A kind of cartilage-like microtissue for treating osteoarthritis and its preparation method and application

Abstract

The application provides a kind of cartilage-like microtissue for treating osteoarthritis and its preparation method and application.The preparation method comprises: mixing mesenchymal stem cells (MSC) with DNA origami to obtain modified cells, mixing the modified cells and culturing to obtain MSC microtissue, and placing the MSC microtissue in a chondrogenic culture medium to obtain cartilage-like microtissue after culture.The application develops a cartilage-like microtissue preparation method based on DNA origami technology, uses DNA origami to realize rapid and controllable assembly of cells, realizes precise control and high uniformity, shortens the preparation period and keeps the cells in an active proliferation state;DNA origami is a highly safe biocompatible material that can be gradually degraded and removed by nucleases, avoiding the risk of residual foreign substances; using chondrogenic culture to initiate the directional differentiation of microtissue makes it have both anti-inflammatory paracrine ability and chondrogenic potential, meeting the needs of clinical treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a cartilage-like microtissue for treating osteoarthritis, its preparation method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with multipotent differentiation potential, widely used in regenerative medicine and cell therapy. Currently, the clinical application of MSCs mainly involves direct injection after expansion through two-dimensional culture. This traditional method first isolates MSCs from donor tissues (such as umbilical cord blood, bone marrow, and adipose tissue), then cultures them in adherent medium containing fetal bovine serum. After multiple passages to obtain a sufficient number of cells, the cells are digested into a single-cell suspension and administered to patients via intravenous or local injection.

[0003] Traditional two-dimensional culture of mesenchymal stem cells has many problems: after long-term culture on the surface of plastic culture containers, cells gradually lose their stem characteristics, resulting in decreased proliferation capacity, weakened differentiation potential, and reduced paracrine function; the retention rate in vivo after single-cell suspension injection is extremely low, with studies showing that more than 90% of cells are cleared within 24-48 hours after injection, making it difficult to exert a sustained therapeutic effect at the site of injury; and the lack of intercellular interactions and three-dimensional microenvironment support severely limits the immunomodulatory function and tissue repair capacity of mesenchymal stem cells.

[0004] To improve the therapeutic effects of mesenchymal stem cells (MSCs), researchers have developed various three-dimensional culture techniques. The hanging drop method involves placing a culture medium containing MSCs onto the lid of a culture dish, allowing gravity to cause the cells to aggregate into spheres at the bottom of the droplet. The low-adhesion culture method uses ultra-low-adhesion culture plates, reducing cell adhesion to the culture surface and promoting cell aggregation. The matrix embedding method involves embedding MSCs in hydrogels, collagen, or other biomaterials to create a three-dimensional culture environment. These methods can, to some extent, maintain the stemness characteristics and biological functions of MSCs. However, existing three-dimensional culture methods also have certain drawbacks. The hanging drop method is cumbersome, each droplet can only form one sphere, making large-scale production difficult, and frequent culture medium changes are required, easily leading to sphere loss or destruction. While the low-adhesion culture method is relatively simple, the sphere formation time is long (3-7 days), and the sphere size is highly inconsistent, with a diameter variation coefficient typically exceeding 20%, making standardized production difficult. The biomaterials used in matrix embedding may hinder direct cell-cell contact and signal transduction, affecting the function of mesenchymal stem cells. At the same time, the degradation products of the materials may cause immune responses, limiting their clinical application.

[0005] Furthermore, existing three-dimensional culture strategies for mesenchymal stem cells (MSCs) still face significant functional limitations in clinical translation. On the one hand, while uninduced MSC microtissues possess strong paracrine activity and can secrete various chondrogenic growth factors and anti-inflammatory factors, their own chondrogenic differentiation capacity is insufficient. After implantation into the cartilage injury site, they often fail to effectively reconstruct cartilage tissue, limiting therapeutic efficacy. On the other hand, fully differentiated cartilage microtissues induced in vitro for extended periods (usually weeks to months) exhibit a well-developed chondrogenic phenotype and extracellular matrix deposition. However, as cells differentiate into mature chondrocytes, their paracrine function significantly weakens, and the secretion levels of chondrogenic growth factors and anti-inflammatory factors decrease dramatically, resulting in insufficient anti-inflammatory and immunomodulatory capabilities. This limits their therapeutic efficacy in inflammatory osteoarthritis settings. Therefore, constructing a three-dimensional cartilage-like microtissue from MSCs that combines high paracrine capacity with cartilage differentiation potential is a core problem urgently needing to be solved in the field of cartilage tissue engineering.

[0006] In summary, the cartilage-like microtissue, while retaining the strong paracrine function of MSCs, has completed the directed initiation of cartilage differentiation. After implantation, it can continue to complete cartilage differentiation in the local microenvironment of the joint, while continuously exerting anti-inflammatory and repair-promoting effects. This is of great significance for the translational application of mesenchymal stem cells in the clinical treatment of osteoarthritis. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a cartilage-like microtissue for treating osteoarthritis, its preparation method, and its application. A method for preparing cartilage-like microtissue based on DNA origami technology has been developed to achieve rapid self-assembly of mesenchymal stem cells, shorten the spheroidization time, construct structurally stable and highly active cartilage-like microtissue, avoid central hypoxic necrosis, and maintain high cell activity and function. Furthermore, a standardized and scalable production process has been developed, providing a new technological platform for the clinical application of mesenchymal stem cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing cartilage-like microtissue for treating osteoarthritis, the method comprising: Mesenchymal stem cells (MSCs) were mixed with DNA origami nucleic acid to obtain modified cells. The modified cells were then cultured to obtain MSC microtissue. The MSC microtissue was placed in a chondrogenic medium for pre-induction of chondrogenic differentiation to initiate MSC cell differentiation toward cartilage. After culture, cartilage-like microtissue was obtained. The DNA origami nucleic acid includes scaffold chains and staple chains, which are assembled into a nanostructure through complementary base pairing. The staple chains include cellular staple chains, connecting staple chains, and fixing staple chains. The structure of the DNA origami nucleic acid is triangular.

[0009] This invention utilizes DNA origami nucleic acid modification of mesenchymal stem cells. The DNA origami nucleic acids can complementarily connect, driving programmed self-assembly of cells through DNA base pairing, shortening the spheroidization time from 3-7 days in traditional methods to 24-48 hours. It precisely controls the size and cell number of cartilage-like microtissues, ensuring batch-to-batch consistency meets clinical application standards. It constructs structurally stable and highly active cartilage-like microtissues (3D microtissues), avoiding central hypoxic necrosis. After assembly, the DNA origami degrades or dissociates from the cell membrane and is removed within approximately 24 hours through the combined action of exogenous / endogenous nucleases and culture medium changes, ensuring that the clinical product contains no (or is below the detection limit) exogenous DNA origami, avoiding long-term residues of unnecessary excipients in the subject's body, reducing immunological and regulatory complexity, and does not involve genome integration.

[0010] It is understood that any chondrogenic induction culture medium commonly used in the art to promote cell differentiation into chondrocytes is applicable to this invention.

[0011] Preferably, the scaffold chain comprises M13mp18 single-stranded DNA.

[0012] Preferably, the nucleic acid sequence of the cell staple chain of the triangular DNA origami nucleic acid includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.12, wherein the 3' end of the cell staple chain is modified with cholesterol, the nucleic acid sequence linking the staple chain includes the sequences shown in SEQ ID NO.13 to SEQ ID NO.24 or SEQ ID NO.25 to SEQ ID NO.36, and the nucleic acid sequence fixing the staple chain includes the sequences shown in SEQ ID NO.37 to SEQ ID NO.220.

[0013] In this invention, the connecting staple chains in the staple chain are divided into two groups (SEQ ID NO.13~SEQ ID NO.24 or SEQ ID NO.25~SEQ ID NO.36). These two groups of chains are complementary through their extended sequences. The DNA origami nucleic acid has one group, which extends from the other side, thus corresponding to the two groups of DNA origami nucleic acids. These two groups of DNA origami nucleic acids can complement each other and pair up. The two groups of DNA origami nucleic acids are used to modify mesenchymal stem cells respectively. When the two modified mesenchymal stem cells are mixed, they can self-assemble.

[0014] Specifically, the two groups of DNA origami nucleic acids can be divided into first DNA origami nucleic acid and second DNA origami nucleic acid. Mesenchymal stem cells are mixed with the first DNA origami nucleic acid and the second DNA origami nucleic acid respectively to obtain two groups of modified cells. The two groups of modified cells are mixed and cultured to obtain MSC microtissue. After chondrocyte differentiation pre-induction, cartilage-like microtissue is obtained.

[0015] Preferably, the method for preparing the DNA origami includes: mixing and incubating the scaffold chain and staple chain to obtain the DNA origami.

[0016] Preferably, the method for preparing the DNA origami nucleic acid includes: mixing the scaffold chain, cell staple chain, connecting staple chain and fixing staple chain with buffer solution and incubating.

[0017] Preferably, the buffer solution contains Tris, acetic acid, EDTA, and magnesium acetate.

[0018] Preferably, the concentration of the scaffold chain is 5~30 nM.

[0019] Preferably, the concentration ratio of the scaffold chain, cell staple chain, connecting staple chain and fixing staple chain is 1:(2~10):(2~10):(2~10).

[0020] Preferably, the incubation procedure includes: incubation at 93~96℃ for 3~6 min, followed by cooling to 20~30℃ at a rate of 0.01~0.02℃ / s.

[0021] Preferably, the chondrogenic induction medium includes a mesenchymal stem cell chondrogenic induction medium, such as MSC chondrogenic induction medium (MdiffCho02), etc.

[0022] Preferably, the cartilage differentiation pre-induction time is 12~72 h, for example, it can be 13, 14, 15, 16, 17, 18, 20, 25, 30, 50, 55, 60, 65, 70 or 71 h.

[0023] Preferably, the mesenchymal stem cells include, but are not limited to, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, or placental mesenchymal stem cells.

[0024] Preferably, the method for preparing the mesenchymal stem cells includes: taking fresh umbilical cord tissue, removing blood vessels and separating Wharton's jelly, culturing it using the tissue adhesion method or the enzyme digestion method, and screening for adherent mesenchymal stem cells.

[0025] Preferably, the concentration of mesenchymal stem cells during the modified cell preparation process is 1×10^ 5 ~5×10^ 6 cells / mL.

[0026] Preferably, the seeding concentration of the modified cells in the cartilage-like microtissue assembly step is 1 × 10^ 4 ~2×10^ 5 cells / mL.

[0027] Preferably, the concentration of the DNA origami nucleic acid is 0.1~50 nM, for example, it can be 0.2, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 17 or 49 nM, etc.

[0028] Preferably, the temperature at which the mesenchymal stem cells are mixed with the DNA origami nucleic acid is 20-30°C, for example, 21, 22, 23, 24, 25, 26, 27, 28 or 29°C, and the time is 20-40 min, for example, 21, 22, 23, 25, 30, 35, 36, 37, 38 or 39 min.

[0029] Preferably, the number of cells in the mixed-modified cells is 1,000 to 20,000.

[0030] In a second aspect, the present invention provides a cartilage-like microtissue for treating osteoarthritis, wherein the cartilage-like microtissue for treating osteoarthritis is prepared by the method for preparing cartilage-like microtissue for treating osteoarthritis described in the first aspect.

[0031] In this invention, the chondrocyte-like microtissue is a three-dimensional microsphere of mesenchymal stem cells that has initiated chondrocyte-directed differentiation. It has the ability to further differentiate into chondrocytes and can secrete high levels of anti-inflammatory factors and various growth factors such as chondrocyte-promoting factors. Thirdly, the present invention provides the use of the cartilage-like microtissue for treating osteoarthritis described in the second aspect in the preparation of a medicament for treating osteoarthritis.

[0032] Preferably, the cartilage-like microtissue is administered via intra-articular injection or local transplantation, allowing it to continue cartilage differentiation in vivo while continuously secreting cartilage growth factors and anti-inflammatory factors to repair damaged cartilage tissue.

[0033] Preferably, the osteoarthritis includes diseases such as knee osteoarthritis or osteoarthritis of the ankle or wrist joints caused by sports injuries.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects: Clinical applications demand high consistency and reproducibility of cell products / tissue-engineered products. The size uniformity of microtissues directly affects their in vivo retention, nutrient / oxygen diffusion, metabolic state, and batch-to-batch quality control. To address these needs, this invention develops a method for preparing cartilage-like microtissues based on DNA origami technology. Utilizing DNA origami with a specific structure, it achieves controllable cell assembly, resulting in precise control and high uniformity. It can form tightly structured mesenchymal stem cell microtissues within 24–48 hours, with highly consistent size and a coefficient of variation (CV) of less than 5%. The cholesterol-oligonucleotide anchoring method is cell membrane-friendly, does not reduce the activity and proliferation capacity of mesenchymal stem cells, maintains stable cell phenotype and function, preserves active proliferation, and maintains multi-lineage differentiation potential. Simultaneously, it does not introduce protein immunogens, facilitating clinical translation. Furthermore, the DNA origami can be progressively degraded by nucleases into physiologically compatible products such as oligonucleotides and nucleotides, which are cleared via intracellular and extracellular pathways, reducing the risk of long-term residue. Furthermore, the cartilage-like microtissues formed after short-term chondrogenic pre-induction have initiated a chondrogenic differentiation process, possessing both the strong anti-inflammatory paracrine capacity of mesenchymal stem cells and the potential to further differentiate into chondrocytes. After implantation into the injury site, they can continue to differentiate into chondrocytes under the continuous induction of the local joint microenvironment, promoting cartilage matrix synthesis and inhibiting inflammatory responses, thus being more conducive to the repair of damaged cartilage tissue. Compared to uninduced MSC microtissues, the cartilage-like microtissues of this invention have a more clearly defined chondrogenic direction; compared to long-term, fully differentiated cartilage microtissues in vitro, they avoid the problem of significantly weakened paracrine function, thereby achieving a better balance between inflammation regulation and cartilage regeneration. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of a triangular DNA origami structure.

[0036] Figure 2 This is a triangular NAC-Linker agarose gel electrophoresis image.

[0037] Figure 3 This is a schematic diagram of the construction of cartilage-like microtissue.

[0038] Figure 4 Bright-field maps of MSC microtissues were constructed with or without NAC-Linker, with a scale bar of 200 μm.

[0039] Figure 5 To construct micro-tissue images of different numbers of MSCs using a triangular NAC-Linker, and to perform live and dead cell staining, the scale bar is 100 μm.

[0040] Figure 6 This is a diagram showing the results of the cartilage-like micro-tissue homogeneity assessment. The scale bar is 100 μm.

[0041] Figure 7 The figure shows the results of detecting the mRNA expression levels of cartilage differentiation-related genes in MSC microtissues before and after 48 h of pre-induction with MSC chondrogenic medium.

[0042] Figure 8 The image shows the H&E and Safranin O staining results of cartilage-like microtissue induced for 42 days. The scale bar is 20 μm.

[0043] Figure 9 The image shows the results of AGG immunofluorescence staining and collagen II and collagen I immunohistochemical staining of chondroid microtissue induced for 42 days. The scale bar is 100 μm.

[0044] Figure 10 The image shows the results of treating a human osteoarthritis cell model with cartilage-like microtissue.

[0045] Figure 11 Image showing the results of Safranin O-Fix Green staining of the knee joint.

[0046] Figure 12 Image showing the results of TUNEL staining of the knee joint, scale bar 100 μm. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0049] This invention designs a method for preparing cartilage-like microtissues based on DNA origami technology. Two nucleic acid connectors with complementary DNA sequences (named NAC-Linker) are used to modify the surface of MSCs, driving programmed self-assembly of cells through DNA base pairing. After assembly, the NAC-Linker is degraded or detached from the cell membrane and removed within approximately 24 hours by exogenous / endogenous nucleases and medium changes, ensuring that the clinically produced MSC microtissues contain no (or are below the detection limit) exogenous NAC-Linker. This strategy avoids long-term residues of unnecessary excipients in the subject's body, reduces immunological and regulatory complexity, and does not involve genome integration. Specific technical solutions include the following aspects: The NAC-Linker was designed and fabricated using DNA origami technology, which includes scaffold chains and staple chains. The scaffold chains and staple chains form a basic origami structure through base pairing hybridization. The staple chains include cellular staple chains (modified with cholesterol at the 5' or 3' end for cell anchoring), connecting staple chains (for structural connection), and fixing staple chains (for structural stability).

[0050] The M13mp18 single-stranded phage DNA (7249 bases) was used as the scaffold strand.

[0051] Cellular staple chain: nucleic acid sequence as shown in SEQ ID NO.1~SEQ ID NO.12 (triangle, 3' end cholesterol).

[0052] Connecting staple chain: nucleic acid sequence such as SEQ ID NO.13~SEQ ID NO.36 (triangle).

[0053] Fixed staple chain: nucleic acid sequence such as SEQ ID NO.37~SEQ ID NO.220 (triangle).

[0054] Using staple chains, scaffolding chains are folded and assembled into origami structures. Furthermore, cellular staple chains and connecting staple chains possess special extension sequences. The cellular staple chains have extension sequences with cholesterol-modified ends, allowing them to extend from one side of the DNA origami nucleic acid and bind to cells. The connecting staple chains are divided into two groups (taking the triangular connecting staple chains with nucleic acid sequences as shown in SEQ ID NO.13~SEQ ID NO.36 as an example, SEQ ID NO.13~SEQ ID NO.24 is one type, and SEQ ID NO.25~SEQ ID NO.36 is the other type). These two groups of chains are complementary through their extension sequences. The DNA origami nucleic acid belongs to one type, extending from the other side. A schematic diagram of the overall structure is shown below. Figure 1 and Figure 2 As shown, this corresponds to two sets of DNA origami nucleic acids (which can be named NAC-Linker A and NAC-Linker B). These two sets of DNA origami nucleic acids are linked by complementary staple chains. Therefore, if these two sets of DNA origami nucleic acids are modified on the cell surface, the cells can aggregate and self-assemble into a three-dimensional structure through the connection between the DNA origami nucleic acids.

[0055] The specific preparation process for DNA origami is as follows: Triangular DNA origami: First, prepare 1× TAE-Mg 2+For the buffer solution (40 mM Tris-acetate, 1 mM EDTA, 12.5 mM magnesium acetate, pH 8.0), add approximately 80% of the target volume of ultrapure water to a clean beaker. Then add Tris, glacial acetic acid, EDTA, and magnesium acetate sequentially, stirring thoroughly until completely dissolved. Adjust the pH to 8.0, transfer to a volumetric flask, and bring to a final volume with ultrapure water. Filter through a 0.22 μm filter for sterilization, then aliquot and store. To prepare the staple chain premix, take 208 staple chains of stock solution (original concentration 100 μM), and pipette 1 μL from each chain into a 1.5 mL enzyme-free centrifuge tube. Gently mix to form the premix. To prepare the reaction system, add 12 μL of M13mp18 single-stranded DNA (0.2 μg / μL), 20.8 μL of premixed staple chain solution (containing only one type of staple chain, thus creating two types of premixed staple chain solution, which can be used to prepare NAC-Linker A and NAC-Linker B respectively), and 67.2 μL of buffer to a final volume of 100 μL, bringing the final concentration of M13mp18 to 10 nM and the final concentration of a single staple chain to 100 nM. The annealing reaction was performed in a PCR instrument, first denaturing at 95℃ for 5 min, then decreasing from 95℃ to 25℃ at a rate of 0.01℃ / s, for a total of approximately 7000 seconds. After the reaction, purification was performed using a 100 kDa ultrafiltration tube to remove unbound short chains, ultimately obtaining high-purity DNA origami structures.

[0056] MSCs were isolated and cultured using umbilical cord tissue. The specific procedure was as follows: 20-30 cm of fresh umbilical cord was collected and rinsed with PBS under aseptic conditions to remove residual blood; the umbilical artery (×2) and umbilical vein (×1) were dissected and removed, Wharton's jelly tissue was separated, and the tissue was cut into 1-3 mm pieces. 3 Small tissue fragments were cultured using the tissue adherence method: The tissue fragments were evenly spread on the bottom of a T75 culture flask, moistened with a small amount of low-glucose DMEM containing 10% fetal bovine serum, and incubated at 37°C and 5% CO2 for 4-6 hours, followed by gentle replenishment of the medium. The medium was changed every 2-3 days thereafter. When the cells reached 80-90% confluence, they were digested with 0.25% trypsin-EDTA and passaged, then expanded at a 1:3 ratio. Cells from passages 3-5 were used for experiments, and flow cytometry identified the phenotype as CD73. + CD90 + CD105 + CD34 - CD45 - CD11b - CD19 - HLA-DR - .

[0057] The MSC surface modification process is simple and efficient: collect MSCs in the logarithmic growth phase and adjust the cell concentration to 1×10⁻⁶. 6cells / mL; take equal amounts of cells and incubate them with 20 nM NAC-Linker A or NAC-Linker B in serum-free medium at 25°C for 30 min; gently mix once every 10 min during the incubation period to promote uniform distribution of NAC-Linker on the cell surface; after incubation, wash twice with medium to remove unbound NAC-Linker.

[0058] Assembly of MSC microtissues was performed in culture plates: NAC-Linker A-modified MSCs and NAC-Linker B-modified MSCs were mixed at a 1:1 ratio, and the total cell concentration was adjusted to 3 × 10⁻⁶. 4 cells / mL; seeded in 96-well culture plates, 100 μL per well (containing 3000 cells); cultured at 37°C and 5% CO2, cells rapidly aggregate through NAC-Linker-mediated DNA hybridization; uniform spheres with a diameter of approximately 200 μm are formed within 24–48 h; the size of MSC micro-tissues can be precisely controlled by adjusting the initial cell number, and 1000–20000 cells can form spheres with a diameter of 100–600 μm.

[0059] The prepared MSC microtissues were transferred into MSC chondrogenic induction medium and cultured statically at 37°C and 5% CO2 for 12–72 h, preferably 48 h, to pre-induce chondrogenic differentiation and obtain cartilage-like microtissues. The obtained cartilage-like microtissues can be used for the clinical treatment of osteoarthritis via intra-articular injection or local transplantation. After administration, the cartilage-like microtissues can further complete directional cartilage differentiation under the continuous induction of the joint microenvironment in vivo. Simultaneously, they inhibit local inflammatory responses and promote cartilage matrix synthesis through paracrine pathways, thereby achieving comprehensive repair of osteoarthritis.

[0060] Example 1 This embodiment describes the synthesis and characterization of DNA origami (NAC-Linker).

[0061] 1. Preparation and characterization of NAC-Linker Preparation of the triangular NAC-Linker: 12 μL of M13mp18 single-stranded DNA (New England Biolabs, 0.2 μg / μL) was used as a scaffold strand and mixed with 208 premixed staple strands for annealing. These 208 staple strands included 12 3' cholesterol-modified cell staple strands (SEQ ID NO. 1-12), 12 linker staple strands (SEQ ID NO. 13-SEQ ID NO. 24 or SEQ ID NO. 25~SEQ ID NO. 36, corresponding to NAC-Linker A or NAC-Linker B respectively), and 184 immobilized staple strands (SEQ ID NO. 37-220). All staple strands were custom-synthesized by Sangon Biotech, with HPLC purification purity >95% and a storage concentration of 100 μM. 1 μL of each staple strand was mixed thoroughly to form a premix. 20.8 μL of this premix was then mixed with 67.2 μL of TAE-Mg... 2+ Mix the buffer solution and 12 μL of M13mp18 to make a final reaction volume of 100 μL. Annealing procedure: denature at 95 °C for 5 min, cool to 25 °C at 0.01 °C / s, and store at 4 °C.

[0062] The triangular NAC-Linker was obtained through a predetermined annealing and purification process. Figure 1 NAC-Linker showed a single band on agarose gel electrophoresis, with a purity >95%. Figure 2 ).

[0063] 2. Isolation, culture, and phenotypic identification of MSCs Collect 20-30 cm of fresh umbilical cord (under sterile conditions and processed within 6 hours), rinse with PBS to remove residual blood; dissect and remove the umbilical artery (×2) and umbilical vein (×1), separate Wharton's jelly tissue, and cut into 1-3 mm pieces. 3 Small tissue fragments were seeded onto the bottom of T75 culture flasks, and 2 mL of low-glucose DMEM containing 10% FBS was added. The flasks were incubated at 37°C with 5% CO2 for 4–6 h to allow the tissue fragments to adhere. The fluid was then gently added to a final volume of 10 mL. The medium was then changed at half volume every 2–3 days. When cells reached 80–90% confluence, they were digested with 0.25% trypsin-EDTA and passaged at a 1:3 ratio. Flow cytometry analysis of P4 cells showed that the positive rates for CD73, CD90, and CD105 were all >95%, while the positive rates for CD34, CD45, CD11b, CD19, and HLA-DR were <2%, consistent with the MSC phenotype.

[0064] Example 2 In this embodiment, DNA origami was used to prepare MSC microtissues, and the controllability of MSC microtissue size was compared with the spheroidization performance of NAC-Linker.

[0065] 1. Collect MSCs in the logarithmic growth phase and incubate them with 20 nM NAC-Linker A or NAC-Linker B at 25°C for 30 min under serum-free conditions (with intermittent gentle mixing). After incubation, wash twice with culture medium to remove unbound linkers, obtaining NAC-Linker A or NAC-Linker B modified cells. Mix A and B cells at a 1:1 ratio and adjust the concentration to 3 × 10⁻⁶ cells / mL. 4 Cells / mL were seeded into 96-well ultra-low adsorption U substrates (100 μL / well). After incubation at 37℃ and 5% CO2 for 24–48 h, homogeneous MSC microstructures were formed. Precise size control could be achieved through initial cell number and A / B surface density. Figure 3 ).

[0066] 2. Using a triangular NAC-Linker, MSC microtissues were prepared under the same culture conditions with initial numbers of 1000, 2000, 5000, 10000, and 20000 MSCs, and cultured for 24 h. Results are shown below. Figure 4 , Figure 5 As shown, all groups mediated by NAC-Linker formed MSC microorganisms with uniform structure and stable morphology within 24 h, and the size of the spheroids increased controllably with the increase of the initial cell number. As a control, cells seeded at the same cell concentration (3000 cells / well) in 96-well ultra-low adsorption U plates without the addition of NAC-Linker failed to form uniform spherical structures after 24 h of culture under the same conditions; the cells were scattered at the bottom of the culture wells and failed to aggregate into spheres. Figure 4 ).

[0067] Example 3 This embodiment assesses the consistency and activity of MSC microtissues.

[0068] MSC microtissue samples cultured for 48 h were collected and stained for live / dead tissue using Calcein-AM (2 μM) and PI (1 μM), and incubated at 37°C in the dark for 30 min. Figure 5 As shown, fluorescence microscopy revealed that the NAC-Linker-mediated MSC microtissue exhibited uniform green fluorescence (live cells), with only occasional red fluorescent spots (dead cells) visible in the outermost layer. The diameter was measured and photographed under an inverted microscope. Figure 6As shown, the 100 MSC micro-tissues formed by the NAC-Linker group exhibited a concentrated size distribution, with a diameter of 204.0 ± 9.5 μm and a coefficient of variation of only 4.7%. This indicates that the NAC-Linker technology can achieve highly consistent preparation of MSC micro-tissues. The coefficient of variation (CV) is a statistical indicator used to characterize the dispersion of cartilage-like micro-tissue diameters, defined as: CV = (standard deviation of diameter / average diameter) × 100%. A smaller CV indicates a more concentrated diameter and better size uniformity of the cartilage-like micro-tissues. The CV described in this invention is based on statistical analysis of cartilage-like micro-tissue diameters.

[0069] Example 4 In this embodiment, in vitro chondrogenesis pre-induction of MSC microtissue was performed to initiate MSC cell-directed differentiation toward cartilage and to further verify the long-term chondrogenesis potential of cartilage-like microtissue.

[0070] 1. Chondrocyte differentiation pre-induction (48 h) and qRT-PCR detection The MSC microtissues obtained in Example 2 or Example 3 were replaced with MSC chondrogenic medium (MdiffCho02) and cultured at 37℃ and 5% CO2 for 48 h to complete chondrogenic pre-induction and obtain chondrogenic microtissues. Real-time quantitative PCR (qRT-PCR) was used to detect changes in the mRNA expression of chondrogenic differentiation-related genes before and after pre-induction. Total RNA was extracted from each group and reverse transcribed into cDNA. Using GAPDH as an internal reference gene, the mRNA expression levels of the major regulatory transcription factors of chondrogenic differentiation, SOX9, type II collagen (COL2A1), aggrecan / ACAN, and COL11A1, were detected. The results are as follows: Figure 7 As shown, after 48 h of pre-induction with MSC chondrogenic induction medium, the mRNA expression levels of ACAN, COL2A1, COL11A1, and SOX9 were significantly increased, with SOX9 showing the most significant upregulation, indicating that the chondrogenic differentiation-related gene program had been successfully initiated. These results demonstrate that 48 h of pre-induction can effectively activate the chondrogenic differentiation pathway of MSCs, and the resulting cartilage-like microtissues are in a state of chondrogenic differentiation initiation, suitable for direct intra-articular injection or local transplantation, and can continue to complete directed cartilage differentiation in the in vivo joint microenvironment.

[0071] 2. Long-term in vitro culture to verify chondrocyte differentiation potential To further verify the chondrogenic differentiation capacity of MSC microtissues, the aforementioned cartilage-like microtissues were cultured long-term in MSC chondrogenic induction medium. Samples were collected on day 42 for histological and related staining analyses. H&E and safranin O staining results are shown below. Figure 8As shown, after long-term induction, the cell morphology changed significantly, with increased cell volume, abundant cytoplasm, more compact arrangement, smooth edges, and a clear distinction between the inner and outer layers. No necrosis was observed inside the spheroids, indicating successful terminal differentiation towards cartilage. Safranin O staining revealed abundant orange-red glycosaminoglycans (the main component of cartilage matrix) evenly distributed throughout the spheroids, confirming a good cartilage differentiation effect. AGG, collagen II, and collagen I staining results are shown below. Figure 9 As shown, AGG and collagen II both showed significant positive expression, while collagen I staining was only observed in the outermost cells of the microtissue, suggesting that the formed tissue has relatively typical chondrogenic matrix characteristics and tends to differentiate into hyaline cartilage-like structures. These results confirm that the chondrogenic microtissue, after 48 hours of pre-induction, possesses excellent chondrogenic differentiation potential and can further differentiate into mature chondrogenic tissue under continuous induction conditions.

[0072] Example 5 This embodiment tests the therapeutic effect of cartilage-like microtissue on a human osteoarthritis model in vitro.

[0073] An in vitro model of human osteoarthritis (OA) inflammation was established to evaluate the therapeutic effect of cartilage-like microtissues. Specifically, human chondrocytes were seeded at a density of 1.5 × 10^5 cells / mL in the lower chamber of a six-well plate, with 2 mL seeded per well. After 24 h of cell adhesion, the following treatments were administered: Control group: cultured in conventional cartilage medium for 48 h; OA model group: cultured in cartilage medium containing IL-1β (10 ng / mL) for 48 h; Cartilage-like microtissue treatment group: in addition to the OA model, 60 cartilage-like microtissues (each containing approximately 5000 cells) were added to the upper chamber and cultured for 48 h. After culture, the culture supernatant was collected from each group, and the levels of IL-6, IL-8, and TNF-α were detected by ELISA. Three biological replicates were performed for each group. The results showed that (…). Figure 10 Compared with the OA model group, the cartilage-like microtissue treatment group significantly reduced the secretion levels of IL-6, IL-8 and TNF-α, suggesting that it has a certain anti-inflammatory effect.

[0074] Example 6 This embodiment tests the in vivo efficacy of cartilage-like micro-tissue in the treatment of knee osteoarthritis.

[0075] 1. Establishment of a rat model of osteoarthritis (OA) Anesthesia was induced by intraperitoneal injection of 10% chloral hydrate. The hair around the right knee joint of SD rats was shaved, and the area was cleaned and disinfected with povidone-iodine and 75% ethanol. The skin and muscles around the knee joint were carefully separated, the anterior cruciate ligament was removed, and a partial meniscus resection was performed (partial meniscectomy). After cleaning the treated area, the wound was sutured, and the rat was injected with penicillin to reduce the risk of infection.

[0076] The blank control group only separated skin and muscle without damaging joint structures. One week after surgery, the rats underwent 3 weeks of daily 1-hour treadmill training. Treatment interventions began after training: the blank control group received no further treatment; the osteoarthritis (OA) group received an intra-articular injection of 100 μL of saline; and the cartilage-like microtissue group (50 cartilage-like microtissues / joint with 50 cells each) received a single intra-articular injection of the corresponding therapeutic drug. All rats were evaluated 4 weeks after treatment.

[0077] 2. Histological analysis: Knee joint specimens were stored in 4% paraformaldehyde solution for 3 days, followed by decalcification in formalin-EDTA solution for 6 weeks. After decalcification, the specimens were dehydrated with ethanol of varying concentrations, then embedded in paraffin and cut into thin sections. The sections were stained with Safranin-O-Fast Green and TUNEL staining, respectively.

[0078] Histological evaluation of the knee joint was performed 4 weeks after treatment. Safranin O-Fix Green staining and TUNEL staining results are as follows: Figure 11 , Figure 12 As shown, the OA model group showed significant cartilage surface damage, a decrease in safranin O positive areas, and an increase in TUNEL positive cells; in contrast, the cartilage damage was significantly reduced in the cartilage microtissue treatment group.

[0079] In summary, this invention provides a method for preparing cartilage-like microtissues based on DNA origami technology. Regarding the speed of 3D microtissue construction, it can form tightly structured MSC microtissues within 24-48 hours, greatly improving production efficiency. In terms of product uniformity, the prepared MSC microtissues exhibit highly consistent dimensions with a diameter variation coefficient of less than 5%. Functional evaluation shows that the MSC microtissues maintain excellent biological characteristics, remain in an active proliferative state, and have well-maintained differentiation potential. Under appropriate induction conditions, they can efficiently initiate cartilage-directed differentiation. Furthermore, in an in vitro human osteoarthritis (OA) model, the cartilage-like microtissues can significantly downregulate inflammatory factors such as IL-6, IL-8, and TNF-α, exhibiting superior anti-inflammatory and therapeutic capabilities. The therapeutic effect in a rat model of osteoarthritis fully demonstrates the clinical application potential of the cartilage-like microtissues. Four weeks after intra-articular injection of the cartilage-like microtissues, Safranin O staining showed a significant increase in cartilage matrix and restoration of surface smoothness.

[0080] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing cartilage-like microtissue for treating osteoarthritis, characterized in that, The preparation method includes: Mesenchymal stem cells were mixed with DNA origami nucleic acid to obtain modified cells. The modified cells were then mixed and cultured to obtain mesenchymal stem cell microtissue. The mesenchymal stem cell microtissue was placed in a chondrogenic medium for chondrogenic pre-induction to initiate the directional differentiation of mesenchymal stem cells toward cartilage. After culture, cartilage-like microtissue was obtained. The DNA origami nucleic acid comprises scaffold chains and staple chains, which are assembled into a nanostructure through complementary base pairing; the staple chains include cellular staple chains, connecting staple chains, and fixing staple chains; the structure of the DNA origami nucleic acid is a triangular structure.

2. The method for preparing cartilage-like microtissue for treating osteoarthritis according to claim 1, characterized in that, The nucleic acid sequences of the triangular DNA origami nucleic acid cell staple chains include the sequences shown in SEQ ID NO.1 to SEQ ID NO.12, wherein the 3' end of the cell staple chain is modified with cholesterol, the nucleic acid sequences linking the staple chains include the sequences shown in SEQ ID NO.13 to SEQ ID NO.24 or SEQ ID NO.25 to SEQ ID NO.36, and the nucleic acid sequences fixing the staple chains include the sequences shown in SEQ ID NO.37 to SEQ ID NO.

220.

3. The method for preparing cartilage-like microtissue for treating osteoarthritis according to claim 1 or 2, characterized in that, The method for preparing the DNA origami includes: mixing and incubating the scaffolding chain and staple chain to obtain the DNA origami.

4. The method for preparing cartilage-like microtissue for treating osteoarthritis according to claim 3, characterized in that, The method for preparing the DNA origami nucleic acid includes: mixing the scaffold chain, cell staple chain, connecting staple chain and fixing staple chain with buffer solution and incubating. Preferably, the buffer solution contains Tris, acetic acid, EDTA, and magnesium acetate; Preferably, the concentration of the scaffold chain is 5~30 nM; Preferably, the concentration ratio of the scaffold chain, the cell staple chain, the connecting staple chain, and the fixing staple chain is 1:(2~10):(2~10):(2~10); Preferably, the incubation procedure includes: incubation at 93~96℃ for 3~6 min, followed by cooling to 20~30℃ at a rate of 0.01~0.02℃ / s.

5. The method for preparing cartilage-like microtissue for treating osteoarthritis according to any one of claims 1-4, characterized in that, The chondrogenic induction medium includes a mesenchymal stem cell chondrogenic induction medium. Preferably, the cartilage differentiation pre-induction time is 12~72 h.

6. The method for preparing cartilage-like microtissue for treating osteoarthritis according to any one of claims 1-5, characterized in that, The method for preparing mesenchymal stem cells includes: taking umbilical cord tissue to isolate, culture, and screen mesenchymal stem cells.

7. The method for preparing cartilage-like microtissue for treating osteoarthritis according to any one of claims 1-6, characterized in that, The concentration of the mesenchymal stem cells was 1×10⁻⁶. 4 ~2×10 5 cells / mL; Preferably, the concentration of the DNA origami nucleic acid is 0.1~50 nM; Preferably, the temperature for mixing the mesenchymal stem cells with the DNA origami nucleic acid is 20-30°C, and the time is 20-40 min. Preferably, the concentration of mesenchymal stem cells during the modified cell preparation process is 1×10^ 5 ~5×10^ 6 cells / mL; Preferably, the seeding concentration of the modified cells in the cartilage-like microtissue assembly step is 1 × 10^ 4 ~2×10^ 5 cells / mL; Preferably, the cartilage-like microtissue contains 1,000 to 20,000 cells.

8. A cartilage-like microtissue for treating osteoarthritis, characterized in that, The cartilage-like microtissue for treating osteoarthritis is prepared by the method for preparing cartilage-like microtissue for treating osteoarthritis according to any one of claims 1-7.

9. The use of the cartilage-like microtissue for treating osteoarthritis as described in claim 8 in the preparation of a medicament for treating osteoarthritis; Preferably, the cartilage-like microtissue is administered via intra-articular injection or local transplantation, and continues to complete the directed differentiation of cartilage in vivo, while continuously secreting cartilage growth factors and anti-inflammatory factors to repair damaged cartilage tissue; Preferably, the osteoarthritis includes knee osteoarthritis or osteoarthritis of the ankle or wrist joint caused by sports injury.

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