Use of stem cells or transgenic stem cells

Abstract

The use of stem cells or transgenic stem cells in the preparation of a stem cell gel or tissue engineered cartilage. An active protein expressed by the finally obtained stem cell gel or tissue engineered cartilage graft can induce stem cells of host tissue to differentiate into chondrocytes, thus accelerating the integration of the stem cell gel or tissue engineered cartilage graft and the host tissue.

Description

Application of stem cells or genetically modified stem cells Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of stem cells or transgenic stem cells. Background Art

[0002] Cartilage injury is a common clinical condition in orthopedics, often occurring in cases of joint sprains and localized external impacts. Its pathological characteristics include degeneration and destruction of articular cartilage, subchondral bone sclerosis, reactive hyperplasia of the joint margin and subchondral bone, and osteophyte formation. Currently, there are seven main clinical treatments for cartilage injury, with the following specific methods and their limitations:

[0003] 1. Palliative treatment: Also known as joint cleansing, this is a common clinical procedure that primarily removes damaged cartilage without replacing it. Its drawbacks include merely removing damaged cartilage fragments and removing excess fat, improving mobility, alleviating symptoms, and alleviating pain. It is considered conservative treatment and has no effect on repairing cartilage damage, resulting in poor efficacy.

[0004] 2. Restorative treatment: Also known as microfracture, this is a common clinical approach. It involves cleaning the cartilage in the defect area and drilling holes in the subchondral bone to induce blood clots, which then trigger spontaneous repair by bone marrow stem cells. However, its drawback is that it merely fills the damaged area with fibrous tissue without repairing the cartilage, resulting in poor efficacy.

[0005] 3. Artificial joint replacement surgery: This is a commonly used clinical procedure involving the surgical replacement of artificial metal or non-metal joints. However, its drawbacks include significant differences between the implanted joint and the human body, making it difficult for the implanted joint to integrate properly with the human body. This procedure can still cause varying degrees of pain, which can be quite painful for the patient. Furthermore, there is a high risk of infection, loosening, fracture, and dislocation of the prosthesis.

[0006] 4. Autologous chondrocyte implantation (ACI): Clinical results have been less than satisfactory. This procedure involves transplanting autologous cartilage tissue to the injured area. Its drawbacks include requiring two joint surgeries, treating an injury with an injury, and requiring at least two surgeries on two joints. The number of chondrocytes is low, and the cells are prone to aging and phenotypic changes. This treatment is individualized and cannot be commercially promoted on a large scale.

[0007] 5. Autologous chondrocytes cultured in vitro engineered cartilage transplantation: The clinical results are also unsatisfactory, such as the MACI product approved for marketing in the United States. Its defects are the same as those in "4".

[0008] 6. Allogeneic cartilage transplantation (OCA): Rarely used in clinical practice, this procedure involves transplanting cartilage from another person. Its drawbacks include difficulty sourcing donors and the inability to inflict injuries on one's own.

[0009] 7. Allogeneic umbilical cord blood-derived mesenchymal stem cell implantation: Due to the high clinical risk, it is rarely used clinically, such as the Cartistem product approved for marketing in South Korea. Its drawbacks include the fact that umbilical cord blood-derived mesenchymal stem cells implanted at the site of cartilage damage are limited in the number of chondrocytes they transform into at the site of cartilage damage. While this demonstrates good cartilage regeneration, it cannot achieve complete repair, and there are also unknown allogeneic reactions.

[0010] Of the seven methods mentioned above, treatments 1 through 3 are traditional treatments widely used both domestically and internationally. They have no repair effect on cartilage damage, resulting in suboptimal results. These conservative, ameliorative treatments present numerous drawbacks. Treatments 4 through 7 are modern biological treatments, offering significant improvements in efficacy over traditional treatments and capable of partially repairing damaged cartilage. However, these treatments also present drawbacks such as significant adverse reactions, low integration, inability to fully repair cartilage damage, and inability to achieve complete cure.

[0011] In addition, Qu Fujun et al. used BMP7 gene-transfected chondrocytes and collagen-fibrin gel as three-dimensional scaffold materials to construct BMP7 gene-transfected tissue-engineered cartilage (Qu Fujun, Hou Yi, Liu Liling, et al. Application of BMP7 gene-transfected chondrocytes to construct tissue-engineered cartilage [J]. Chinese Journal of Tissue Engineering Research, 2006, 10(13): 59-61). They also used BMP7 gene-transfected tissue-engineered cartilage transplantation to repair rabbit knee cartilage defects (Qu Fujun, Sun Hua, Feng Nianping, et al. Observation on application of BMP7 gene-transfected tissue-engineered cartilage to repair rabbit knee cartilage defects [J]. Chinese Journal of Tissue Engineering Research, 2008, 27(1): 48-52). However, this transplantation method for repairing cartilage damage has the problem of limited sources of tissue-engineered cartilage tissue seed cells, making it unsuitable for repairing large-area cartilage damage. Whether the seed cells are derived from autologous or allogeneic sources, they are used to treat injuries with injuries, and are also unsuitable for large-scale factory production. Therefore, it is difficult to use it as a drug in clinical practice. Summary of the Invention

[0012] In view of this, the purpose of the present invention is to provide the application of stem cells or genetically modified stem cells in the treatment of cartilage damage, so as to solve a series of problems such as the lack of seed cell sources for existing tissue-engineered cartilage, the inability to expand in large quantities in vitro and the aging of expanded cells, as well as the integration of tissue-engineered cartilage grafts with host tissues. The technical solutions of the present invention are as follows:

[0013] The present invention provides the use of stem cells or transgenic stem cells in preparing stem cell gel liquid or tissue engineering cartilage.

[0014] Preferably, the stem cells are derived from umbilical cord blood, umbilical cord, placenta, bone marrow or fat. More preferably, the stem cells are umbilical cord blood mesenchymal stem cells or bone marrow mesenchymal stem cells.

[0015] Preferably, the gene is selected from at least one of bone morphogenetic protein (BMP) gene, TGFβ gene, insulin-like growth factor (IGF) gene, fibroblast growth factor (FGF) gene and SOX9 gene; the BMP gene is preferably BMP7 and / or BMP2.

[0016] Preferably, the method of using stem cells to prepare stem cell gel or construct tissue-engineered cartilage includes:

[0017] Step 1: Isolation and culture of stem cells;

[0018] Step 2: Using active proteins to induce stem cells to differentiate and culture;

[0019] Step three, preparing a stem cell gel solution with the stem cells obtained in step two, or constructing a type III collagen 3D culture scaffold in vitro with the stem cells obtained in step two until tissue-engineered cartilage is formed.

[0020] Preferably, the method of using transgenic stem cells to prepare stem cell gel or construct tissue-engineered cartilage includes:

[0021] Step 1, constructing a recombinant plasmid containing the target gene;

[0022] Step 2, isolation and culture of stem cells;

[0023] Step 3, using the recombinant plasmid constructed in step 1 to transfect the stem cells cultured in step 2 to obtain stem cells transfected with the target gene;

[0024] Step 4, using active protein to induce differentiation culture of stem cells transfected with target gene;

[0025] Step 5: Prepare a stem cell gel solution using the stem cells obtained in step 4, or construct a type III collagen 3D culture scaffold in vitro using the stem cells obtained in step 4 until tissue-engineered cartilage is formed.

[0026] Preferably, the construction of a recombinant plasmid containing the target gene in step 1 comprises:

[0027] Step 1-1, obtain pBlue-target gene plasmid;

[0028] Step 1-2: Double-digest the pBlue-target gene plasmid and extract and purify it;

[0029] Steps 1-3: Double-digest the pcDNA3.1 vector and extract and purify it;

[0030] Step 1-4, ligating the enzyme-digested gene fragment obtained in step 1-2 with the enzyme-digested DNA fragment obtained in step 1-3 to obtain the pcDNA3.1-target gene vector plasmid;

[0031] Steps 1-5: transform the pcDNA3.1-target gene vector plasmid into DH5a competent cells and amplify it. The amplified product is extracted and purified to obtain the product.

[0032] Preferably, the target gene is selected from at least one of: bone morphogenetic protein (BMP) gene, TGFβ gene, insulin-like growth factor (IGF) gene, fibroblast growth factor (FGF) gene and SOX9 gene; the BMP gene is preferably BMP7 and / or BMP2.

[0033] Preferably, the stem cells used in step 1 or step 2 are derived from umbilical cord blood, umbilical cord, placenta, bone marrow or fat. More preferably, the stem cells are umbilical cord blood mesenchymal stem cells or bone marrow mesenchymal stem cells.

[0034] Preferably, the isolation and culture process of the stem cells includes:

[0035] Step 2-1, adding the obtained stem cell-derived tissue to DMEM culture medium, mixing and centrifuging;

[0036] Step 2-2, after removing the fat layer from the centrifuge, mix with Percoll separation solution with a density of 1.073 g / ml at a volume ratio of 1:2 and centrifuge;

[0037] Step 2-3: After the mononuclear cells are separated and washed in sequence, the cells are suspended in stem cell culture medium and the cells are concentrated at a concentration of 5×10 6 The cells were inoculated into a culture flask at a density of 3 ml / ml. The culture was placed in a 5% CO2, 37°C environment for 3-4 days. After that, the non-adherent cells were discarded and the culture medium was replaced with fresh one and the culture was continued for 7-10 days.

[0038] Steps 2-4: Use 0.25% trypsin solution to digest the cells in the culture flask, wash them in sequence, add stem cell culture medium to suspend the cells, and use 5×10 4 The cells were seeded into culture flasks at a density of cells / ml for subculture. The cells were subcultured for 2 to 5 generations for later use. The stem cell culture medium was replaced every time the cells were subcultured.

[0039] Preferably, the DMEM culture medium and the stem cell culture medium both contain fetal bovine serum (FBS) with a final concentration of 10% (v / v) and 150 u / mL heparin.

[0040] Preferably, the active protein-induced differentiation culture of stem cells in step 2 or the active protein-induced differentiation culture of stem cells transfected with the target gene in step 4 includes:

[0041] Active protein is added to the differentiation culture medium containing stem cells or target gene-transfected stem cells, and cultured at 5% CO2 and 37°C for 3 to 5 generations, with the same differentiation culture medium replaced every generation.

[0042] Preferably, the differentiation culture medium is DMEM culture medium.

[0043] Preferably, the active protein is at least one of the following components: TGFβ, platelet-rich plasma, SOX9; further preferably, the active protein is at least two of the following components: TGFβ, platelet-rich plasma, SOX9.

[0044] Further preferably, the active protein is composed of TGFβ, platelet-rich plasma, and SOX9, and the final concentration of each component in the differentiation culture system is: TGFβ, 4.0-16.0 μg / L; platelet-rich plasma, 0.5-2.0%, v / v; SOX9, 2.5-10 μg / L; more preferably, the active protein is composed of the final concentration of each component in the differentiation culture system as follows: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L.

[0045] Preferably, in step 3 or step 5, the process of preparing the stem cell gel solution from the stem cells includes:

[0046] The stem cells were added to normal saline to prepare 0.5×10 7 ~1×10 7 The sodium hyaluronate gel solution was prepared by adding sodium hyaluronate to a saline solution with an albumin concentration of 1.5% to 2.5%. The sodium hyaluronate gel solution was mixed with the cell suspension at a volume ratio of 1:0.5 to 2 to obtain a stem cell gel solution.

[0047] Preferably, in step 3 or step 5, the stem cells are used to construct a type III collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed, comprising:

[0048] The stem cells obtained in the previous step were prepared into a mixture containing 2.5×10 6 ~5×10 6 A type III collagen 3D culture scaffold containing stem cells was added with DMEM cell culture medium containing the active protein, and cultured under 5% CO2 and 37°C conditions. The DMEM cell culture medium containing the active protein was replaced every 7 days, and cultured continuously for 20 to 24 days to obtain tissue-engineered cartilage.

[0049] Preferably, the 2.5×10 6 ~5×10 6The preparation method of the type III collagen 3D culture scaffold for individual stem cells is as follows: the type III collagen solution and the stem cell suspension obtained in the previous step are mixed, a fibrinogen solution is added, and then thrombin is added. After mixing evenly, the scaffold is placed for 8 to 12 hours, and then cultured at 5% CO2 and 37°C for 14 to 21 days.

[0050] Preferably, the final concentration of the active protein in DMEM cell culture medium is as follows: TGFβ, 4.0-16.0 μg / L; platelet-rich plasma, 0.5-2.0%, v / v; SOX9, 2.5-10 μg / L; more preferably, the final concentration of the active protein in DMEM cell culture medium is as follows: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L.

[0051] The present invention further provides the stem cell gel solution or tissue engineering cartilage obtained by the above application.

[0052] The present invention also provides the use of the stem cell gel solution or the tissue engineered cartilage in preparing bone grafts.

[0053] This invention addresses the issues of insufficient seed cell sources, inability to expand in large quantities in vitro, and susceptibility to post-expansion aging in stem cell gel solutions or tissue-engineered cartilage, and is of significant scientific significance. The resulting stem cell gel solution or tissue-engineered cartilage contains a sufficient number of functional seed cells and has the ability to regulate seed cell proliferation and maintain a stable phenotype.

[0054] Furthermore, the present invention addresses the integration of stem cell gels or tissue-engineered cartilage grafts with host tissue. The active protein expressed by the stem cell gels or tissue-engineered cartilage grafts of the present invention can induce stem cells in the host tissue to differentiate into chondrocytes, accelerating their integration with the host tissue. This technology is expected to be further applied in humans. Preclinical studies are currently underway, and clinical studies will commence after obtaining clinical research approval. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 is a map of the pCDNA3.1 plasmid and the NotⅠ and KpnⅠ double enzyme digestion sites in Example 1 of the present invention.

[0056] FIG2 is a cell morphology diagram of umbilical cord blood mesenchymal stem cells after primary culture for 7 days in Example 1 of the present invention.

[0057] FIG3 is a cell morphology diagram of umbilical cord blood mesenchymal stem cells cultured for 7 days after passage one in Example 1 of the present invention.

[0058] FIG4 is a cell morphology diagram of the umbilical cord blood mesenchymal stem cells cultured for 7 days at the second passage in Example 1 of the present invention.

[0059] FIG5 is a cell morphology diagram of umbilical cord blood mesenchymal stem cells cultured for 7 days after passage three in Example 1 of the present invention.

[0060] Figure 6 shows cell growth charts after 28 days of continuous screening using MesenCult MSC Basal Medium stem cell culture medium containing G418 in Example 1 of the present invention, wherein Figure 6-1 shows the cell growth chart of the BMP7-transfected cell group, and Figure 6-2 shows the cell growth chart of the non-BMP7-transfected cell group.

[0061] FIG7 is a morphological diagram of a 3D blank stent in Example 1 of the present invention.

[0062] FIG8 is a diagram showing the cartilage morphology obtained by transferring the BMP7 gene in Example 1 of the present invention without adding mixed induction factors.

[0063] FIG9 is a diagram showing the cartilage morphology in Example 1 of the present invention without BMP7 gene transfer but with the addition of mixed induction factors.

[0064] FIG10 is a diagram showing the cartilage morphology of the BMP7 gene plus mixed induction factors transferred in Example 1 of the present invention.

[0065] FIG11 is a diagram of a rabbit knee cartilage defect model in Example 2 of the present invention.

[0066] FIG12 is a diagram showing the repair effect of the model group in Example 2 of the present invention after 8 weeks.

[0067] FIG13 is a diagram showing the repair effect of the 3D stent assembly in Example 2 of the present invention after 8 weeks.

[0068] FIG14 is a diagram showing the effect of BMP7 genome repair after 8 weeks of treatment in Example 2 of the present invention.

[0069] FIG15 is a diagram showing the repair effect of the BMP7 gene plus mixed induction factor group in Example 2 of the present invention after 8 weeks.

[0070] FIG16 is a diagram of the rabbit knee cartilage defect model in Example 3 of the present invention.

[0071] FIG17 is a diagram showing the effect of tissue-engineered cartilage repair after 8 weeks obtained in Example 3 of the present invention.

[0072] FIG18 is a morphological diagram of the blank injection solution in Example 4 of the present invention.

[0073] FIG. 19 is a morphological diagram of the stem cell gel solution in Example 4 of the present invention in which the BMP7 gene is transferred but no mixed induction factors are added.

[0074] FIG. 20 is a morphological diagram of the stem cell gel solution containing the transferred BMP7 gene and mixed induction factors in Example 4 of the present invention.

[0075] FIG21 is a diagram of the rabbit knee cartilage defect model in Example 4 of the present invention.

[0076] FIG22 is a diagram showing the repair effect of the model group after 8 weeks in Example 4 of the present invention.

[0077] FIG23 is a diagram showing the repair effect of the blank gel solution group in Example 4 of the present invention after 8 weeks.

[0078] FIG24 is a diagram showing the repair effect of the BMP7 gene stem cell gel solution group transferred in Example 4 of the present invention after 8 weeks.

[0079] FIG25 is a diagram showing the repair effect of the BMP7 gene plus mixed induction factor stem cell gel solution group in Example 4 of the present invention after 8 weeks.

[0080] FIG26 is a morphological diagram of the stem cell gel solution containing umbilical cord blood mesenchymal stem cells and mixed induction factors in Example 5 of the present invention.

[0081] FIG27 is a diagram of the rabbit knee cartilage defect model in Example 5 of the present invention.

[0082] FIG28 is a diagram showing the repair effect of the stem cell gel solution group in Example 5 of the present invention after 8 weeks.

[0083] FIG29 is a diagram of the rabbit knee cartilage defect model in Example 6 of the present invention.

[0084] FIG30 is a diagram showing the repair effect of the bone marrow mesenchymal stem cell gel solution obtained in Example 6 of the present invention after 8 weeks.

[0085] FIG31 is a diagram showing the effect of 8-week tissue-engineered cartilage repair constructed by bone marrow mesenchymal stem cells transfected with the BMP7 target gene obtained in Example 6 of the present invention. DETAILED DESCRIPTION

[0086] In the examples of the present invention, pBlue-BMP7 plasmid was purchased from ATCC.

[0087] In the examples of the present invention, the pcDNA3.1 vector was purchased from Shanghai Boya Biotechnology Co., Ltd.

[0088] In an embodiment of the present invention, the stem cell culture medium is MesenCult MSC Basal Medium, purchased from Shanghai Huayasichuang Biotechnology Co., Ltd. This stem cell culture medium is suitable for culturing bone marrow, umbilical cord, umbilical cord blood, placenta, and adipose stem cells. The isolation and culture of stem cells derived from umbilical cord blood includes: collecting 2-3 ml of umbilical cord blood from a newborn rabbit, adding DMEM culture medium, and centrifuging; after removing the upper fat layer and part of the supernatant from the centrifuge, adding it to a Percoll separation medium with a density of 1.073 g / ml and centrifuging again; separating the middle layer of mononuclear cells and washing them in sequence, adding MesenCult MSC Basal Medium stem cell culture medium to suspend the cells, and culturing them in a 5% CO2, 37°C incubator; after culturing for 3-4 days, discarding non-adherent cells, replacing MesenCult MSC Basal Medium stem cell culture medium and continuing to culture; after about 7-10 days, the primary cells cover the bottom of the culture flask; then digesting them with 0.25% trypsin solution and continuing to culture them in MesenCult MSC Basal Medium stem cell culture medium. Twelve hours after inoculation, some primary umbilical cord blood mesenchymal stem cells begin to adhere to the culture medium. Newly adhered cells are rounded and gradually extend pseudopodia, forming cell-cell connections. The cells adopt a spindle- or fusiform appearance resembling fibroblasts. The nucleus is centrally located, and may have two or more nuclei. After attachment, the cells increase in size, forming colonies and developing protruding protrusions. Some cells adopt triangular or polygonal shapes, with good reflectivity and a strong three-dimensional appearance. After three days of culture and medium exchange, the cells are predominantly spindle-shaped, with a few exhibiting polygonal shapes. Pseudopodia connect the cells, forming clonal clusters. These cells are mostly long, thin spindles, arranged radially in concentric circles, with no contact inhibition. The cells grow in parallel or in a swirling pattern. By seven days of culture, the cell fusion rate can reach over 95%.

[0089] The isolation and culture of umbilical cord-derived stem cells include: taking a fresh umbilical cord from a healthy rabbit, washing it with PBS and then with normal saline to rinse the blood out of the umbilical cord, cutting the umbilical cord into small segments of approximately 0.5 cm with scissors, cutting open each segment of the umbilical cord, removing two arteries and one vein with scissors and forceps, and then peeling out the Wharton's jelly tissue inside with forceps; cutting the Wharton's jelly tissue of the umbilical cord into pieces of 1 mm × 1 mm × 1 mm in size, and then transferring the cut tissue into a culture bottle and spreading it evenly (approximately 2 to 3 pieces / cm 2) and placed in a 37°C, 5% CO2 incubator for approximately 2 hours, inverted. MesenCult MSC Basal Medium was slowly added along the wall of the flask and incubated in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cell confluency exceeded 90%, the cells were digested and passaged using 0.25% trypsin, washed with PBS, and continued to be passaged in MesenCult MSC Basal Medium. The primary umbilical cord tissue explants were cultured for 3 days with the medium changed. During the medium change, some cells were observed emerging from the periphery of the tissue explants. These cells exhibited a small, spindle-shaped morphology, with a small number exhibiting polygonal shapes. After approximately 7 days of culture, cells began to proliferate rapidly, forming colonies of varying sizes. Newly attached cells were rounded and gradually extended pseudopodia, forming cell-cell connections, forming clonal clusters. These cells were mostly slender, spindle-shaped, and distributed radially in concentric circles, without contact inhibition. They grew in parallel or in a swirling pattern. The cell nucleus is centrally located and may have two or more nuclei. The cells have good light reflection and a strong three-dimensional effect. After about 14 days of culture, the cell fusion rate can reach over 90%.

[0090] The isolation and culture of placenta-derived stem cells include: taking the rabbit placenta subamniotic tissue, washing it with PBS and then with normal saline to rinse the blood inside the placenta, and then using surgical scissors and forceps to remove the arterial and venous blood vessels in the placenta; fully chopping the placenta tissue into 1mm×1mm×1mm size, and then transferring the chopped tissue into a culture bottle and spreading it evenly (about 2 to 3 pieces / cm 2 ) and incubated upside down in a 37°C, 5% CO2 incubator for approximately 2 hours. MesenCult MSC Basal Medium stem cell culture medium was slowly added along the wall, and the cells were incubated in a 37°C, 5% CO2 incubator, with the culture medium replaced every three days. Once the cell confluence exceeded 90%, the cells were digested and passaged: 0.25% trypsin was used to digest the cells, washed with PBS, and continued to be passaged in MesenCult MSC Basal Medium stem cell culture medium. After approximately three days of culture, a small number of cells emerged from the periphery of the primary placental tissue block, forming a short, spindle-like shape with a strong three-dimensional effect. Subsequently, the cells slowly extended outward, mostly forming a thin, long spindle-like shape, distributed radially in concentric circles, without contact inhibition. The cells arranged themselves in parallel or grew in a spiral pattern, gradually forming spiral-shaped colonies. As the cell population increased, the tissue blocks gradually detached. After approximately 21 days of culture, only a small amount of tissue blocks remained, and the cells reached approximately 90% confluence.

[0091] The isolation and culture of adipose-derived stem cells include: in the animal experimental operating room, after anesthetizing a healthy New Zealand rabbit by intraperitoneal injection of an appropriate amount of 10% chloral hydrate, the skin is prepared, disinfected, and an appropriate amount of subcutaneous adipose tissue is removed from the groin under aseptic conditions. The adipose tissue is placed in a beaker in the clean bench, and the adipose tissue is washed three times with PBS solution and normal saline solution, and small blood vessels visible to the naked eye are removed to obtain adipose tissue; the adipose tissue is cut into particles of approximately 1mm×1mm×1mm in size with scissors, and then an equal volume of 0.1% type I collagenase is added to the adipose tissue, and the cells are shaken and digested at a constant temperature of 37°C for about 60 minutes; then centrifuged at 3500 rpm for 10 minutes, the upper lipid droplets and supernatant are discarded, and the precipitate is resuspended in MesenCult MSC Basal Medium stem cell culture medium, filtered through a 200-mesh sieve, and the cell suspension is counted using a cell counting plate; 2×10 5 cells / cm 2 The cells were seeded at a density of 100 μg / cm2 in a culture flask and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. After 10-14 days of culture, the cells were digested and passaged when the confluence rate exceeded 90%. The primary cell culture medium was aspirated, and 0.125% trypsin-EDTA digestion solution was added. The digested cells were washed with PBS and continued to be passaged in MesenCult MSC Basal Medium. Primary isolated rabbit adipose-derived mesenchymal stem cells are small, spherical in shape. They begin to adhere to the surface of the culture medium approximately 4 hours after seeding. After approximately 24 hours, the cells begin to extend and grow, forming a short spindle shape with clear cell outlines and a strong three-dimensional sense, growing in parallel or swirling patterns. The cell confluence rate can reach over 90% after 7-10 days. After passage, the cells proliferate rapidly, become evenly distributed, and adopt a fibroblast-like morphology. The cell confluence rate reaches 90% after approximately 6 days before passage.

[0092] In addition, the stem cells used in the present invention are not limited to animal sources. For cartilage damage in a certain type of animal, stem cells from the same type of animal are preferably used. For example, the experimental animals used in some embodiments of the present invention are rabbits, so the source of stem cells is also rabbits.

[0093] In the embodiment of the present invention, the transfection culture medium is OPTI-MEM TM I Reduced Serum Medium was purchased from Shanghai Mituo Biotechnology Co., Ltd.

[0094] In the examples of the present invention, TGFβ was purchased from Promega Corporation of the United States, platelet-rich plasma was purchased from Shanghai Huzheng Biotechnology Co., Ltd., and SOX9 was purchased from Shanghai Huzheng Biotechnology Co., Ltd.

[0095] In the examples of the present invention, type III recombinant collagen was purchased from Shanxi Nanba Biotechnology Co., Ltd.

[0096] In the examples of the present invention, sodium hyaluronate was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0097] In the embodiment of the present invention, the method for isolating and culturing chondrocytes in vitro is as follows: 2-week-old large-eared pure white rabbit knee cartilage is isolated under sterile conditions, 0.1% type II collagenase is added for digestion, the digestion solution is centrifuged and the supernatant is discarded, and DMEM culture medium containing 10% (v / v) calf serum is added to suspend the cells, and 5×10 4 Cells were inoculated into culture flasks for 7 days and cultured when the cell confluence reached 80% to 90%. The cells were subcultured for 3 generations, about 21 days, and then used. DMEM culture medium was purchased from Heilongjiang Jiufeng Bioengineering Co., Ltd.

[0098] In the examples of the present invention, the big-eared pure white rabbits and baby rabbits were purchased from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University.

[0099] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0100] The present invention provides a method for preparing a stem cell gel solution or constructing tissue-engineered cartilage using transgenic stem cells. The transgenic stem cells contain target genes for inducing stem cell transformation into chondrocytes. These target genes may be at least one of BMP7, BMP2, TGFβ, IGF, FGF, and SOX9. BMP7 and / or BMP2 are preferred. The stem cell gel solution or tissue-engineered cartilage construction method comprises the following steps:

[0101] Step 1, constructing a recombinant plasmid containing the target gene;

[0102] Step 2, isolation and culture of bone marrow, umbilical cord, umbilical cord blood, placenta, or adipose-derived stem cells;

[0103] Step 3, using the recombinant plasmid constructed in step 1 to transfect the stem cells cultured in step 2 to obtain stem cells transfected with the target gene;

[0104] Step 4, using active protein to induce differentiation culture of target gene-transfected stem cells; alternatively, differentiating culture of target gene-transfected stem cells;

[0105] Step 5: Prepare the stem cells obtained in step 4 into a stem cell gel solution.

[0106] Step 6: The stem cells obtained in step 4 are constructed into a type III recombinant collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed.

[0107] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. In addition, the following examples are merely illustrative of the present invention and are not intended to limit the present invention.

[0108] Example 1

[0109] This embodiment provides a method for constructing tissue-engineered cartilage using genetically modified umbilical cord blood mesenchymal stem cells, specifically comprising:

[0110] Step 1, constructing a recombinant plasmid containing the target gene BMP7; specifically comprising:

[0111] Step 1-1, double-enzyme digestion of the pBlue-BMP7 plasmid and extraction and purification;

[0112] The pBlue-BMP7 prokaryotic expression vector plasmid was double-digested with Not I and Kpn I. The reaction system was: 5 μl of pBlue-BMP7, 0.5 μl of Not I, 0.5 μl of Kpn I, 2 μl of 10× Buffer, and 12 μl of distilled water, for a total reaction volume of 20 μl. Reaction conditions were: 37°C in a water bath for 2 hours. Separate 20 μl of the digestion reaction product by agarose gel electrophoresis, and observe the separated DNA fragments under UV light. Use a scalpel to excise the approximately 1.3 kb DNA fragment observed under UV light. Extract and purify the BMP7 gene DNA fragment according to the instructions of the gel extraction kit (Shanghai Boya Biotechnology Co., Ltd.). Store in a refrigerator at -20°C until ready for use.

[0113] Step 1-2: Double-enzyme digestion and extraction and purification of the pcDNA3.1 vector;

[0114] The pcDNA3.1(+) eukaryotic expression vector was double-digested with Not I and Kpn I. The reaction system was: 5 μl (approximately 500 ng) of pcDNA3.1(+), 0.5 μl of Not I, 0.5 μl of Kpn I, 2 μl of 10× Buffer, and 12 μl of distilled water, for a total reaction volume of 20 μl. The reaction conditions were: 37°C water bath for 2 hours. After the reaction, 20 μl of the digested product was separated by agarose gel electrophoresis, and the separated DNA fragments were observed under UV light. Using a scalpel, the approximately 5.4 kb DNA fragment observed under UV light was excised. The pcDNA3.1 eukaryotic expression vector DNA fragment was extracted and purified according to the instructions of the gel extraction and purification kit (Shanghai Boya Biotechnology Co., Ltd.) and stored in a refrigerator at -20°C until further use. The digestion site was 912-970, as shown in Figure 1.

[0115] Step 1-3, ligating the enzyme-digested gene fragment obtained in step 1-2 with the enzyme-digested DNA fragment obtained in step 1-3 to obtain the pcDNA3.1-BMP7 vector plasmid;

[0116] Ligate the BMP7 gene fragment extracted and purified in Steps 1-2 with the DNA fragment from the pcDNA3.1 eukaryotic expression vector using T4 DNA ligase. The reaction system is: 6 μl of BMP7, 2 μl of pcDNA3.1, 1 μl of 10× Buffer, 0.5 μl of T4 DNA ligase, and 0.5 μl of distilled water, for a total reaction volume of 10 μl. Reaction conditions: in a 6°C water bath for 8 hours. Store the ligation product at -20°C until needed.

[0117] Steps 1-4: The pcDNA3.1-BMP7 vector plasmid was transformed into DH5a competent cells and amplified. The pcDNA3.1-BMP7 vector plasmid was extracted and purified according to the instructions of the plasmid extraction kit (Shanghai Boya Biotechnology Co., Ltd.), and the target gene BMP7 in the pcDNA3.1-BMP7 vector plasmid was sequenced and identified. Sequencing method: Using the pcDNA3.1-BMP7 eukaryotic expression vector plasmid as a template, two PCR amplification reactions were performed using T3 (5'-TAA TAC GAC TCA CTA TAG GG-3') and T7 (5'-ATT AAC CCT CAC TAA AG-3') sequencing primers. The reaction products were sequenced, and the sequencing results of the above two reactions were spliced ​​using computer software (determined by Shanghai Boya Biotechnology Co., Ltd.). The results showed that the spliced ​​DNA sequence was completely consistent with the full-length sequence of the BMP7 gene reported in the gene bank, and no mutations occurred.

[0118] Step 2, isolation and culture of stem cells; specifically including:

[0119] Step 2-1: Immediately after birth, umbilical cord blood was collected from the rabbit and placed in a sterile tube containing 3.8% sodium citrate anticoagulant. 2-3 ml of the umbilical cord blood was transferred to a 50 ml sterile centrifuge tube containing 10 ml of DMEM culture medium and centrifuged at 300 g for 5 minutes. The DMEM culture medium also contained 10% (v / v) FBS and 150 u / ml heparin.

[0120] Step 2-2: After removing the upper fat layer and part of the supernatant from the centrifuge, add the centrifuge liquid to a Percoll separation solution with a density of 1.073 g / ml at a ratio of 1:2 and centrifuge at 900 g for 30 min.

[0121] Step 2-3: Separate the middle layer of mononuclear cells and wash them sequentially. Add MesenCult MSC Basal Medium stem cell culture medium to suspend the cells. 6 Primary cells were seeded into culture flasks at a density of 100 cells / ml in a 3 ml incubator and cultured in a 5% CO2, 37°C incubator. After 3-4 days of culture, the cells were rinsed three times with PBS, non-adherent cells were discarded, and MesenCult MSC Basal Medium was replaced with stem cell culture medium for continued culture. After approximately 7 days, the primary cells completely covered the bottom of the culture flask, as shown in Figure 2.

[0122] The MesenCult MSC Basal Medium stem cell culture medium also contains fetal bovine serum (FBS) with a final concentration of 10% (v / v) and heparin at 150 u / mL.

[0123] In steps 2-4, the culture medium was digested with 0.25% trypsin solution and 5 × 10 4Cells were seeded at a density of 100 cells / ml in MesenCult MSC Basal Medium. The seeded cells were designated as P1. Cultures were continued for approximately 7 days, with each passage designated as P2 and P3. Twelve hours after inoculation, some primary umbilical cord blood mesenchymal stem cells began to adhere. These newly adhered cells were rounded and gradually extended pseudopodia, forming cell-cell connections. The cells exhibited a spindle- or fusiform fibroblast-like appearance. The nucleus was centrally located, but dual or multiple nuclei may be present. After attachment, the cells increased in size, formed colonies, and developed protruding protrusions. Some cells were triangular or polygonal in shape, exhibiting good reflectivity and a strong three-dimensional appearance. After 3 days of culture and medium exchange, the cells were predominantly spindle-shaped, with a few showing polygonal shapes. Pseudopodia formed cell connections, forming clonal clusters. The cells were mostly long, thin spindles, distributed radially in concentric circles, with no contact inhibition. The cells grew in parallel or in a swirling pattern. After 7 days of culture, the cell fusion rate exceeded 95%. Passage P1 cells formed colonies similar to those formed in primary cultures. Passage P2 cells formed fewer colonies, and from P3 onwards, no colonies formed. With increasing passage number, adherence and adaptability increased, and cells quickly adhered and proliferated after passage. Their morphology became flat, and their reflectivity and three-dimensionality decreased. The results are shown in Figures 3-5.

[0124] Step 3: The recombinant plasmid constructed in step 1 is used to transfect the stem cells cultured in step 2 to obtain target gene-transfected stem cells (BMP7 transfected cell group). At the same time, a non-BMP7 transfected cell group is set up, as follows:

[0125] For BMP7 cell transfection, sterile test tubes A and B were prepared. To tube A, add 250 μl of transfection medium and 1.5 μl of pcDNA3.1-BMP7 plasmid, respectively, and mix thoroughly. To tube B, add 250 μl of transfection medium and 10 μl of Lipofectamin™ 2000 (Gibco), respectively, and mix thoroughly. Incubate for 5 minutes. To form the DNA-liposome complex, combine the solutions in tubes A and B, gently mix thoroughly, and let stand for 20 minutes. Discard the MesenCult MSC Basal Medium culture medium from cells in the P3 exponential growth phase of a six-well plate. Add 500 μl of serum-free high-glucose DMEM and 500 μl of the DNA-liposome complex to each well. Gently mix the six-well plate and incubate in a 5% CO2, 37°C incubator. After 6 hours, discard the serum-free high-glucose DMEM and DNA-liposome complex, and add 2 ml of MesenCult MSC Basal Medium. 24 hours after transfection, replace the medium containing 350 μg / ml G 418Cells were screened using MesenCult MSC Basal Medium, changing the medium every four days. Cell activity and growth were observed under a microscope. Appropriate amounts of the culture medium were collected 7, 14, and 28 days after screening to measure BMP7 expression.

[0126] Non-BMP7 transfected cell group: Follow the method for the BMP7 transfected cell group described above, with the following differences from the BMP7 transfected cell group: Tube A: Add 250 μl of transfection medium and 1.5 μl of pcDNA3.1 plasmid, respectively, and mix well; other steps are the same as those for the BMP7 transfected cell group. Take appropriate amounts of culture medium on days 7, 14, and 28 after screening for BMP7 expression detection.

[0127] Five parallel samples were set for all experimental groups, and the BMP7 expression level was taken as the mean of the measured values ​​of the five parallel samples.

[0128] BMP7 expression was detected by ELISA after 7, 14, and 28 days of culture. The results showed that both the BMP7-transfected and non-BMP7-transfected cell groups continued to grow after 28 days of continuous screening (see Figures 6-1 and 6-2). These cells were transfected with the pcDNA3.1-BMP7 plasmid. BMP7 expression was significantly increased in cells transfected with the BMP7 gene (p < 0.05) (see Table 1), indicating that the transfection was stable.

[0129] Table 1 Average values ​​of BMP7 expression (pg / ml, n=5)

[0130] Step 4: The active protein induces the stem cells transfected with the target gene to differentiate and culture;

[0131] This step also examined the experimental results of different active proteins inducing the differentiation of stem cells transfected with the BMP7 gene into chondrocytes. The specific experimental process is as follows:

[0132] (1) The present invention has found three active proteins: TGFβ, platelet-rich plasma and SOX9, and used TGFβ, platelet-rich plasma and SOX9 to induce the differentiation of stem cells transfected with BMP7 gene into chondrocytes.

[0133] The specific method is as follows: take a 96-well culture plate and add 1×10 5100 μl of stem cells transfected with the pcDNA3.1-BMP7 plasmid at a concentration of 100 μl / ml were placed in a 5% CO2, 37°C incubator and cultured for 24 hours before the culture medium was discarded. Three active proteins were added, each at different concentrations, and diluted with DMEM culture medium, specifically: TGFβ (4 μg / l, 10 μg / l, and 16 μg / l) DMEM culture medium, platelet-rich plasma (0.5%, 1.0%, and 2.0%) DMEM culture medium, and SOX9 (2.5 μg / l, 5 μg / l, and 10 μg / l) DMEM culture medium. Three replicate wells were set for each concentration of each induction factor. Separate wells were set up for chondrocyte control cells (see above for details on chondrocyte isolation and in vitro culture; no BMP7 gene transfection, no induction factors, and only DMEM culture medium), stem cell control wells (stem cells obtained in step 2, no BMP7 gene transfection, no induction factors, and only DMEM culture medium), control wells for stem cells transfected with the BMP7 gene (no active protein added, only DMEM culture medium added), wells for stem cells not transfected with the BMP7 gene and with active protein (stem cells obtained in step 2, no BMP7 gene transfection, only DMEM culture medium containing active protein added), and blank control wells (no cells added, only DMEM culture medium containing active protein added). All culture wells were cultured in an incubator, and culture medium was collected on days 7 (P1), 14 (P2), and 21 (P3) to determine the content of collagen II and glycosaminoglycans. The ELISA method for measuring collagen II and glycosaminoglycans is a conventional method in the field and will not be explained in detail here. The experimental results are shown in Tables 2 and 3, indicating that: ① The TGFβ group, platelet-rich plasma group and SOX9 group can increase the amount of collagen II and glycosaminoglycan secreted by stem cells transfected with the BMP7 gene, which are significantly higher than the control group of stem cells without BMP7 gene transfection plus active protein, the stem cell control group and the control group of stem cells transfected with the BMP7 gene. The medium-dose effect of the TGFβ group and the SOX9 group reached a plateau (reaching the plateau means there was no difference between the medium-dose group and the high-dose group), while the high-dose effect of the platelet-rich plasma group was the strongest; ② The secretion of collagen II and glycosaminoglycans in the control group without BMP7 gene transfection plus active protein increased, which was significantly higher than that in the control group with BMP7 gene transfection and the stem cell control group; ③ The secretion of collagen II and glycosaminoglycans in the control group with BMP7 gene transfection plus active protein increased, which was significantly higher than that in the stem cell control group; ④ The secretion levels of collagen II and glycosaminoglycans in the control group without BMP7 gene transfection plus active protein, the stem cell control group, the control group with BMP7 gene transfection plus active protein, and the group with BMP7 gene transfection plus active protein were all lower than those in the chondrocyte control group.These results indicate that BMP7 protein expressed by BMP7-transfected stem cells can induce chondrogenic differentiation. Furthermore, the addition of TGFβ, platelet-rich plasma, and SOX9 inducer can induce chondrogenic differentiation. The optimal induction concentrations of TGFβ, platelet-rich plasma, and SOX9 were 10.0 μg / L, 2%, and 5.0 μg / L, respectively.

[0134] Table 2 shows the average secretion level of collagen II in each group (ng / ml)

[0135] Table 3 shows the average secretion levels of glycosaminoglycans in each group (μg / ml)

[0136] (2) Based on the results of (1), the results of the experiments on the combined induction of BMP7 gene-transfected stem cells into chondrocytes by TGFβ+platelet-rich plasma, TGFβ+SOX9 and platelet-rich plasma+SOX9 were further investigated.

[0137] The specific method is as follows: take a 96-well culture plate and add 1×10 5100 μl of stem cells transfected with the pcDNA3.1-BMP7 plasmid at a concentration of 100 μl were cultured in a 5% CO2, 37°C incubator for 24 hours. The culture medium was discarded. Three different DMEM-diluted mixed culture media containing TGFβ and platelet-rich plasma, TGFβ and SOX9, and platelet-rich plasma and SOX9 were added. The mixed culture medium of TGFβ and platelet-rich plasma includes three different concentration compositions, specifically: ① The TGFβ concentration in the mixed culture medium is 10.0 μg / l, and the platelet-rich plasma concentration is 2%; ② The TGFβ concentration in the mixed culture medium is 10.0 μg / l, and the platelet-rich plasma concentration is 1%; ③ The TGFβ concentration in the mixed culture medium is 4.0 μg / l (since there is no difference between the medium-dose group and the high-dose group, the effect of the medium-dose group reaches a platform, so the concentration below the medium-dose group is selected for screening, the same below), and the platelet-rich plasma concentration is 2%. The mixed culture medium containing TGFβ and SOX9 consisted of three different concentrations: ① 10.0 μg / L TGFβ and 5.0 μg / L SOX9; ② 10.0 μg / L TGFβ and 2.5 μg / L SOX9; and ③ 4.0 μg / L TGFβ and 5.0 μg / L SOX9. The mixed culture medium containing platelet-rich plasma and SOX9 consisted of three different concentrations: ① 2% platelet-rich plasma and 5.0 μg / L SOX9; ② 2% platelet-rich plasma and 2.5 μg / L SOX9; and ③ 1% platelet-rich plasma and 5.0 μg / L SOX9. Three replicate wells were prepared for each combination of each mixed culture medium. In addition, chondrocyte control wells (same as (1)), stem cell control wells (same as (1)), stem cell control wells containing transfected BMP7 gene wells (same as (1)), stem cell plus active protein wells without transfected BMP7 gene wells (same as (1)) and blank control wells (same as (1)) were set up. All culture wells were cultured in an incubator, and the culture medium was taken to determine the content of collagen II and glycosaminoglycans on the 7th day (P1 generation), 14th day (P2 generation) and 21st day (P3 generation) of culture. The determination method of collagen II and glycosaminoglycans was ELISA. The results are shown in Tables 4 and 5, indicating that: ① TGFβ+platelet-rich plasma group, TGFβ+SOX9 group and platelet-rich plasma+SOX9 group can further increase the secretion of collagen II and glycosaminoglycans by stem cells transfected with BMP7 gene, which are significantly higher than the control group of stem cells without transfected BMP7 gene plus active protein, stem cell group and stem cell group containing transfected BMP7 gene.The effects of the mixed culture medium TGFβ (10.0μg / l) + platelet-rich plasma (1%) and TGFβ (10.0μg / l) + SOX9 (2.5μg / l) reached a plateau, while the concentration of platelet-rich plasma (2%) + SOX9 (5.0μg / l) had the strongest effect; ② The secretion of collagen II and glycosaminoglycans in the control group without BMP7 gene transfection plus active protein increased, which was significantly higher than that in the control group with BMP7 gene transfection and the stem cell control group. The effects of the mixed culture medium TGFβ (10.0 μg / l) + platelet-rich plasma (1%) and TGFβ (10.0 μg / l) + SOX9 (2.5 μg / l) reached a plateau, while the platelet-rich plasma concentration (2%) + SOX9 (5.0 μg / l) had the strongest effect; ③ The secretion of collagen II and glycosaminoglycans in the control group containing the transfected BMP7 gene stem cells increased, significantly higher than that of the stem cell control group; ④ The secretion levels of collagen II and glycosaminoglycans in the stem cell control group, the control group containing the transfected BMP7 gene stem cells, the control group containing the non-transfected BMP7 gene stem cells plus active protein control group, and the group containing the transfected BMP7 gene plus active protein group were all lower than those in the chondrocyte group. From the above results, it can be concluded that the BMP7 protein expressed by the BMP7 gene transfected stem cells of the present invention can induce stem cells to differentiate into chondrocytes, and on this basis, the addition of the mixed induction factors TGFβ + platelet-rich plasma, TGFβ + SOX9, and platelet-rich plasma + SOX9 can all induce stem cells to differentiate into chondrocytes. The optimal induction concentrations of the combination of TGFβ and platelet-rich plasma were 10.0 μg / l and 1%, respectively; the optimal induction concentrations of the combination of TGFβ and SOX9 were 10.0 μg / l and 2.5 μg / l, respectively; the optimal induction concentrations of the combination of platelet-rich plasma and SOX9 were 2 and 5.0 μg / l, respectively.

[0138] Table 4 shows the average secretion level of collagen II in each group (ng / ml)

[0139] Table 5 shows the average secretion levels of glycosaminoglycans in each group (μg / ml)

[0140] (3) Based on the results of (2), we further investigated the use of TGFβ+platelet-rich plasma+SOX9 to induce BMP7 gene-transfected stem cells to differentiate into chondrocytes. The specific method is: take a 96-well culture plate and add 1×10 5100 μl of stem cells transfected with the pcDNA3.1-BMP7 plasmid (100 μl / ml) were cultured in a 5% CO2, 37°C incubator. After 24 hours, the culture medium was discarded. Three mixed culture solutions containing varying concentrations of TGFβ, platelet-rich plasma, and SOX9, diluted in DMEM, were added. The mixed culture solutions were as follows: ① 10.0 μg / l TGFβ, 2% platelet-rich plasma, and 5.0 μg / l SOX9; ② 10.0 μg / l TGFβ, 1% platelet-rich plasma, and 2.5 μg / l SOX9; and ③ 4.0 μg / l TGFβ, 2% platelet-rich plasma, and 5.0 μg / l SOX9. Each mixed culture solution combination was plated in triplicate. In addition, chondrocyte control wells (same as (1)), stem cell control wells (same as (1)), stem cell control wells containing transfected BMP7 gene (same as (1)), stem cell plus active protein wells without transfected BMP7 gene (same as (1)) and blank control wells (same as (1)) were set up. All culture wells were cultured in an incubator, and the culture medium was taken to determine the content of collagen II and glycosaminoglycans on the 7th day (P1 generation), 14th day (P2 generation) and 21st day (P3 generation) of culture. The determination method of collagen II and glycosaminoglycans was ELISA. The results are shown in Tables 6 and 7, indicating that: ① TGFβ+platelet-rich plasma+SOX9 mixed culture can significantly increase the amount of collagen II and glycosaminoglycans secreted by stem cells transfected with BMP7 gene, which are significantly higher than the control group of stem cells without transfected BMP7 gene plus active protein, stem cell group and stem cell group containing transfected BMP7 gene. The effect of the mixed culture medium TGFβ (10.0μg / l) + platelet-rich plasma (1%) + SOX9 (2.5μg / l) reached a plateau, with no difference in secretion level from chondrocytes, and was the best induction combination; ② The secretion of collagen II and glycosaminoglycans in the control group without BMP7 gene transfection plus active protein increased, which was significantly higher than that in the control group with BMP7 gene transfection and the stem cell control group. The effect of the mixed culture medium TGFβ (10.0μg / l) + platelet-rich plasma (1%) + SOX9 (2.5μg / l) reached a platform, slightly lower than the secretion level of chondrocytes, and was the best induction combination; ③ The secretion of collagen II and glycosaminoglycans in the control group containing BMP7 gene transfection increased, which was significantly higher than that in the stem cell group; ④ The collagen II and glycosaminoglycan secretions in the stem cell control group, the control group containing BMP7 gene transfection, the control group without BMP7 gene transfection plus active protein, and the TGFβ (4.0μg / l) + platelet-rich plasma (2%) + SOX9 (5.0μg / l) group containing BMP7 gene transfection plus active protein were all lower than those in the chondrocyte group.These results indicate that BMP7 protein expressed by BMP7-transfected stem cells can induce chondrogenic differentiation. Furthermore, the addition of a mixed induction factor (TGFβ, platelet-rich plasma, and SOX9) significantly induced chondrogenic differentiation. The optimal induction concentrations for the TGFβ, platelet-rich plasma, and SOX9 combination were 10.0 μg / L, 1%, and 2.5 μg / L, respectively.

[0141] Table 6 shows the average secretion level of collagen II in each group (ng / ml)

[0142] Table 7 shows the average secretion level of glycosaminoglycans in each group (μg / ml)

[0143] Therefore, the active protein of the present invention is composed of the following final concentrations of each component in the differentiation culture system: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L.

[0144] Step 5: Inducing the stem cells obtained in step 4 with active proteins at final concentrations of: TGFβ, 10.0 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L, was used to construct a type III recombinant collagen 3D culture scaffold in vitro until tissue-engineered cartilage was formed (target gene transfection + induction factor group and non-target gene transfection + induction factor group). At the same time, a blank type III recombinant collagen 3D scaffold group and a target gene transfection group were set up, specifically including:

[0145] Transfection target gene + induction factor group: 1×10 7 The stem cells (obtained in step 4) transfected with the target gene and induced by active protein were constructed to contain about 2×10 6 The cells were cultured on a 3D collagen scaffold containing type III recombinant cells at 37°C in a 5% CO2 incubator for 12 hours to allow the cells to be evenly distributed within the scaffold space and fully bonded to the scaffold material. The 3D collagen scaffold containing stem cells was transferred from a 12-well culture plate to a 6-well culture plate. 3 ml of DMEM culture medium containing a mixed active protein [final concentration: TGFβ1 (10.0 μg / l) + platelet-rich plasma (1%) + SOX9 (2.5 μg / l)] and 10% fetal bovine serum was added. The cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was replaced every 3 days. The appearance of the cells was observed after 0, 7, and 14 days of culture, and collagen II and glycosaminoglycan secretion were measured by ELISA.

[0146] The non-transfected target gene + induction factor group is different from the transfected target gene + induction factor group in that the number of cells used is 1×107 cells / ml of stem cells that have not been transfected with the target gene and have been induced with active protein (obtained in step 4).

[0147] Blank type III recombinant collagen 3D scaffold group: The blank type III recombinant collagen 3D scaffold without cells prepared in a 12-well culture plate was transferred to a 6-well culture plate and added with DMEM culture medium. The appearance was observed and the secretion of collagen II and glycosaminoglycans was determined by ELISA after 0, 7, and 14 days of culture.

[0148] Transfection of target genome: The difference from the transfection of target gene + induction factor group is that there is no step 4 in which active protein is used to induce the differentiation culture of the target gene-transfected stem cells. Instead, the target gene-transfected stem cells obtained in step 3 are directly used for step 5. The operation is the same as that of the transfection of target gene + induction factor group.

[0149] Contains 2×10 6 The preparation method of type III recombinant collagen 3D culture scaffold for stem cells is as follows: 1 ml of 200 mg / ml type III recombinant collagen solution (solvent is normal saline for injection) and about 1×10 7 Place 0.5 ml of stem cell suspension in a 12-well culture plate; slowly add 0.8 ml of 40 mg / ml fibrinogen solution and mix thoroughly, then add 0.2 ml of 1000 U / ml thrombin; shake the plate and mix thoroughly; after culturing for 12 hours, transfer the plate to a 6-well culture plate and culture for 14 days. 6 The preparation method of blank type III recombinant collagen 3D culture scaffold is basically the same as the above method, except that 1×10 7 Replace 0.5 ml of cells / ml stem cell suspension with 0.5 ml of PBS solution.

[0150] The experimental results showed that the blank type III recombinant collagen 3D scaffold was dull white, lacking cell population (as shown in Figure 7), and secreting no collagen II or glycosaminoglycans. The cartilage in the BMP7 gene-transfected group was white, exhibiting a cartilage-like appearance (as shown in Figure 8), with significantly increased collagen II and glycosaminoglycan secretion. The cartilage in the group not transfected with the target gene plus induction factor was white, exhibiting a cartilage-like appearance (as shown in Figure 9), with significantly higher levels of collagen II and glycosaminoglycan secretion than the group transfected with the target gene plus induction factor. The cartilage in the group transfected with the target gene plus induction factor was white, exhibiting a cartilage-like appearance with a denser structure (as shown in Figure 10), with significantly higher levels of collagen II and glycosaminoglycan secretion than the groups transfected with the target gene and the group not transfected with the target gene plus induction factor. The secretion levels of collagen II and glycosaminoglycans are shown in Tables 8 and 9. Therefore, transfection with the target gene plus induction factor is the optimal method for constructing cartilage tissue.

[0151] Table 8 Average values ​​of collagen II secretion (ng / ml)

[0152] Table 9 Mean values ​​of glycosaminoglycan secretion in each group (μg / ml)

[0153] Example 2

[0154] The tissue engineering cartilage component (BMP7 gene plus mixed induction factor group) was constructed using the technical route of transfecting the BMP7 target gene in Example 1 in combination with the active protein induction factor of the three-component compound (TGFβ (10.0 μg / L) + platelet-rich plasma (1%, v / v) + SOX9 (2.5 μg / L) and constructing the cartilage tissue of the type III recombinant collagen 3D culture scaffold in vitro. The model group, the blank type III recombinant collagen 3D scaffold group, and the BMP7 gene group were set to conduct the following animal comparative experiments:

[0155] Thirty-six healthy, large-eared, pure white male rabbits weighing 20-2.5 kg were randomly divided into four groups, with nine rabbits in each group. The experiments were divided into a model group, a blank type III recombinant collagen 3D scaffold group, a BMP7 gene transfection group, and a BMP7 gene transfection plus mixed induction factor group.

[0156] The cartilage injury model was created by injecting 30 mg / kg of 1% sodium barbital into the rabbit's ear vein. After anesthesia, the rabbit was immobilized on an operating table. An incision was made on the medial side of the patella, and the lateral side of the patella was dislocated to expose the articular cartilage on the patellar surface of the distal femur. A full-thickness cartilage defect with a diameter of 5.0 mm and a depth to the subchondral bone plate was created using an orthopedic manual drill. The defect, with minimal blood exudation, was then flushed with physiological saline to remove tissue debris and blood clots. This created the rabbit cartilage injury model (see Figure 11).

[0157] Model group: rabbit cartilage injury model site was not treated in any way;

[0158] Blank type III recombinant collagen 3D scaffold group: The cartilage injury model site was filled with blank type III recombinant collagen 3D scaffold;

[0159] Transfection of BMP7 genome: The cartilage injury model site is filled with cartilage tissue containing the transfected BMP7 gene and cultured in vitro for 14 days; the detailed method for obtaining cartilage tissue containing the transfected BMP7 gene and cultured in vitro for 14 days is as follows: refer to steps 1-3 of Example 1 to construct a recombinant plasmid and transfect the stem cells in step 2 to obtain cells transfected with BMP7, and then directly construct the cells into a type III recombinant collagen 3D culture scaffold, and construct a type III recombinant collagen 3D culture scaffold in vitro with the same transfection target genome as step 5 in Example 1 to obtain cartilage tissue.

[0160] BMP7 gene transfection plus mixed induction factor group: The cartilage injury model site was filled with cartilage tissue containing transfected BMP7 gene + active protein induction factor and cultured in vitro for 14 days (i.e., the cartilage tissue obtained by the in vitro culture method of the target gene transfection + induction factor group in Example 1).

[0161] After each experimental group was treated separately, the rabbit patella was repositioned, and the knee joint and skin were sutured. After surgery, the rabbits were returned to the breeding cage for breeding. The hind limbs were not fixed after surgery and they were allowed to move freely. Eight weeks after transplantation, the rabbits were euthanized, the joints at the transplanted site were removed, and the cartilage damage repair effect was evaluated. The results 8 weeks after surgery are shown in Figures 12-15 (only one of the effect pictures is given in each experimental group, and the results of the same experimental group are similar), indicating that: in the model group, the bottom of the cartilage defect of the rabbits was dark red, the wound surface was sunken, and the boundary with the normal tissue was clear. The cartilage damage of 9 animals was not repaired, and the model was successful (as shown in Figure 12); the filling of the 3D scaffold group was slightly lower than the normal joint surface, the middle of the graft was white, and the surrounding area was dark red, with a clear boundary with the surrounding normal articular cartilage. The cartilage damage of 9 animals was not repaired, and the 3D scaffold had no repair effect on cartilage damage (as shown in Figure 13); the cartilage defect of the rabbits in the BMP7 genome was filled with a milky white graft, which was dark red in color compared with the normal joint. The repaired tissue was shallow, the joint and articular surface of the repaired tissue were at the same height, without gaps or cracks, and well integrated with the surrounding tissue. Cartilage damage was repaired in all nine animals, and the BMP7 gene transfection group had a significant repair effect on cartilage damage (as shown in Figure 14). In the rabbits in the BMP7 gene transfection plus mixed induction factor group, the cartilage defects were filled with cartilage-like grafts, with little color difference from the normal joint. The repaired tissue joint and articular surface were at the same height, with a boundary between the repaired tissue and the surrounding normal articular cartilage, without gaps or cracks, and fully integrated with the surrounding tissue. Cartilage damage was repaired in all nine animals, and the BMP7 gene transfection plus mixed induction factor group had a significant repair effect on cartilage damage, which was superior to the BMP7 gene transfection group (as shown in Figure 15). These results indicate that the BMP7 gene transfection plus mixed induction factor group can completely repair cartilage defects in rabbits, surpassing the other groups.

[0162] Example 3

[0163] This embodiment provides a method for constructing tissue-engineered cartilage using umbilical cord blood mesenchymal stem cells, which specifically includes:

[0164] Step 1, isolation and culture of stem cells; the specific process is the same as the isolation and culture process of stem cells in Example 1;

[0165] Step 2: Differentiation and culture of stem cells induced by active proteins. The specific process is as follows:

[0166] Step 2-1: Take a 6-well culture plate and add 1×10 52 ml of the third generation stem cells with a concentration of 100 cells / ml were placed in a 5% CO2, 37°C incubator for culture. After culturing for 24 hours, the culture medium was discarded.

[0167] Step 2-2: Add DMEM culture medium containing active proteins (final concentration composition: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L), culture under 5% CO2, 37°C conditions, and culture for three generations, replacing the same culture medium after each generation.

[0168] Step 3, using the stem cells cultured for three generations in Step 2 to construct a type III recombinant collagen 3D culture scaffold in vitro, the specific process is as follows: the construction process of the type III recombinant collagen 3D culture scaffold in Example 1;

[0169] Step 3-1, construct a 2×10 6 Type III recombinant collagen 3D culture scaffold for stem cells: the specific construction process is the same as that in Example 1.

[0170] In step 3-2, the type III recombinant collagen 3D scaffold containing stem cells was transferred from a 12-well culture plate to a 6-well culture plate, and 3 ml of DMEM culture medium containing active protein [final concentration composition: TGFβ1 (10.0 μg / l) + platelet-rich plasma (1%) + SOX9 (2.5 μg / l)] and 10% fetal bovine serum was added. The cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was replaced every 3 days to obtain tissue-engineered cartilage.

[0171] A cartilage injury model (nine rabbits) was obtained using the method described in Example 2. The results are shown in Figure 16. The tissue-engineered cartilage was then implanted at the site of the cartilage injury. The patella was then repositioned, and the knee joint and skin were sutured. Postoperatively, the rabbits were returned to their cages for care. Their hind limbs were not immobilized and allowed to move freely. Eight weeks after transplantation, the rabbits were euthanized, and the joints at the transplanted sites were removed to evaluate the cartilage repair effect.

[0172] Figure 17 shows the repair effect of tissue-engineered cartilage obtained in this example for 8 weeks. It can be seen that the present invention successfully repaired rabbit articular cartilage defects, proving that direct induction of tissue-engineered cartilage by stem cells plus active protein (TGFβ (10.0 μg / L) + platelet-rich plasma (1%, v / v) + SOX9 (2.5 μg / L) can also accelerate the repair of rabbit articular cartilage defects, but the effect is slightly worse than the repair effect of tissue-engineered cartilage induced by transfection of BMP7 combined with active protein.

[0173] Example 4

[0174] Stem cell gel solution (BMP7 gene transfection plus mixed induction factor group) was prepared using the technical route of BMP7 target gene transfection combined with three components (TGFβ (10.0 μg / L) + platelet-rich plasma (1%, v / v) + SOX9 (2.5 μg / L)) active protein induction factor and gel injection in Example 1. Preparation method of stem cell gel solution: The stem cells induced by target gene transfection and active protein induction factor were added to physiological saline to prepare 5×10 6 A cell suspension of 100 cells / ml was prepared. Sodium hyaluronate was then added to a 2.0% albumin saline solution to prepare a sodium hyaluronate gel solution. The sodium hyaluronate and 2% human albumin saline solution were mixed at a volume ratio of 1:30 (m / v). The cell suspension and sodium hyaluronate gel solution were mixed at a volume ratio of 1:1 to obtain a stem cell gel solution. The following comparative experiments were conducted on animals with a model group, a blank gel group, and a BMP7 gene transfection group:

[0175] Thirty-six healthy, large-eared, pure white male rabbits weighing 20-2.5 kg were randomly divided into four groups, with nine rabbits in each group. The experiments were divided into a model group, a blank gel group, a BMP7 gene transfection group, and a BMP7 gene transfection plus mixed induction factor group.

[0176] The method for preparing the cartilage injury model is as follows: the cartilage injury model is obtained by the method of Example 2, and the result is shown in FIG21 .

[0177] Model group: rabbit cartilage injury model site was not treated in any way;

[0178] Blank gel group: The cartilage injury model site was filled with blank gel solution (containing no stem cells, 2.0% albumin saline sodium hyaluronate solution mixed with an equal amount of saline) (as shown in FIG18 ).

[0179] Transfection of BMP7 genome: The cartilage injury model site is filled with a stem cell gel solution containing the transfected BMP7 gene and cultured in vitro for 14 days; the detailed method for obtaining the stem cell gel solution containing the transfected BMP7 gene and cultured in vitro for 14 days is as follows: refer to steps 1-3 of Example 1 to construct a recombinant plasmid and transfect the stem cells of step 2 to obtain cells transfected with BMP7, and then culture the stem cells in vitro for 14 days, collect the stem cells, suspend the cells with physiological saline, and then mix them with equal amounts of 2.0% albumin physiological saline sodium hyaluronate solution to obtain a stem cell gel solution (as shown in Figure 19).

[0180] BMP7 gene transfection + mixed induction factor group: the cartilage injury model site was filled with stem cell gel containing transfected BMP7 gene + active protein induction factor and cultured in vitro for 14 days; the detailed method for obtaining the stem cell gel containing transfected BMP7 gene + active protein induction factor and cultured in vitro for 14 days was as follows: refer to the in vitro culture method of the target gene transfection + induction factor group in Example 1, culture in vitro for 14 days, collect the cells, suspend the cells with physiological saline, and then mix them with equal amounts of 2.0% albumin physiological saline sodium hyaluronate solution to obtain stem cell gel (as shown in Figure 20).

[0181] After each experimental group was treated separately, the rabbit patella was repositioned, and the knee joint and skin were sutured. After surgery, the rabbits were returned to the breeding cage for breeding. The hind limbs were not fixed after surgery and they were allowed to move freely. Eight weeks after transplantation, the rabbits were euthanized, the joints at the transplanted site were removed, and the cartilage damage repair effect was evaluated. The results 8 weeks after surgery are shown in Figures 22-25 (only one of the effect pictures is given for each experimental group, and there is no significant difference in the results of the same experimental group), indicating that: in the model group, the bottom of the cartilage defect of the rabbits was dark red, the wound surface was sunken, and there was no tissue filling. The cartilage damage of the 9 animals was not repaired (as shown in Figure 22); the filling of the blank gel group was lower than the normal joint surface, the middle of the graft was white, and the surrounding area was dark red. The boundary with the surrounding normal articular cartilage was clear. The cartilage damage of the 9 animals was not repaired, and the blank gel group had no repair effect on cartilage damage (as shown in Figure 23); the cartilage defect of the rabbits in the BMP7 genome was filled with a milky white graft, which was lighter in color than the normal joint, and the repair The repaired joint was at the same height as the articular surface, without gaps or cracks, and well integrated with the surrounding tissue. Cartilage damage was repaired in all nine animals, and the BMP7 gene transfection group had a significant repair effect on cartilage damage (as shown in Figure 24). In the rabbits in the BMP7 gene transfection plus mixed induction factor group, the cartilage defects were filled with cartilage-like grafts, with little color difference from the normal joint. The repaired joint was at the same height as the articular surface, with a boundary between the repaired tissue and the surrounding normal articular cartilage, without gaps or cracks, and fully integrated with the surrounding tissue. Cartilage damage was repaired in all nine animals, and the BMP7 gene transfection plus mixed induction factor group had a significant repair effect on cartilage damage, which was superior to the BMP7 gene transfection group (as shown in Figure 25). Based on the above results, it can be concluded that the BMP7 gene transfection plus mixed induction factor group can completely repair cartilage defects in rabbits, which is superior to the other groups.

[0182] Example 5

[0183] This embodiment provides a method for preparing a stem cell gel solution from umbilical cord blood mesenchymal stem cells, which specifically includes:

[0184] Step 1, isolating and culturing stem cells according to the method of Example 3;

[0185] Step 2, performing differentiation culture of stem cells induced by the active protein according to the method of Example 3;

[0186] Step 3: Prepare stem cell gel solution in vitro using the stem cells cultured for three generations in step 2. The specific method is as follows: prepare 5×10 6 10 cells / ml cell suspension, and 2.0% albumin saline solution was used to prepare sodium hyaluronate gel. The feed ratio of sodium hyaluronate and 2% human albumin saline was 1:30 (m / v). The sodium hyaluronate gel was mixed with the cell suspension (1:1, v / v) to prepare the stem cell gel. The final cell number was about 2.5×10 6 The stem cell gel solution was prepared (as shown in FIG26 ).

[0187] A cartilage injury model (nine rabbits) was obtained using the method described in Example 2, with the results shown in Figure 27. The cartilage injury model was then filled with the stem cell gel solution. The rabbit patella was then repositioned, and the knee joint and skin were sutured. Postoperatively, the rabbits were returned to their cages for care, with their hind limbs left unfixed and allowed to move freely. Eight weeks after transplantation, the rabbits were euthanized, and the joints at the transplanted sites were removed for evaluation of cartilage repair.

[0188] Figure 28 shows the effect of tissue-engineered cartilage repair obtained in this example for 8 weeks. It can be seen that the present invention successfully repaired rabbit articular cartilage defects, proving that stem cells directly induced by active protein (TGFβ (10.0 μg / L) + platelet-rich plasma (1%, v / v) + SOX9 (2.5 μg / L) to prepare a stem cell gel solution can also accelerate the repair of rabbit articular cartilage defects, but the effect is slightly worse than the repair effect of the stem cell gel solution obtained by transfection with BMP7 combined with active protein induction.

[0189] Example 6

[0190] This embodiment provides a method for preparing a stem cell gel solution or constructing tissue-engineered cartilage using bone marrow-derived mesenchymal stem cells transfected with the BMP7 target gene, which specifically includes:

[0191] Step 1: construct a recombinant plasmid containing the target gene BMP7; (same as Example 1)

[0192] Step 2, isolating and culturing bone marrow mesenchymal stem cells; specifically, the steps include: puncturing the proximal femur of a rabbit with a bone marrow puncture needle, and obtaining 2-3 ml of bone marrow for isolating and culturing bone marrow mesenchymal stem cells, using the same method as that for isolating and culturing umbilical cord blood mesenchymal stem cells.

[0193] Step 3, using the recombinant plasmid constructed in step 1 to transfect the stem cells cultured in step 2 to obtain stem cells transfected with the target gene; (same as Example 1)

[0194] Step 4: Using active proteins to induce differentiation culture of target gene-transfected stem cells; (same as in Example 1, the active proteins used are composed of the following final concentrations of each component in the differentiation culture system: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L);

[0195] Step 5: Prepare the stem cells obtained in step 4 into a stem cell gel solution (same as in Example 5).

[0196] Step 6: The stem cells obtained in step 4 are used to construct a type III recombinant collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed (same as in Example 1).

[0197] The method of Example 2 was used to obtain a cartilage injury model (a total of 18 rabbits), and the results are shown in Figure 29; the method of Example 4 was used to fill the cartilage injury model site with stem cell gel liquid (a total of 9 rabbits); the method of Example 2 was used to fill the cartilage injury model site with tissue-engineered cartilage cultured with type III recombinant collagen 3D (a total of 9 rabbits).

[0198] Figure 30 shows the repair effect of the stem cell gel solution obtained in this example for 8 weeks, and Figure 31 shows the repair effect of the tissue-engineered cartilage obtained in this example for 8 weeks. It can be seen that the stem cell gel solution prepared in this example and the constructed tissue-engineered cartilage both successfully repaired the rabbit articular cartilage defect. Therefore, stem cells derived from bone marrow and umbilical cord blood can be used for the preparation of the stem cell gel solution and the construction of tissue-engineered cartilage of the present invention.

[0199] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of stem cells or genetically modified stem cells in the preparation of stem cell gel or tissue-engineered cartilage.

2. The use according to claim 1, characterized in that: The stem cells are selected from: umbilical cord blood, umbilical cord, placenta, bone marrow or fat; and / or the genes are selected from: at least one of bone morphogenetic protein (BMP) gene, TGFβ gene and SOX9 gene.

3. The use according to claim 1 or 2, characterized in that: The method of preparing stem cell gel solution or constructing tissue-engineered cartilage using stem cells comprises: Step 1: Isolation and culture of stem cells; Step 2: Using active proteins to induce stem cells to differentiate and culture; Step three, preparing a stem cell gel solution with the stem cells obtained in step two, or constructing a type III collagen 3D culture scaffold in vitro with the stem cells obtained in step two until tissue-engineered cartilage is formed.

4. The use according to claim 1 or 2, characterized in that: A method for preparing a stem cell gel solution or constructing tissue-engineered cartilage using genetically modified stem cells comprises: Step 1, constructing a recombinant plasmid containing the target gene; Step 2, isolation and culture of stem cells; Step 3, using the recombinant plasmid constructed in step 1 to transfect the stem cells cultured in step 2 to obtain stem cells transfected with the target gene; Step 4, using active protein to induce differentiation culture of stem cells transfected with target gene; Step 5: Prepare a stem cell gel solution using the stem cells obtained in step 4, or construct a type III collagen 3D culture scaffold in vitro using the stem cells obtained in step 4 until tissue-engineered cartilage is formed.

5. The use according to claim 4, characterized in that: The step 1 of constructing a recombinant plasmid containing the target gene comprises: Step 1-1, obtain pBlue-target gene plasmid; Step 1-2: Double-digest the pBlue-target gene plasmid and extract and purify it; Steps 1-3: Double-digest the pcDNA3.1 vector and extract and purify it; Step 1-4, ligating the enzyme-digested gene fragment obtained in step 1-2 with the enzyme-digested DNA fragment obtained in step 1-3 to obtain the pcDNA3.1-target gene vector plasmid; Steps 1-5: transform the pcDNA3.1-target gene vector plasmid into DH5a competent cells and amplify it. The amplified product is extracted and purified to obtain the product.

6. The use according to claim 3, characterized in that: In step 2, the active protein is used to induce stem cells to differentiate and culture, including: Active proteins are added to the differentiation culture medium containing stem cells or target gene-transfected stem cells, and cultured at 5% CO2 and 37°C for 3 to 5 generations, with the same differentiation culture medium replaced every generation.

7. The use according to claim 4, characterized in that: In step 4, the active protein is used to induce the stem cells transfected with the target gene to undergo differentiation culture, including: Active proteins are added to the differentiation culture medium containing stem cells or target gene-transfected stem cells, and cultured at 5% CO2 and 37°C for 3 to 5 generations, with the same differentiation culture medium replaced every generation.

8. The use according to claim 6 or 7, characterized in that: The active protein is at least one of the following components: TGFβ, platelet-rich plasma, and SOX9.

9. The use according to claim 8, characterized in that: The active protein consists of TGFβ, platelet-rich plasma and SOX9, and the final concentrations of the components in the differentiation culture system are: TGFβ, 4.0-16.0 μg / L; platelet-rich plasma, 0.5-2.0%, v / v; SOX9, 2.5-10 μg / L.

10. The use according to claim 3, characterized in that: In step 3, the process of preparing stem cell gel solution from stem cells includes: The stem cells were added to normal saline to prepare 0.5×10 7 ~1×10 7 The sodium hyaluronate gel solution was prepared by adding sodium hyaluronate to a saline solution with an albumin concentration of 1.5% to 2.5%. The sodium hyaluronate gel solution was mixed with the cell suspension at a volume ratio of 1:0.5 to 2 to obtain a stem cell gel solution.

11. The use according to claim 4, characterized in that: In step 5, the process of preparing stem cell gel solution from stem cells includes: The stem cells were added to normal saline to prepare 0.5×10 7 ~1×10 7 The sodium hyaluronate gel solution was prepared by adding sodium hyaluronate to a saline solution with an albumin concentration of 1.5% to 2.5%. The sodium hyaluronate gel solution was mixed with the cell suspension at a volume ratio of 1:0.5 to 2 to obtain a stem cell gel solution.

12. The use according to claim 3, characterized in that: In the step 3, the stem cells construct a type III collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed, including: The stem cells obtained in the previous step were prepared into a mixture containing 2.5×10 6 ~5×10 6 A type III collagen 3D culture scaffold containing stem cells is added with a DMEM cell culture medium containing the active protein, and cultured under 5% CO2 and 37°C conditions. The DMEM cell culture medium containing the active protein is replaced every 7 days, and cultured continuously for 20 to 24 days to obtain tissue engineered cartilage.

13. The use according to claim 4, characterized in that: In step 5, the stem cells are used to construct a type III collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed, including: The stem cells obtained in the previous step were prepared into a mixture containing 2.5×10 6 ~5×10 6 A type III collagen 3D culture scaffold containing stem cells is added with a DMEM cell culture medium containing the active protein, and cultured under 5% CO2 and 37°C conditions. The DMEM cell culture medium containing the active protein is replaced every 7 days, and cultured continuously for 20 to 24 days to obtain tissue engineered cartilage.

14. The use according to claim 12 or 13, characterized in that: The content of 2.5×10 6 ~5×10 6 The preparation method of the type III collagen 3D culture scaffold for stem cells is as follows: mixing the type III collagen solution and the stem cell suspension obtained in the previous step, adding a fibrinogen solution, and then adding thrombin, mixing evenly and then standing for 8 to 12 hours, and then culturing at 5% CO2 and 37°C for 14 to 21 days; and / or, The final concentration of the active protein in the DMEM cell culture medium is as follows: TGFβ, 4.0-16.0 μg / L; platelet-rich plasma, 0.5-2.0%, v / v; SOX9, 2.5-10 μg / L; preferably, the final concentration of the active protein in the DMEM cell culture medium is as follows: TGFβ, 10 μg / L; platelet-rich plasma, 1%, v / v; SOX9, 2.5 μg / L.

15. The stem cell gel solution or engineered cartilage obtained by the use according to any one of claims 1 to 14.

16. Use of the stem cell gel solution or engineered cartilage according to claim 15 in preparing bone grafts.

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