A hydrogel loaded with dkk3-overexpressing mscs chondrogenic spheroids and preparation method and application thereof

Abstract

The present application belongs to the technical field of tissue engineering and regenerative medicine and stem cell therapy, and particularly relates to a DKK3-overexpressing MSC chondrogenic sphere-loaded hydrogel, a preparation method and application thereof.The hydrogel comprises: (1) a hydrogel carrier; and (2) DKK3-highly-expressing chondrogenic-induced mesenchymal stromal cells loaded in the hydrogel carrier.The present application constructs a novel cell-material composite system with biological activity and tissue engineering support function, can provide a stable three-dimensional microenvironment for cells, improve cell survival rate and chondrocyte phenotype stability, promote cartilage-related gene expression and extracellular matrix deposition, can effectively inhibit inflammation factor-induced cartilage matrix degradation, promote cartilage tissue regeneration and functional recovery, and shows excellent cartilage protection and repair effect.The present application can be used for the treatment of osteoarthritis, cartilage defects and other degenerative joint diseases, and has good clinical conversion application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of tissue engineering and regenerative medicine and stem cell therapy. Specifically, it relates to a hydrogel loaded with DKK3-overexpressing MSCs to form chondrocytes, its preparation method, and its application. Background Technology

[0002] Articular cartilage is a highly specialized connective tissue covering the surface of joints, mainly composed of chondrocytes and their secreted extracellular matrix. It plays a crucial role in cushioning impact, distributing load, and reducing friction during joint movement. However, because articular cartilage lacks a blood vessel, nerve, and lymphatic system, its nutrition supply relies primarily on synovial fluid diffusion. Therefore, once cartilage defects are caused by trauma, sports injuries, degenerative changes, or disease, effective self-repair is difficult to achieve.

[0003] Cartilage defects are a common clinical problem in orthopedics and sports medicine, caused by a variety of factors including acute trauma, sports injuries, joint instability, osteochondral lesions, and long-term mechanical wear. Untreated cartilage defects can further lead to cartilage degeneration, destruction of osteochondral structure, and decreased joint function, and in severe cases, even progress to osteoarthritis. Therefore, achieving effective regeneration and repair of cartilage tissue has always been an important research direction in regenerative medicine and tissue engineering.

[0004] Currently, clinical treatments for cartilage defects mainly include microfracture surgery, autologous cartilage transplantation, autologous chondrocyte transplantation, and osteochondral transplantation. While these methods can improve patients' symptoms to some extent, they still have drawbacks such as limited donor availability, significant surgical trauma, insufficient mechanical properties of the repaired tissue, and unstable long-term efficacy. Furthermore, the newly formed tissue is often predominantly fibrocartilage, making it difficult to restore the structure and function of natural hyaline cartilage.

[0005] In recent years, tissue-engineered cartilage repair technology based on mesenchymal stromal cells (MSCs) has attracted widespread attention. MSCs have advantages such as wide availability, strong self-renewal capacity, high multi-lineage differentiation potential, and low immunogenicity, making them ideal seed cells for cartilage tissue engineering. However, significant heterogeneity exists among MSCs from different tissue sources, resulting in considerable differences in their cartilage differentiation capacity and repair efficacy. Furthermore, transplanted MSCs are prone to reduced survival rates, unstable cartilage phenotypes, and insufficient repair efficiency in the damaged microenvironment, limiting their clinical application.

[0006] Dickkopf-3 (DKK3), a member of the Dickkopf family, is involved in the regulation of the Wnt / β-catenin signaling pathway, cell proliferation and differentiation, and tissue regeneration. Previous studies have shown that DKK3 is closely related to chondrogenesis and the maintenance of cartilage homeostasis; however, the relationship between DKK3 expression levels and chondrogenesis capacity in MSCs from different sources remains insufficiently elucidated. Furthermore, how to enhance the chondrogenesis capacity of MSCs by regulating DKK3 expression and construct a stable and efficient cartilage repair system using suitable biomaterials remains a key technical challenge in the field of cartilage tissue engineering.

[0007] Therefore, developing engineered MSCs with high cartilage-forming capacity and their biomaterial delivery system is of great theoretical significance and clinical application value for promoting cartilage defect repair and improving the regeneration effect of tissue-engineered cartilage. Summary of the Invention

[0008] The purpose of this invention is to provide a hydrogel loaded with DKK3-overexpressing MSCs to form cartilage spheres, its preparation method, and its application. This allows for the selection of MSC subsets with high cartilage differentiation potential and the acquisition of engineered cells with enhanced cartilage regeneration capabilities through DKK3 gene regulation technology. Furthermore, a cell-biomaterial composite system is constructed using PEG-SM@CD hydrogel to promote cartilage tissue regeneration and treat osteoarthritis, cartilage defects, and other degenerative joint diseases.

[0009] This invention first screened MSCs from different sources to identify a subset with the strongest chondrogenic differentiation capacity, and then, through gene and protein level detection, found that this subset had a high DKK3 expression level. Furthermore, DKK3-overexpressing MSCs were constructed using genetic engineering techniques and subjected to three-dimensional chondrogenic induction culture to obtain engineered cells with enhanced chondrogenic capacity.

[0010] Based on this, the engineered cells were loaded into PEG-SM@CD hydrogel to construct a cell-biomaterial composite system. The hydrogel exhibits good biocompatibility, injectability, self-healing ability, and three-dimensional support, providing a suitable microenvironment for cells and improving cell survival rate and cartilage phenotypic stability.

[0011] Therefore, the present invention provides the following technical solution.

[0012] The first aspect of the present invention provides a hydrogel loaded with DKK3-overexpressing MSCs to form chondrocytes, comprising: (1) Hydrogel carrier; and (2) DKK3 loaded in the hydrogel carrier is highly expressed as chondrogenic mesenchymal matrix cells.

[0013] In a preferred embodiment of the present invention, the hydrogel carrier is formed by a cross-linking reaction of four-armed polyethylene glycol-succinimide ester, four-armed polyethylene glycol-amino ester, and a β-cyclodextrin derivative loaded with cartilage damage repair drugs.

[0014] In a preferred embodiment of the present invention, the mesenchymal stromal cells are derived from any one of human bone marrow mesenchymal stromal cells, adipose mesenchymal stromal cells, umbilical cord mesenchymal stromal cells, or decidual mesenchymal stromal cells. The subtype with the strongest chondrogenic differentiation capacity was obtained through screening, and the expression level of DKK3 protein in the MSCs was significantly higher than that of other MSC subtypes.

[0015] In a preferred embodiment of the present invention, the high expression of DKK3 is achieved through any one of viral vectors, plasmid vectors, transposon systems, or gene editing technologies. After overexpression of the DKK3 gene, its chondrogenic differentiation capacity is further enhanced.

[0016] In a preferred embodiment of the present invention, the DKK3 overexpression chondrogenic mesenchymal stromal cells are obtained by constructing DKK3 overexpression mesenchymal stromal cells through genetic engineering technology, followed by three-dimensional chondrogenic induction culture.

[0017] In a preferred embodiment of the present invention, the final concentration of the β-cyclodextrin derivative loaded with the cartilage repair drug in the hydrogel carrier is 20-50 nM.

[0018] In a preferred embodiment of the present invention, the final concentration of the β-cyclodextrin derivative loaded with the cartilage repair drug in the hydrogel carrier is 30 nM.

[0019] In a preferred embodiment of the present invention, the mass concentrations of the four-armed polyethylene glycol-succinimide ester and the four-armed polyethylene glycol-amino ester in the hydrogel carrier are each 10% to 20% (w / v).

[0020] In a preferred embodiment of the present invention, the mass concentrations of the four-armed polyethylene glycol-succinimide ester and the four-armed polyethylene glycol-amino ester in the hydrogel carrier are each 10% (w / v).

[0021] In a preferred embodiment of the present invention, the concentration of DKK3 highly expressed in the hydrogel carrier to induce chondrocyte mesenchymal matrix cells is 1 × 10⁻⁶. 5 ~1×10 8 cells / mL.

[0022] In a preferred embodiment of the present invention, the concentration of DKK3 highly expressed in the hydrogel carrier to induce chondrocyte mesenchymal matrix cells is 1 × 10⁻⁶. 7cells / mL.

[0023] In a preferred embodiment of the present invention, the hydrogel carrier has a three-dimensional porous network structure with a pore size of 50–200 μm.

[0024] In a preferred embodiment of the present invention, the overexpression of DKK3 into cartilage induces a 2-10 fold increase in DKK3 protein expression, a 1.5-3 fold increase in SOX9 expression, and a 2-4 fold increase in COL2A1 expression in mesenchymal matrix cells.

[0025] A second aspect of the present invention provides a method for preparing the hydrogel as described above, comprising the following steps: (1) DKK3 overexpression vector was introduced into mesenchymal stromal cells using genetic engineering technology, and mesenchymal stromal cells that stably overexpress DKK3 were screened to obtain them. (2) The obtained DKK3-overexpressing mesenchymal stromal cells were subjected to three-dimensional chondrogenic induction culture to obtain DKK3-overexpressing chondrogenic induced mesenchymal stromal cells; (3) Preparation of hydrogel precursor solution; (4) DKK3 overexpression to induce chondrocyte mesenchymal stromal cells were mixed with hydrogel precursor solution and cross-linked to form hydrogel loaded with DKK3 overexpressing MSCs into chondrocytes.

[0026] In a preferred embodiment of the present invention, in step (1), the genetic engineering technology is lentivirus-mediated gene transfection.

[0027] In a preferred embodiment of the present invention, in step (2), the three-dimensional chondrogenic induction culture involves seeding mesenchymal matrix cells between generations P3 and P7 into ultra-low adhesion wells, with a cell seeding amount of not less than 10,000 per well and a total seeding amount of not less than 400,000; after the cells are seeded into spheres, they are induced and cultured in chondrogenic differentiation solution for 21-28 days.

[0028] In a preferred embodiment of the present invention, the cartilage differentiation-related indicators include: Alcian Blue staining to detect glycosaminoglycan deposition, ELISA to detect cartilage formation-related secretory factors COMP and TSP-2, and Western Blot to detect cartilage-related proteins SOX9 and COL2A1.

[0029] In a preferred embodiment of the present invention, step (3) involves the following steps in preparing the hydrogel precursor solution: A cartilage injury repair drug was mixed with a β-cyclodextrin derivative to obtain an inclusion complex solution; A four-armed polyethylene glycol-succinimide ester solution, a four-armed polyethylene glycol-amino solution, and an inclusion complex solution are mixed to form a hydrogel precursor solution.

[0030] In a preferred embodiment of the present invention, the molar ratio of the cartilage damage repair drug to the β-cyclodextrin derivative is 1:1 to 1:3.

[0031] In a preferred embodiment of the present invention, the molar ratio of the cartilage damage repair drug to the β-cyclodextrin derivative is 1:2.

[0032] In a preferred embodiment of the present invention, the mass concentrations of the four-arm polyethylene glycol-succinimide ester solution and the four-arm polyethylene glycol-amino solution are both 10% to 20% (w / v).

[0033] In a preferred embodiment of the present invention, the mass concentration of both the four-arm polyethylene glycol-succinimide ester solution and the four-arm polyethylene glycol-amino solution is 10% (w / v).

[0034] In a preferred embodiment of the present invention, the volume ratio of the four-arm polyethylene glycol-succinimide ester solution to the four-arm polyethylene glycol-amino solution is 1:1.

[0035] In a preferred embodiment of the present invention, in step (4), the loading concentration of DKK3 overexpression to induce chondrocyte mesenchymal stromal cells is 1×10⁻⁶. 5 ~1×10 8 cells / mL.

[0036] A third aspect of the invention provides the use of the hydrogel as described above in the preparation of a drug that promotes bone injury repair.

[0037] In a preferred embodiment of the present invention, the bone injury includes osteoarthritis, traumatic / focal cartilage defects, degenerative cartilage lesions, articular cartilage damage, or other degenerative joint diseases.

[0038] In a preferred embodiment of the present invention, the administration method of the drug includes intra-articular injection, cartilage defect filling, or tissue engineering transplantation.

[0039] The hydrogel of this invention can promote the formation of extracellular matrix in chondrocytes, increase the expression levels of COL2A1 and SOX9, and inhibit the degradation of cartilage matrix mediated by MMP9 and MMP13. It exhibits significant cartilage protection and repair effects in an in vitro IL-1β-induced cartilage injury model and can inhibit IL-1β-induced cartilage matrix degradation. It also improves the inflammatory microenvironment and promotes cartilage tissue regeneration.

[0040] By employing the above technical solution, the present invention has at least the following advantages: (1) This invention is the first to screen out a subpopulation of MSCs with high chondrogenic potential (DMSCs) from MSCs of different tissue sources, and establishes the correlation between the chondrogenic differentiation capacity of MSCs and the expression level of DKK3, providing a new theoretical basis and technical path for the screening of high-functioning MSCs.

[0041] (2) This invention significantly enhances the cartilage differentiation ability of MSCs through DKK3 gene overexpression technology, increases the expression of cartilage-specific genes such as SOX9 and COL2A1, promotes extracellular matrix synthesis and cartilage phenotype maintenance, and effectively solves the problem of low cartilage differentiation efficiency of MSCs.

[0042] (3) The present invention uses a three-dimensional chondrogenic induction culture method to obtain engineered MSCs that stably express cartilage phenotype. Compared with traditional two-dimensional culture, it can better simulate the microenvironment of cartilage development in vivo, thereby improving the cartilage formation ability and therapeutic effect of cells.

[0043] (4) The present invention utilizes PEG-SM@CD injectable hydrogel to construct a cell delivery platform. The hydrogel has good biocompatibility, injectability, self-healing ability, three-dimensional porous structure and suitable degradation performance, which can provide cells with a good three-dimensional microenvironment and improve cell survival rate, colonization ability and tissue repair efficiency.

[0044] (5) In this invention, the small molecule drug SM04690 is loaded in a hydrogel. Through the inhibitory effect of the Wnt signaling pathway of SM04690 and the synergistic effect of DKK3, the cartilage repair capacity of the composite system is further enhanced.

[0045] (6) In the IL-1β-induced cartilage injury model, the cell-hydrogel composite system of the present invention can effectively inhibit the inflammatory response and cartilage matrix degradation (MMP9 / MMP13), promote cartilage regeneration and functional recovery, and show excellent anti-inflammatory and cartilage protective effects.

[0046] (7) This invention realizes the organic combination of "high-functioning seed cell screening + gene enhancement strategy + small molecule drug regulation + biomaterial delivery system", forming a tissue-engineered cartilage construction system with synergistic repair effect.

[0047] (8) The cell-biomaterial composite system constructed in this invention has good biocompatibility, safety and clinical translation potential, and can be widely used in the treatment of osteoarthritis, traumatic cartilage defects, degenerative cartilage lesions and other joint diseases.

[0048] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0049] Figure 1 The results of flow cytometry identification of bone marrow-derived mesenchymal stromal cells (BMMSCs), adipose-derived mesenchymal stromal cells (ADMSCs), umbilical cord-derived mesenchymal stromal cells (UCMSCs), and decidual-derived mesenchymal stromal cells (DMSCs).

[0050] Figure 2 Figure 1 shows the comparison of chondrogenic differentiation capacity of MSCs from different tissue sources after chondrogenic induction. BMMSCs, ADMSCs, UCMSCs, and DMSCs were chondrogenic induction cultures. In Figure 2, A shows Alcian Blue staining to evaluate the deposition of glycosaminoglycans in the chondrogenic matrix of each group. B shows the quantitative analysis results of chondrogenic secretory factors COMP and TSP-2 (TSP-2) detected by enzyme-linked immunosorbent assay (ELISA). C shows the expression levels of chondrogenic proteins SOX9 and type II collagen (COL2A1) detected by Western blotting.

[0051] Figure 3 Figure 1 shows the results of DKK3 expression level detection in MSCs from different tissue sources. A shows the results of DKK3 gene transcription level detection in MSCs from different tissue sources by real-time quantitative PCR (RT-qPCR); B shows the quantitative analysis results of DKK3 protein secretion level in MSCs from different tissue sources by ELISA; and C shows the results of DKK3 protein expression level detection in ADMSCs and DMSCs by Western Blot.

[0052] Figure 4 Figure 1 shows the construction and identification results of DKK3-overexpressing DMSCs; where A shows the fluorescence expression of cells after transfection with the DKK3 overexpression vector; B shows the analysis results of transfection efficiency detected by flow cytometry; C shows the results of DKK3 protein expression level detected by Western Blot; and D shows the results of DKK3 secretion level quantitatively detected by ELISA.

[0053] Figure 5 The image shows the chondrogenic capacity evaluation results of DKK3-overexpressing DMSCs after three-dimensional chondrogenic induction. In the image, A is Alcian Blue staining, used to evaluate the cartilage matrix formation; B is the result of Western Blot detection of the expression levels of cartilage-related proteins SOX9 and COL2A1; and C is the result of ELISA detection of the quantitative analysis of the levels of cartilage formation-related secretory factors COMP and TSP-2.

[0054] Figure 6Figure 1 shows the preparation and performance characterization results of PEG-SM@CD hydrogel; where A is a schematic diagram of the formation process of PEG-SM@CD hydrogel; B is the result of scanning electron microscopy (SEM) observation of the internal microstructure of the hydrogel; C is the result of rheological property testing; D is the result of hydrogel swelling performance analysis; and E is the result of hydrogel degradation performance analysis.

[0055] Figure 7 Figure 1 shows the cell viability and proliferation results after DKK3 overexpressing DMSCs were loaded onto PEG-SM@CD hydrogel; where A represents the cell viability result detected by CCK-8 assay; and B represents the cell proliferation result detected by CCK-8 assay.

[0056] Figure 8 Figure 1 shows the results of establishing an IL-1β-induced cartilage injury model and evaluating its repair effects. In this figure, A shows the results of Western blotting analysis of the expression levels of cartilage-related proteins SOX9 and COL2A1, as well as cartilage matrix degradation-related proteins MMP9 and MMP13; B shows the quantitative analysis results of ELISA analysis of the levels of cartilage formation-related secretory factors COMP and TSP-2; and C shows the results of the detection of inflammatory factor expression levels.

[0057] Figure 9 Figure 1 shows the comparison of cartilage repair effects in different treatment groups: The experimental groups included a blank control group, a hydrogel group, a hydrogel-loaded MSCs group, a hydrogel-loaded three-dimensional chondrogenic MSCs group, and a hydrogel-loaded DKK3-overexpressing three-dimensional chondrogenic MSCs group; A shows the gross observation results of cartilage defect repair in each group; B shows the microCT analysis results, used to evaluate the cartilage defect repair effect and tissue regeneration in each group; C shows the histological scoring results. Detailed Implementation

[0058] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0059] Example 1: Isolation, culture and identification of mesenchymal stromal cells from different tissue sources Human bone marrow, adipose tissue, umbilical cord tissue, and decidual tissue samples were obtained. Bone marrow tissue was used to isolate mononuclear cells using density gradient centrifugation and then cultured in an adherent culture medium. Adipose tissue, umbilical cord tissue, and decidual tissue were cultured using a tissue block adherent method. The resulting cells were seeded in DMEM / F12 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics and cultured at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2–3 days during culture. Cells were passaged when confluence reached 80%–90%. The fifth generation (P5) cells were used for subsequent experiments.

[0060] Phenotypic identification of cells from various sources was performed using flow cytometry, and the results are shown in [Figure number missing]. Figure 1 .

[0061] like Figure 1 The test results showed that all groups of cells highly expressed the mesenchymal stromal cell surface markers CD73, CD90 and CD105, while the expression levels of hematopoietic cell-related markers CD34 and CD45 were low, which met the identification criteria of the International Society for Cell and Gene Therapy (ISCT) for mesenchymal stromal cells, indicating that mesenchymal stromal cells (MSCs) from different tissue sources were successfully obtained.

[0062] Example 2: Screening of chondrogenic differentiation capacity and DKK3 expression level of MSCs from different tissue sources 1. Screening of chondrogenic differentiation capacity of MSCs from different tissue sources To compare the chondrogenic differentiation potential of MSCs from different tissue sources, this embodiment selected human umbilical cord mesenchymal stromal cells (UCMSCs), human bone marrow mesenchymal stromal cells (BMSCs), human adipose-derived mesenchymal stromal cells (ADMSCs), and human decidual mesenchymal stromal cells (DMSCs) for three-dimensional chondrogenic induction culture. The specific steps included: P5 generation MSCs cultured to a confluence of 75%–85% were digested, centrifuged, resuspended, and counted before being seeded into 96-well ultra-low adsorption U-shaped culture plates for three-dimensional chondrogenic induction culture. The specific steps included: firstly, seeding 2 × 10⁶ MSCs into each well... 4Cells were inoculated at a volume of 100–200 μL and cultured for 24–48 h. The cells gradually aggregated to form stable cell spheroids, with one spheroid per well. The culture medium was then replaced with osteogenic induction medium (formula as follows: 1 L of low-glucose MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL ascorbic acid, 1.8 mmol / L potassium dihydrogen phosphate, and 100 nmol / L dexamethasone). The cells were continuously induced and cultured at 37°C in a 5% CO2 incubator for 21 days. The chondrogenic induction medium was replaced every 3 days during the culture period to obtain chondrogenic spheroids.

[0063] After induction culture, the following tests were performed: ① Alcian Blue staining was used to evaluate glycosaminoglycan (GAG) deposition; ② ELISA was used to detect the expression levels of chondrogenesis-related factors COMP and TSP-2 in the culture supernatant; ③ Western Blot was used to detect the expression levels of chondrogenesis-related proteins SOX9 and COL2A1. Results are shown below. Figure 2 .

[0064] like Figure 2 The results showed that the DMSCs group had the highest Alcian Blue staining intensity, significantly higher levels of COMP and TSP-2 secretion than other groups, and significantly increased expression levels of SOX9 and COL2A1; the ADMSCs group had the lowest levels of all indicators. These results indicate that there are significant differences in the chondrogenic differentiation capacity of MSCs from different tissue sources. DMSCs (human decidual mesenchymal stromal cells) have the best chondrogenic differentiation potential, while ADMSCs (human adipose-derived mesenchymal stromal cells) have relatively weaker chondrogenic differentiation capacity.

[0065] 2. Detection of DKK3 expression level and screening of highly chondrogenic differentiated MSCs This embodiment aims to detect the expression level of DKK3 in mesenchymal stromal cells (MSCs) from different tissue sources and analyze its correlation with chondrogenic capacity. Specifically, it includes the following: Cell samples were collected from UCMSCs, BMSCs, ADMSCs, and DMSCs (each group derived from three donors). Total RNA and total protein were extracted and analyzed as follows: ① DKK3 gene transcription level was detected by real-time quantitative PCR (RT-qPCR); ② DKK3 protein secretion level in cell culture supernatant was detected by ELISA; ③ Intracellular DKK3 protein expression level was detected by Western blotting. Results are shown below. Figure 3 .

[0066] like Figure 3The results showed that RT-qPCR revealed significant differences in DKK3 mRNA expression levels among MSCs from different sources. The DMSCs group exhibited the highest DKK3 expression level, significantly higher than the BMMSCs, ADMSCs, and UCMSCs groups (P<0.05). ELISA analysis showed that the DKK3 protein concentration was highest in the culture supernatant of DMSCs, consistent with the RT-qPCR results. Western blot analysis further confirmed that the DKK3 protein expression level in DMSCs was significantly higher than that in ADMSCs.

[0067] The results of the above cartilage differentiation experiments show that human decidual mesenchymal stromal cells (DMSCs) exhibit the strongest cartilage differentiation capacity, including the highest Alcian Blue staining intensity, the highest COMP and TSP-2 secretion levels, and the highest SOX9 and COL2A1 expression levels.

[0068] The results above show that the expression level of DKK3 is closely related to the chondrogenic ability of MSCs, and high expression of DKK3 may be an important molecular basis for the excellent chondrogenic ability of DMSCs.

[0069] Therefore, this invention selects DMSCs as seed cells for subsequent gene modification and cartilage repair research.

[0070] Example 3: Construction of DKK3-overexpressing DMSCs and verification of their chondrogenic differentiation capacity This embodiment aims to construct decidual-derived mesenchymal stromal cells (DMSCs) stably overexpressing DKK3 and evaluate the effect of DKK3 overexpression on the chondrogenesis ability of DMSCs. Specifically, it includes the following: 1. Construction of DKK3 stably overexpressing DMSCs DMSCs cultured to passage P3 were seeded into 6-well plates and cultured. When the cell confluence reached 50%–60%, lentiviral infection was performed. The experiment was divided into a negative control group (EV-DMSC group) and a DKK3 overexpression group (DKK3-DMSC group). The EV-DMSC group was infected with an empty lentiviral vector, while the DKK3-DMSC group was infected with a DKK3 overexpressing lentivirus. The DKK3 overexpressing lentivirus was constructed by Shanghai Jikai Gene Medical Technology Co., Ltd., using the GV492 cloning vector and the Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin packaging vector. The nucleotide sequence of the DKK3 gene is shown in SEQ ID NO. 1.

[0071] SEQ ID NO. 1: To enhance transfection efficiency, 8 μg / mL Polybrene was added during infection, and infection was performed at a multiplicity of infection (MOI) of 100. After 24 h of infection, the culture medium was replaced with fresh medium, and the culture was continued for another 48 h before observing the expression of green fluorescent protein. Results are shown below. Figure 4 AB.

[0072] like Figure 4 The results of AB analysis showed that under bright-field conditions, DMSCs exhibited typical spindle-shaped or elongated spindle-shaped adherent growth, with intact cell morphology, uniform arrangement, and no obvious cell death or morphological abnormalities. Under the FITC fluorescence channel, most cells emitted a strong green fluorescence signal, indicating that DKK3-overexpressing lentivirus could effectively infect DMSCs, and the cells maintained good growth status and morphological characteristics after viral infection. Further flow cytometry analysis revealed that the proportion of green fluorescent protein (GFP) positive cells reached 96.9%, indicating high lentiviral infection efficiency, which can meet the requirements for subsequent functional studies and stable cell line construction.

[0073] Subsequently, selection was performed using 2 μg / mL puromycin for 7–10 days to obtain DMSCs cell lines stably overexpressing DKK3. DKK3 expression was validated using ELISA and Western Blot; results are shown below. Figure 4 CD.

[0074] like Figure 4 The results of CD showed that the expression level of DKK3 protein in the DKK3-DMSC group was significantly higher than that in the EV-DMSC group, with an increase of about 2 to 10 times, indicating that DKK3 stably overexpressing DMSCs were successfully constructed.

[0075] 2. Three-dimensional chondrogenic induction culture of DKK3 stably overexpressing DMSCs Three-dimensional chondrogenic induction culture of DKK3 stably overexpressing DMSCs was performed as described in Part 1 of Example 2.

[0076] After 21 days of induction culture, the following assays were performed: ① Alcian Blue staining was used to detect glycosaminoglycan deposition; ② ELISA was used to detect the expression levels of COMP and TSP-2; ③ Western Blot was used to detect the protein expression levels of SOX9 and COL2A1. Results are shown below. Figure 5 .

[0077] like Figure 5The results showed that, compared with the negative control group (EV-ch-DMSC), the DKK3 overexpression group (DKK3-ch-DMSC) exhibited stronger Alcian Blue staining signal and higher glycosaminoglycan deposition; COMP and TSP-2 secretion levels were significantly increased. p <0.0001); the expression levels of SOX9 and COL2A1 proteins were significantly increased, with SOX9 expression increasing by about 1.5 to 3 times and COL2A1 expression increasing by about 2 to 4 times.

[0078] The results show that DKK3 overexpression can significantly promote the differentiation of DMSCs into chondrocytes, increase the expression levels of cartilage-related genes and proteins, and enhance the cartilage matrix formation capacity, indicating that DKK3 is an important functional molecule regulating the cartilage differentiation of DMSCs.

[0079] Example 4: Preparation and performance characterization of PEG-SM@CD hydrogel This embodiment aims to prepare a PEG-SM@CD hydrogel loaded with the small molecule drug SM04690, and to characterize its microstructure, rheological properties, swelling properties, and degradation properties. Specifically, it includes the following: 4arm-PEG-NHS (four-arm polyethylene glycol-succinimide ester) and 4arm-PEG-NH2 (four-arm polyethylene glycol-amino) powders were weighed separately and prepared into 10% (w / v) precursor solutions using PBS buffer. β-cyclodextrin derivative (CD) derivatives were weighed and prepared into a 100 mg / mL CD solution using PBS buffer. The small molecule drug SM04690 and CD solution were mixed at a molar ratio of 1:2, and stirred at room temperature to obtain an SM@CD inclusion complex solution. Then, equal volumes of 4arm-PEG-NHS and 4arm-PEG-NH2 solutions were taken and added to a final concentration of 30 nM SM@CD solution, and rapidly mixed at room temperature. A stable gel formed within approximately 300 s after mixing, yielding the PEG-SM@CD hydrogel. The gel formation time was recorded using the inverted method.

[0080] (1) The obtained hydrogel was freeze-dried for 48 h, subjected to liquid nitrogen embrittlement, and then sputter-coated with gold. Its microstructure was observed using a scanning electron microscope (SEM). The results are shown in the figure. Figure 6 B.

[0081] like Figure 6 The results of B show that the PEG-SM@CD hydrogel forms a uniform and interconnected three-dimensional porous network structure with a pore size of approximately 50–200 μm, which is conducive to nutrient exchange, metabolite diffusion, and cell adhesion and growth.

[0082] (2) The rheological properties of the hydrogel were tested using a rheometer with a scanning frequency range of 0.1–100 rad / s. The results are shown in [Figure number missing]. Figure 6 C.

[0083] like Figure 6 The results of C show that in the initial stage of the test (0–250 s), the storage modulus (G') was lower than the loss modulus (G''), indicating that the system mainly exhibited viscous behavior and had not yet formed a stable three-dimensional cross-linked network. As the reaction time increased, the storage modulus gradually increased and intersected with the loss modulus at approximately 280–300 s, at which point G'≈G'', indicating that the system had reached the gel point, signifying that the PEG-SM@CD system had completed the transition from a solution state to a gel state. With continued reaction, the storage modulus continued to increase rapidly, while the loss modulus remained relatively stable, and G' was consistently higher than G''. When the reaction proceeded to 600 s, the storage modulus was approximately 330–340 Pa, significantly higher than the loss modulus (approximately 140 Pa), indicating that the hydrogel had formed a stable three-dimensional cross-linked network structure and possessed good elastic mechanical properties and structural stability.

[0084] (3) The swelling test was used to evaluate the liquid absorption performance of the hydrogel. The initial dry weight W0 of the lyophilized hydrogel was recorded. It was then immersed in PBS buffer at 37℃. The wet weight Wt was measured at the preset time point, and the swelling rate was calculated according to the following formula: Swelling rate (%) = (Wt) / (Wt) W0) / W0×100%, the result is shown in [the table]. Figure 6 D.

[0085] like Figure 6 The results showed that the PEG-SM@CD hydrogel reached swelling equilibrium in about 24 hours and had good water absorption and retention capabilities.

[0086] (4) Evaluation of hydrogel degradation performance using in vitro degradation experiments: The prepared PEG-SM@CD hydrogels were placed in PBS buffer and PBS buffer containing proteinase K, respectively, and incubated at 37℃. The remaining mass was measured on days 1, 4, 7, 10, 14, 21, 28, 35, and 42. The results are shown in the figure. Figure 6 E.

[0087] like Figure 6 The results from E showed that the degradation rates of both hydrogels gradually increased with prolonged culture time, with the degradation rate of the proteinase K group being significantly higher than that of the PBS group. At 42 days, the degradation rate of the PBS group was approximately 20%, while that of the proteinase K group was approximately 55%, indicating that the PEG-SM@CD hydrogel possesses good enzyme-responsive degradation characteristics and long-term structural stability.

[0088] The results above show that the PEG-SM@CD hydrogel prepared in this embodiment has good injectability, rapid gelation ability, three-dimensional porous structure, excellent liquid absorption performance and suitable degradation characteristics, and can be used as a cell delivery carrier and repair scaffold material in the field of cartilage tissue engineering.

[0089] Example 5: Construction and biological evaluation of PEG-SM@CD-loaded DKK3-overexpression 3D chondrogenic induced DMSCs This embodiment aims to construct a composite system of PEG-SM@CD-loaded DKK3-overexpressing three-dimensional chondrogenic decidual-derived mesenchymal stromal cells (DKK3-ch-DMSCs) and evaluate its cell compatibility and cell biological properties. Specifically, it includes the following: The DKK3-overexpressing DMSCs obtained in Example 3 were cultured for three-dimensional chondrogenic induction according to the method described in Part 1 of Example 2. After 21 days of induction, DKK3-overexpressing DMSCs formed chondrocytes, abbreviated as DKK3-ch-DMSCs. The DKK3-ch-DMSCs were collected, washed twice with PBS, and resuspended in 10% (w / v) 4arm-PEG-NHS precursor solution, adjusting the final cell concentration to 1×10⁻⁶. 7 cells / mL. Then, an equal volume of 10% (w / v) 4arm-PEG-NH2 solution and 30 nM SM@CD solution (preparation process as in Example 4) were added. After gentle mixing, the mixture was allowed to stand at room temperature for about 5 min to form a PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs (Gel+DKK3-ch-DMSC), denoted as the Gel+DKK3-ch-DM group.

[0090] The cell compatibility of the materials was evaluated using the hydrogel extraction method. The PEG-SM@CD hydrogel prepared in Example 4 was immersed in serum-free DMEM basal medium at an extraction ratio of 200 mg / mL for 24 h at 37 ℃. The supernatant was collected and filtered through a 0.22 μm sterile filter to obtain the PEG-SM@CD hydrogel extraction solution. This extraction solution was added to the DMSCs culture system as the experimental group (PEG-SM@CD group), while the control group consisted of DMSCs culture system containing an equal volume of serum-free DMEM basal medium as the experimental group extraction solution. After culturing for 24 h and 48 h, respectively, the effect of the materials on cell survival was evaluated using the CCK8 assay. The results are shown in [Figure 1]. Figure 7 A.

[0091] like Figure 7 As shown in Figure A, after 24 h of culture, the cell viability of the PEG-SM@CD group was significantly higher than that of the control group. p<0.0001), indicating that PEG-SM@CD hydrogel can maintain a high cell viability and promote early cell growth. After 48 h of culture, there was no significant difference in cell viability between the two groups ( p The result was >0.05, indicating that the PEG-SM@CD hydrogel did not produce significant toxicity to cells and had good cell compatibility.

[0092] The effect of PEG-SM@CD hydrogel on the proliferation of DMSCs was further investigated using the CCK-8 assay. The absorbance (OD450) of each group of cells was measured at 450 nm on days 1, 3, and 5 of culture. The results are shown below. Figure 7 B.

[0093] like Figure 7 As shown in Figure B, the OD values ​​of cells in each group gradually increased with the extension of culture time, indicating that the cells maintained a good proliferative state. Compared with the control group, the OD450 values ​​of the PEG-SM@CD group were significantly increased on days 1, 3, and 5 of culture. p The result (<0.01) indicates that the PEG-SM@CD hydrogel can promote the proliferation activity of DMSCs and provide a suitable growth microenvironment for cells.

[0094] The results show that the PEG-SM@CD hydrogel prepared in this invention can effectively load DKK3-overexpressing 3D chondrogenic DMSCs and provide them with a favorable three-dimensional growth microenvironment. This composite system exhibits excellent cell compatibility, not only avoiding cytotoxicity to DMSCs but also promoting early cell survival and continuous proliferation. This is beneficial for the long-term survival and functional maintenance of the loaded cells within the scaffold material, providing a favorable cellular microenvironment for subsequent cartilage tissue repair.

[0095] Example 6: Evaluation of the repair effect in an IL-1β-induced cartilage injury model This embodiment aims to evaluate the repair effect of the PEG-SM@CD loaded DKK3-ch-DMSCs composite system (Gel+DKK3-ch-DM) on inflammatory cartilage damage.

[0096] An in vitro cartilage injury model was established using the mouse ATDC5 chondrocyte cell line or primary chondrocytes. Cells were seeded in 6-well plates and cultured. When cell confluence reached approximately 80%, IL-1β at a final concentration of 20 ng / mL was added for 48 hours to induce a chondrocyte inflammatory injury model.

[0097] The experimental groups and treatments are as follows: (1) Normal control group (Vehicle): The Transwell non-contact co-culture system was used for the experiment; normal ATDC5 chondrocytes were seeded in the lower chamber and DMEM basal medium was added to the upper chamber for co-culture for 48 h. (2) Model group (IL-1β): The Transwell non-contact co-culture system was used for the experiment; the lower chamber was seeded with IL-1β-induced chondrocytes, and the upper chamber was added with DMEM basal medium and co-cultured for 48 h. (3) Gel group (PEG-SM@CD hydrogel): The experiment was conducted using the Transwell non-contact co-culture system; wherein, IL-1β-induced chondrocytes were seeded in the lower chamber, and 40 μL of PEG-SM@CD hydrogel (prepared in Example 4) and DMEM basal medium were added to the upper chamber, and co-cultured for 48 h; (4) Gel+DM group (PEG-SM@CD hydrogel loaded with DMSCs): The experiment was carried out using the Transwell non-contact co-culture system; wherein, IL-1β-induced chondrocytes were seeded in the lower chamber, and 40 μL of PEG-SM@CD hydrogel loaded with DMSCs (the only difference from the PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs prepared in Example 5 is that the DMSCs were not infected with DKK3 overexpression lentivirus and three-dimensional chondrogenic induction) and DMEM basal medium were added to the upper chamber, and co-cultured for 48 h; (5) Gel+ch-DM group (PEG-SM@CD hydrogel loaded with three-dimensional chondrogenic DMSCs): The experiment was carried out using the Transwell non-contact co-culture system; wherein, IL-1β-induced chondrocytes were seeded in the lower chamber, and 40 μL of PEG-SM@CD hydrogel loaded with three-dimensional chondrogenic DMSCs (the only difference from the PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs prepared in Example 5 is that the DMSCs were not infected with DKK3 overexpression lentivirus) and DMEM basal medium were added to the upper chamber, and co-cultured for 48 h; (6) Gel+DKK3-ch-DM group (PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs): The experiment was carried out using the Transwell non-contact co-culture system; wherein, IL-1β-induced chondrocytes were seeded in the lower chamber, and 40 μL of PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs (prepared in Example 5) and DMEM basal medium were added to the upper chamber, and co-cultured for 48 h.

[0098] The following tests were performed after the culture was completed: (1) Lower chamber ATDC5 chondrocytes were collected, and total protein was extracted for Western blot analysis. Results are shown in […]. Figure 8 A.

[0099] like Figure 8 The results of A showed that, compared with the normal group, the expression of SOX9 and COL2A1 in the model group was significantly reduced, while the expression of MMP9 and MMP13 was significantly increased. Compared with the model group, all treatment groups showed a certain degree of repair effect, among which the Gel+DKK3-ch-DMSC group showed the most significant repair effect, with significant recovery of SOX9 and COL2A1 expression levels and significant reduction of MMP9 and MMP13 expression.

[0100] (2) The levels of COMP and TSP-2 in the culture supernatant were detected by ELISA. The results are shown in the figure. Figure 8 B.

[0101] like Figure 8 The results of B showed that the expression levels of COMP and TSP-2 in the Gel+DKK3-ch-DM group were significantly higher than those in other experimental groups.

[0102] (3) The levels of inflammatory factors TNF-α, IFN-γ, IL-12(p70), and IL-6 were detected. The results are shown in […]. Figure 8 C.

[0103] like Figure 8 The results showed that the Gel+DKK3-ch-DM group had the lowest expression level of inflammatory factors, which was significantly lower than that of the model group and other treatment groups.

[0104] The results show that PEG-SM@CD loaded with DKK3 overexpression in three-dimensional chondrogenic DMSCs can effectively restore chondrocyte phenotype, promote cartilage matrix formation, and inhibit inflammatory response and matrix degradation, thereby exerting excellent cartilage damage repair effects.

[0105] Example 7: Evaluation of Repair Effect in Rabbit Cartilage Defect Model This embodiment aims to evaluate the therapeutic effect of the PEG-SM@CD loaded DKK3-ch-DMSCs composite system (Gel+DKK3-ch-DM) in in vivo cartilage defect repair. Specifically, it includes the following: Male New Zealand white rabbits aged 4–6 months, weighing 2.0–2.5 kg, were selected. After anesthesia, the knee joint was exposed, and a full-thickness cartilage defect model with a diameter of 3 mm and a depth of 2 mm was established in the center of the trochlear groove of the femur.

[0106] Each group contains 6 animals, randomly divided into the following groups: (1) Blank group (Defect only): The defective area was not processed; (2) Gel group: PEG-SM@CD hydrogel was implanted into the defect area (prepared in Example 4); (3) Gel+DM group: PEG-SM@CD hydrogel loaded with DMSCs was implanted into the defect area (the only difference between the PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs prepared in Example 5 is that the DMSCs were not infected with DKK3 overexpression lentivirus and three-dimensional chondrogenic induction was not performed). (4) Gel+ch-DM group: PEG-SM@CD hydrogel loaded with three-dimensional chondrogenic DMSCs was implanted into the defect area (the only difference between this and the PEG-SM@CD hydrogel loaded with DKK3 overexpression three-dimensional chondrogenic DMSCs prepared in Example 5 is that the DMSCs were not infected with DKK3 overexpression lentivirus). (5) Gel+DKK3-ch-DM group: PEG-SM@CD hydrogel loaded with DKK3-overexpressing three-dimensional chondrogenic DMSCs was implanted into the defect area (prepared in Example 5).

[0107] Animals were fed routinely for 6 weeks post-surgery. At the end of the experiment, animals were sacrificed, and distal femoral specimens were collected for gross observation and micro-CT examination. The International Cartilage Repair Society (ICRS) scoring system was used for evaluation. Results are shown below. Figure 9 .

[0108] like Figure 9 The results showed that the defect area in the blank group still had obvious depressions; the Gel group and the Gel+DM group showed partial tissue repair; the defect area in the Gel+ch-DM group was basically covered by repaired tissue. Among them, the defect area in the Gel+DKK3-ch-DM group formed continuous and smooth hyaline cartilage-like tissue, which was tightly integrated with the surrounding normal cartilage, and the tissue structure was close to that of natural cartilage. Micro-CT analysis further confirmed that the Gel+DKK3-ch-DM group had the most complete defect filling and the best cartilage continuity. ICRS scoring results showed that the Gel+DKK3-ch-DM group was significantly better than the other experimental groups (…). p <0.0001).

[0109] The results above show that the PEG-SM@CD hydrogel loaded with DKK3-overexpressing three-dimensional chondrogenic DMSCs prepared in this invention can significantly promote the repair of cartilage defects and the regeneration of cartilage tissue, and has good prospects for clinical application.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A hydrogel loaded with DKK3-overexpressing MSCs to form chondrocytes, characterized in that, include: (1) Hydrogel carrier; as well as (2) DKK3 loaded in the hydrogel carrier is highly expressed as chondrogenic mesenchymal matrix cells.

2. The hydrogel according to claim 1, characterized in that, The hydrogel carrier is formed by a cross-linking reaction of four-arm polyethylene glycol-succinimide ester, four-arm polyethylene glycol-amino ester, and β-cyclodextrin derivative loaded with cartilage damage repair drugs.

3. The hydrogel according to claim 1, characterized in that, The mesenchymal stromal cells are derived from any one of human bone marrow mesenchymal stromal cells, adipose mesenchymal stromal cells, umbilical cord mesenchymal stromal cells, or decidual mesenchymal stromal cells.

4. The hydrogel according to claim 1, characterized in that, The DKK3 high expression is achieved through any one of the following: viral vector, plasmid vector, transposon system, or gene editing technology.

5. The hydrogel according to claim 1, characterized in that, The DKK3 overexpression chondrogenic mesenchymal stromal cells were obtained by constructing DKK3 overexpression mesenchymal stromal cells using genetic engineering technology, followed by three-dimensional chondrogenic induction culture.

6. The hydrogel according to claim 2, characterized in that, The final concentration of the β-cyclodextrin derivative loaded with cartilage repair drugs in the hydrogel carrier is 20–50 nM.

7. The hydrogel according to claim 2, characterized in that, The mass concentrations of the four-arm polyethylene glycol-succinimide ester and the four-arm polyethylene glycol-amino ester in the hydrogel carrier are each 10% to 20%.

8. The hydrogel according to claim 1, characterized in that, The hydrogel carrier loaded with DKK3 highly expressed chondrogenic mesenchymal stromal cells at a concentration of 1×10⁻⁶. 5 ~1×10 8 cells / mL.

9. The hydrogel according to claim 2, characterized in that, The hydrogel carrier has a three-dimensional porous network structure with a pore size of 50–200 μm.

10. The hydrogel according to claim 1, characterized in that, The DKK3 overexpression induced a 2-10 fold increase in DKK3 protein expression, a 1.5-3 fold increase in SOX9 expression, and a 2-4 fold increase in COL2A1 expression in mesenchymal matrix cells.

11. A method for preparing a hydrogel according to any one of claims 1-10, characterized in that, Includes the following steps: (1) DKK3 overexpression vector was introduced into mesenchymal stromal cells using genetic engineering technology, and mesenchymal stromal cells that stably overexpress DKK3 were screened to obtain them. (2) The obtained DKK3-overexpressing mesenchymal stromal cells were subjected to three-dimensional chondrogenic induction culture to obtain DKK3-overexpressing chondrogenic induced mesenchymal stromal cells; (3) Preparation of hydrogel precursor solution; (4) DKK3 overexpression to induce chondrocyte mesenchymal stromal cells were mixed with hydrogel precursor solution and cross-linked to form hydrogel loaded with DKK3 overexpressing MSCs into chondrocytes.

12. The preparation method according to claim 11, characterized in that, In step (1), the genetic engineering technology is lentivirus-mediated gene transfection.

13. The preparation method according to claim 11, characterized in that, In step (2), the three-dimensional chondrogenic induction culture involves seeding mesenchymal matrix cells between generations P3 and P7 into ultra-low adhesion wells, with a cell seeding amount of no less than 10,000 per well and a total seeding amount of no less than 400,000; after the cells form spheroids after plating, they are induced and cultured in chondrogenic differentiation solution for 21-28 days. The cartilage differentiation-related indicators include: Alcian Blue staining to detect glycosaminoglycan deposition, ELISA to detect cartilage-related secretory factors COMP and TSP-2, and Western Blot to detect cartilage-related proteins SOX9 and COL2A1.

14. The preparation method according to claim 11, characterized in that, In step (3), the preparation method of the hydrogel precursor solution includes the following steps: A cartilage injury repair drug was mixed with a β-cyclodextrin derivative to obtain an inclusion complex solution; A four-armed polyethylene glycol-succinimide ester solution, a four-armed polyethylene glycol-amino solution, and an inclusion complex solution are mixed to form a hydrogel precursor solution.

15. The preparation method according to claim 14, characterized in that, The molar ratio of the cartilage damage repair drug to the β-cyclodextrin derivative is 1:1 to 1:

3.

16. The preparation method according to claim 14, characterized in that, The mass concentrations of the four-arm polyethylene glycol-succinimide ester solution and the four-arm polyethylene glycol-amino solution are both 10% to 20% (w / v). The volume ratio of the four-arm polyethylene glycol-succinimide ester solution to the four-arm polyethylene glycol-amino solution is 1:

1.

17. The preparation method according to claim 10, characterized in that, In step (4), the loading concentration of DKK3 overexpression to induce chondrocyte mesenchymal stromal cells is 1×10⁻⁶. 5 ~1×10 8 cells / mL.

18. The use of a hydrogel as described in any one of claims 1-10 in the preparation of a drug for promoting bone injury repair.

19. The application according to claim 18, characterized in that, The bone injury includes osteoarthritis, traumatic / focal cartilage defects, degenerative cartilage lesions, articular cartilage damage, or other degenerative joint diseases.

20. The application according to claim 18, characterized in that, The drug can be administered via intra-articular injection, cartilage defect filling, or tissue engineering transplantation.

Images