttttgatgtcacccccgctgtgatgcggtggactgcacagggacacgccaacc

[0033] atggattcgtggtggaagtggcccacttggaggagaaacaaggtgtctccaagagacatgttaggat

[0034] aagcaggtctttgcaccaagatgaacacagctggtcacagataaggccattgctagtaacttttggc

[0035] catgatggaaaagggcatcctctccacaaaagagaaaaacgtcaagccaaacacaaacagcggaaac

[0036] gccttaagtccagctgtaagagacaccctttgtacgtggacttcagtgacgtggggtggaatgactg

[0037] gattgtggctcccccggggtatcacgccttttactgccacggagaatgcccttttcctctggctgat

[0038] catctgaactccactaatcatgccattgttcagacgttggtcaactctgttaactctaagattccta

[0039] aggcatgctgtgtcccgacagaactcagtgctatctcgatgctgtaccttgacgagaatgaaaaggt

[0040] tgtattaaagaactatcaggacatggttgtggagggttgtgggtgtcgctag

[0041] Preferably, in said S102, the BMP-2 protein is expressed on the surface of MSCs by connecting the 3' end of the BMP-2 gene to the PDGFR transmembrane polypeptide gene on the pDisplay expression vector.

[0042] Preferably, in said S103, the method for preparing MSCdBMP2Exo comprises the following steps:

[0043] S1031. Cultivate MSCdBMP2 cells;

[0044] S1032. Use the collected MSCdBMP2 cell culture supernatant as raw material to prepare MSCdBMP2Exo.

[0045] The method for promoting chondrocyte growth disclosed in the present invention has the following characteristics: (1) simple process: it is only necessary to culture MSCdBMP2 cells before use, without the need to purify the protein and without worrying about the protein refolding problem; (2) long half-life: the action period of BMP-2 on its surface is equivalent to the survival period of MSCdBMP2 cells. As long as the MSCdBMP2 cells are alive, the BMP-2 protein can play a role, so that the half-life of BMP-2 is as long as several weeks, just like that of MSCdBMP2 cells; (3) dual function of a cell: MSCdBMP2 cells have the dual functions of MSC and BMP-2, that is, BMP-2 gives MSC new functions; (4) MSC acts as a carrier of BMP-2, which, on the one hand, limits the distribution of BMP-2 in the body, avoiding its diffusion throughout the body and causing other unnecessary side effects, while maintaining its high concentration locally; (5) the membrane surface display method is used in gene recombination to anchor BMP-2 on the surface of MSC. Unlike proteins expressed within cells, BMP-2 displayed on the membrane surface provides a good platform for it to exert its effects; it is equivalent to immobilizing the BMP-2 protein, which is more conducive to its binding to the receptor and exerting a better effect. (6) Exosomes derived from MSCdBMP2 cells (MSCdBMP2Exo) have similar biological functions to MSCdBMP2 cells, and BMP-2 is also anchored on the surface of MSCdBMP2Exo. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0047] Figure 1 A technical roadmap for a method of promoting chondrocyte growth provided by an embodiment of the present invention;

[0048] Figure 2 A schematic diagram of the structure of the pDisplay expression vector provided in an embodiment of the present invention;

[0049] Figure 3 Figure 3 shows the effect of recombinant MSCdBMP2 cells and their exosomes on the activity of chondrocytes (CHs) provided in an embodiment of the present invention.

[0050] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0052] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the various components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0053] In addition, the descriptions of "first", "second", etc. in the present invention are for descriptive purposes only and should not be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between the various embodiments can be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in this field. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0054] In the embodiment of the present invention, referring to Figure 1 , this method of promoting cartilage cell growth includes:

[0055] 1. Preparation of hUC-MSC cells

[0056] In this embodiment, although hUC-MSC cells are used, the present invention does not limit the source of MSCs, and can be bone marrow MSCs (BM-MSCs), umbilical cord MSCs (UC-MSCs), umbilical cord blood MSCs (UCB-MSCs), adipose MSCs (Ad-MSCs), dental pulp MSCs (Dental Pulp MSCs, Dp-MSCs), etc.

[0057] 1.1. The umbilical cord was obtained from a healthy pregnant woman who had undergone cesarean section at full term, and the maternal test results for HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, mycoplasma, and anti-cytomegalovirus antibody were all negative. All maternal tests during pregnancy, including TORCH and Down syndrome screening, were normal.

[0058] 1.2. Aseptically collect the umbilical cord and cut it into small segments, each approximately 1 cm. Remove the arteries and veins (two arteries and one vein). Mince the umbilical cord in a glass bottle and place it in a 15 ml centrifuge tube (two segments, approximately 2 cm per tube). Add 8 ml of collagenase (type IV) for digestion. After digestion for 3 h, filter through a 100-mesh filter (or centrifuge at 500 rpm for 3-5 minutes) and discard any undigested tissue fragments. Transfer the supernatant to a 15 ml centrifuge tube, approximately 5 ml per tube. Add PBS to 10 ml and centrifuge at high speed (3000-4000 rpm). Discard the supernatant and transfer the pellet to a culture dish. Incubate in STEMPRO hMSC SFM human mesenchymal stem cell culture medium at 37°C in a 5% CO2 saturated humidity incubator. Replace half of the medium after 48 h and replace the full medium after 4 days. Discard any excess suspended cells.

[0059] 1.3. Change the culture medium every 3-4 days according to the color of the culture medium. When the primary cells cover 60-80% of the culture dish, perform a 1:2 subculture.

[0060] 1.4. UC-MSC Identification and Usage Criteria: Use cells from passages 3-10. When the cell count reaches 108, rinse twice with PBS and digest with 0.05% Tris-EDTA solution. Digestion is terminated by adding culture medium to prepare a 1-3 × 106 cell suspension. Flow cytometry analysis reveals negative results for CD14 or CD11b, CD79a or CD19, CD34, CD45, CD109, and HLA-DR (<2%); positive rates for CD29, CD44, CD73, CD90, and CD105 (>95%); and negative results for Epstein-Barr virus, cytomegalovirus, HIV, hepatitis B virus, mycoplasma, bacterial culture, and fungal culture.

[0061] 1.5. Use directly or store in liquid nitrogen for later use.

[0062] 2. Construction of BMP-2 membrane surface display plasmid pdBMP2

[0063] 2.1. BMP-2 protein is characterized by being composed of 396 amino acids, and its sequence is shown in SEQ ID NO: 1.

[0064] Sequence 1: BMP-2 amino acid sequence

[0065] "MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLL

[0066] SMFGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEES

[0067] LEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNSSFHHRINIYEI

[0068] IKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEE

[0069] KQGVSKRHVRISRSLHQDEHSWSQIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKS

[0070] SCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR"

[0071] 2.2. The gene for the BMP-2 protein is characterized by:

[0072] 1) The sequence is shown in 2. Its DNA consists of 1191 bases, and its coding sequence is the 1st to 1191th bases from the 5' end;

[0073] 2) a DNA sequence that has more than 90% homology with the DNA sequence defined by SEQ ID NO: 2 and encodes the amino acid sequence of SEQ ID NO: 1;

[0074] 3) A nucleotide sequence that can hybridize with the DNA defined by sequence 2 under highly stringent conditions (after hybridization, the membrane is washed with a solution containing 0.1X SSPE or 0.1X SSC, 0.1% SDS at 65°C).

[0075] Sequence 2: DNA sequence of BMP-2

[0076] atggtggccgggacccgctgtcttctagcgttgctgcttccccaggtcctcctgggcggcgcggctg

[0077] gcctcgttccggagctgggccgcaggaagttcgcggcggcgtcgtcgggccgcccctcatcccagcc

[0078] ctctgacgaggtcctgagcgagttcgagttgcggctgctcagcatgttcggcctgaaacagagaccc

[0079] acccccagcagggacgccgtggtgcccccctacatgctagacctgtatcgcaggcactcaggtcagc

[0080] cgggctcacccgccccagaccaccggttggagagggcagccagccgagccaacactgtgcgcagctt

[0081] ccaccatgaagaatctttggaagaactaccagaaacgagtgggaaaacaacccggagaattcttcttt

[0082] aatttaagttctatccccacggaggagtttatcacctcagcagagcttcaggttttccgagaacaga

[0083] tgcaagatgctttaggaaacaatagcagtttccatcaccgaattaatatttatgaaatcataaaacc

[0084] tgcaacagccaactcgaaattccccgtgaccagacttttggacaccaggttggtgaatcagaatgca

[0085] agcaggtgggaaagttttgatgtcacccccgctgtgatgcggtggactgcacagggacacgccaacc

[0086] atggattcgtggtggaagtggcccacttggaggagaaacaaggtgtctccaagagacatgttaggat

[0087] aagcaggtctttgcaccaagatgaacacagctggtcacagataaggccattgctagtaacttttggc

[0088] catgatggaaaagggcatcctctccacaaaagagaaaaacgtcaagccaaacacaaacagcggaaac

[0089] gccttaagtccagctgtaagagacaccctttgtacgtggacttcagtgacgtggggtggaatgactg

[0090] gattgtggctcccccggggtatcacgccttttactgccacggagaatgcccttttcctctggctgat

[0091] catctgaactccactaatcatgccattgttcagacgttggtcaactctgttaactctaagattccta

[0092] aggcatgctgtgtcccgacagaactcagtgctatctcgatgctgtaccttgacgagaatgaaaaggt

[0093] tgtattaaagaactatcaggacatggttgtggagggttgtgggtgtcgctag

[0094] 2.3. Cell membrane expression vector

[0095] The core approach of this invention is to express BMP-2 on the cell membrane. This unique structure not only adds new functions to MSCs, but also provides an immobilized surface for BMP-2, helping it better promote cartilage growth. Figure 2 Figure 2 shows a schematic diagram of the pDisplay expression vector structure. The present invention expresses BMP-2 protein on the surface of MSCs by linking the 3' end of the BMP-2 gene to the PDGFR transmembrane polypeptide gene on the pDisplay expression vector. However, the present invention is not limited to this transmembrane region and this vector; other expression vectors with any transmembrane region structure are within the scope of protection of the present invention.

[0096] Primer design

[0097] 1) Sequence 3: BMP2upBglII primer: GACGTCAGATCTatggtggccgggacccgctgtctt;

[0098] 2) Sequence 4: BMP2dnSalI primer: GACGTCGTCGACctagcgacacccacaaccctccac;

[0099] 2.5. RNA Preparation: Collect 50 ml of peripheral blood from volunteers, separate peripheral blood mononuclear cells (PBMCs) using lymphocyte separation medium, and prepare total RNA using TRIzol reagent.

[0100] 2.6. RT-PCR: Using total RNA as a template and primers 1 and 2 as primers, the BMP-2 gene fragment was prepared using the Invitrogen SuperScript IV One-Step RT-PCR Kit. After agarose gel electrophoresis, the gel containing the digested BMP-2 gene DNA fragment was excised and purified using the EZ Spin column-based PCR product purification kit.

[0101] 2.7. Restriction Enzyme Digestion of pDisplay Vector and PCR Product: Digest the pDisplay vector with BglII / SalI restriction enzymes, perform agarose gel electrophoresis, and cut the gel containing the digested pDisplay vector DNA fragment; the pDisplay BglII / SalI digestion product was purified using the EZ-10 Spin Column DNA PAGE Gel Extraction Kit.

[0102] 2.8. Connect: Use DNA ligase to connect pDisplay BglII / SalI and BMP-2 gene BglII / SalI fragment to obtain pdBMP2 product;

[0103] 2.9. Transfection of competent bacteria:

[0104] DH5α competent bacteria were transfected with pdBMP2 product;

[0105] 2.10. Colony picking and verification:

[0106] Pick positive colonies, culture them in small quantities, extract plasmid DNA, perform preliminary screening with corresponding restriction endonucleases, and finally confirm by sequencing. Save the pdBMP2 recombinant bacteria;

[0107] 2.11. Cultivate pdBMP2 recombinant bacteria and prepare pdBMP2 recombinant plasmid.

[0108] 3. Preparation of BMP-2 gene recombinant MSC (MSCdBMP2) cells

[0109] 3.1. Prepare cells: Take each well of a 6-well plate as an example. One day before transfection, add an appropriate amount (5×10 5 MSC cells were cultured in 2 ml of culture medium and transfected when the cell density reached 50-80% confluence.

[0110] 3.2. Take 1 μg of pdBMP2 plasmid;

[0111] Prepare transfection reagent: Add 1 μg of plasmid DNA to 600 μl of Opti-MEM® Reduced Serum Medium and mix thoroughly. Gently mix Lipofectamine LTX reagent and add 5 μl to the DNA tube. Gently mix thoroughly and incubate at room temperature for 30 min.

[0112] Add approximately 100 μL of DNA-Lipofectamine LTX complex to each well of cells and gently shake the plate back and forth to mix.

[0113] 3.5. After culturing for 24 hours in a 37°C, 5% CO2 incubator with STEMPRO hMSC SFM human mesenchymal stem cell medium, transfected MSCdBMP2 cells were harvested and incubated with mouse anti-human BMP2 monoclonal antibody (Abcam) at 37°C for 1 hour. After washing twice, FITC-labeled rabbit anti-mouse secondary antibody (Abcam) was added and incubated at 30°C for 30 minutes. After washing twice, the cells were suspended in 50 μl of saline.

[0114] 3.6. Fluorescence microscopy (Olympus) showed that the positive rate of BMP2 protein was approximately 55%.

[0115] 3.7. Cultivation of MSCs and MSCdBMP2 Cells

[0116] 1) Culture MSCs and MSCdBMP2 cells in STEMPRO hMSC SFM human mesenchymal stem cell medium in a 37°C, 5% CO2 incubator. When the cells reach 80% confluence, digest them with 0.05% Tris-EDTA and subculture them for no more than 10 passages.

[0117] 2) Collect MSC cell and MSCdBMP2 cell culture supernatants as raw materials for preparing MSC exosomes (MSC-Exo) and MSCdBMP2 exosomes (MSCdBMP2Exo).

[0118] 4. Enrichment of exosomes from MSC and MSCdBMP2 cell culture supernatants

[0119] The total exosome isolation (TEI) kit from Life Technology was used.

[0120] 4.1. Collect the MSC and MSCdBMP2 cell culture supernatants, divide them into 50 ml conical-bottom centrifuge tubes, and centrifuge at 2000 x g for 30 minutes.

[0121] Transfer the supernatant to two new centrifuge tubes, add 0.5 volumes of TEI reagent, mix thoroughly, incubate overnight at 4°C, and centrifuge at 10,000 x g for 1 hour the next day.

[0122] 4.3. Aspirate the supernatant and resuspend the pellet in 5 ml of PBS.

[0123] Centrifuge at 10,000 x g for 1 hour. Aspirate the supernatant and resuspend the pellet in PBS. Adjust the concentration to 1 mg / ml, resulting in 80-150 nm MSC-Exo and MSCdBMP2Exo. Aliquot and store at -20°C until needed.

[0124] 5. Application of MSCdBMP2 Cells and MSCdBMP2Exo

[0125] 1. Test samples: MSCdBMP2 cells and MSCdBMP2Exo, with MSC cells, MSC-Exo, and BMP-2 protein as controls.

[0126] 2. Purpose of test: To evaluate the effect of the test sample in promoting chondrocyte growth.

[0127] 3. Experimental Animals: Male SD rats, weighing approximately 50 g, clean grade, were purchased from the Experimental Animal Center of the Fourth Military Medical University and used in the experiments after conventional feeding for one week.

[0128] 4. Isolation and Culture of Chondrocytes (CHs): Rats were sacrificed by dislocation, and the hair on both lower limbs and abdomen was shaved with a shaver. After spraying with 75% alcohol, the rats were moved to the animal room's clean bench. In the clean bench, 500ml of PBS and 2.5ml of double-antibody were prepared. Alcohol was added to a small beaker. The double-antibody was added to the PBS and pipetted into two 50cm culture dishes using a dropper. 10ml was then transferred to a 50ml centrifuge tube. The skin of the legs was cut open to expose the knee joints. The muscle was dissected and the knee joints were soaked in 75% alcohol for 30 seconds. The joints were then washed in sterile PBS containing double-antibody. This was repeated three times with PBS and placed in a centrifuge tube containing double-antibody PBS and stored on ice. The rats were moved to the cell room's clean bench. An appropriate amount of PBS was added to a glass culture dish. Excess muscle and tendon were removed using ophthalmic scissors. The synovium was dissected from the joints, and the femur and tibia were separated. The hyaline cartilage from the femoral and tibial surfaces was removed using a scalpel. Hyaline cartilage was washed twice in PBS, minced, and transferred to a centrifuge tube containing 10ml of 0.3% collagenase II. The cells were shaken for 60 beats and then placed in an incubator for digestion. The shaking was repeated 60 times every 30 minutes. After 3 hours, the cells were passed through a 200-mesh cell sieve and centrifuged at 1200rpm for 10 minutes. The cells were resuspended in DMEM medium containing 20% ​​FBS and plated into a cell culture dish. The dish was incubated in a 5% CO2, 37°C incubator. The next day, the cells were washed several times with PBS and the medium was replaced with fresh medium. When the cells reached 80% confluency, the medium was replaced. The next morning, the cells were washed with PBS, covered with 1ml of 0.25% trypsin-EDTA, and digested in an incubator for 5 minutes. The digestion was terminated by adding 3ml of culture medium. The cells were harvested, centrifuged at 500rpm for 5 minutes, and the pellet was resuspended in complete culture medium for passage.

[0129] 5. Test of chondrocyte growth and the effect of test substances on chondrocyte growth: CCK-8 method was used to detect chondrocyte proliferation.

[0130] 1) MSC cells and MSCdBMP2 cells cultured in DMEM medium containing 20% ​​FBS were trypsin-EDTA digested as described above when the chondrocyte density reached 80%, and resuspended in DMEM medium containing 20% ​​FBS for cell counting;

[0131] 2) Experimental groups: A (chondrocyte CHs) normal control group, B (CHs+MSC cells), C (CHs+MSCdBMP2), D (CHs+MSC-Exo), E (CHs+MSCdBMP2Exo), F (CHs+BMP-2), G (CHs+MSC+BMP-2) six experimental groups, H (MSC), I (MSCdBMP2), J (blank Med) three control groups.

[0132] 3) Sample loading: Add samples to a 96-well plate as shown in Table 1. Set up 7 replicate plates:

[0133] Table 1. Sample template for chondrocyte growth assay

[0134]

[0135] (1) 100ul contains 2X10 3 Chondrocytes (CHs, passage 4, 7 groups A, B, C, D, E, F, G, x 3 replicates, totaling 21 wells);

[0136] (2) 100ul contains 2X10 3 MSC cells (passage 4, 3 groups of B, G, and H, x 3 replicates, 9 wells in total);

[0137] (3) 100ul contains 2X10 3 MSCdBMP2 cells (passage 4, 2 groups C and I, etc., x 3 replicates, a total of 6 wells);

[0138] (4) 100 μl MSC-Exo (Group D x 3 replicates, 3 wells in total);

[0139] (5) 100 μl MSCdBMP2Exo (Group E x 3 replicates, 3 wells in total);

[0140] (6) 100 μl BMP-2 (100 ng / ml, 2 groups F and G, x 3 replicates, 6 wells in total);

[0141] (7) 200 μl of culture medium as blank control;

[0142] (8) Use culture medium to make up the volume of each well to 200ul.

[0143] 4) Culture the cells in a 37°C, 5% CO2 incubator, changing the medium every two days. Remove one plate daily on days 1, 3, 5, 7, 9, 11, and 13. Add 10 μL of CCK-8 to each well and mix thoroughly with shaking for 5 minutes. Incubate the plates in the incubator for 2 hours. Measure the absorbance (OD) at 450 nm using a microplate reader, using the blank control as the background. Plot a line graph to depict cell growth.

[0144] 5) Take the average value of 3 replicate wells in each group;

[0145] Normal control: OD in group A represents the normal proliferation of chondrocytes (CHs);

[0146] Background control: The OD value of group H represents the background proliferation of MSC, the OD value of group I represents the background proliferation of MSCdBMP2, and the OD value of group J serves as the culture medium blank control; these background OD values ​​need to be subtracted from the OD values ​​of the corresponding experimental groups;

[0147] Groups B, C, D, E, F, and G are experimental groups:

[0148] OD in group B and OD in group H represent the effects of MSCs on CHs proliferation;

[0149] OD of group C-OD of group I is the effect of MSCdBMP2 on CHs proliferation;

[0150] OD in group D represents the effect of MSC-Exo on CHs proliferation;

[0151] OD in group E represents the effect of MSCdBMP2Exo on CHs proliferation;

[0152] The OD of group F is the effect of BMP-2 protein (100 ng / ml) on CHs proliferation;

[0153] OD of group G-OD of group H represent the effect of MSC+BMP-2 protein (100 ng / ml) on CHs proliferation.

[0154] Calculate cell viability:

[0155] CHs cell viability (%) = [OD(test) - OD(background)] / OD(normal) × 100

[0156] CHs cell viability: CHs cell proliferation activity

[0157] The chondrocyte growth curve of each group was drawn with time as the horizontal axis and CHs cell activity as the vertical axis.

[0158] Results: See Table 2 and Figure 3 .

[0159] As can be seen from Table 2, based on the proliferation of normal CHs, MSC, MSC-Exo, and BMP-2 all have a certain effect on stimulating CHs proliferation, and the effect of MSC combined with BMP-2 is better than that of a single component; the recombinant MSCdBMP2 cells of the present invention have the best effect, and the effect of their exosomes is comparable to that of the cells, which is better than the effect of MSC combined with BMP-2 and has a significant difference.

[0160] Table 2. Effects of recombinant MSCdBMP2 cells and their exosomes on CHs activity

[0161]

[0162]

[0163] The growth curve of chondrocytes (CHs) was S-shaped. The cells reached the logarithmic growth phase on the 4th day and reached the plateau phase after the 7th day (see Figure 3 ).

[0164] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention description and drawings under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

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