Cell gel formulations that reduce cell shear damage and methods for reducing cell shear damage

Abstract

The present invention relates to a cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells for the treatment and / or prevention of acute or chronic osteoarthritis and / or symptoms thereof by intra-articular injection; a method for treating or preventing acute and chronic osteoarthritis and associated inflammation-derived symptoms (particularly bone joint pain, reduced mobility or function) using said cell gel formulation; and a method for reducing shear damage to cells in a shear environment using said nucleic acid hydrogel.

Description

[Technical field]

[0001] The present invention relates to a cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells for the treatment and / or prevention of acute or chronic osteoarthritis and / or symptoms thereof by intra-articular injection; a method for treating or preventing acute and chronic osteoarthritis and associated inflammation-derived symptoms (particularly bone joint pain, reduced mobility or function) using said cell gel formulation; and a method for reducing shear damage to cells in a shear environment using said nucleic acid hydrogel. [Background technology]

[0002] Osteoarthritis (OA) is a chronic disease characterized by degenerative changes in articular cartilage and secondary bone hyperplasia. The regenerative ability of chondrocytes is limited, so the degenerative changes in cartilage are irreversible. Mesenchymal stem cells (MSCs) have been shown to have some effect in repairing cartilage and delaying degenerative changes, and are of great significance in the treatment of osteoarthritis and cartilage defects.

[0003] Currently, cell therapy is one of the treatments that are expected to be clinically applied, and in conventional cell therapy, an aqueous solution is used as a cell delivery medium. For example, in conventional cell therapy for bone joints, a cell suspension is injected into the joint cavity with a syringe to exert a biological therapeutic effect. However, in the process of delivering cells in an aqueous solution, a large number of cells die within a few hours to a few days after injection. Currently, three mechanisms are widely accepted as the cause of the large number of cell deaths in the metastasis process: cell death caused by the shear force that cells experience during the injection process; death of anchorage-dependent cells due to a lack of growth factors; and cell death caused by a lack of nutritional supply from the host tissue, because the injected foreign cells have difficulty accessing blood vessels in the host tissue. Among them, mechanical shear force during the injection process is an important cause of cell death. When cells are injected into a Newtonian fluid such as saline, the cells are subjected to shear and tensile forces within the syringe. This is due to the flow resistance at the interface between the inner wall of the syringe and the fluid, because the flow rate at the center of the syringe is faster than the flow rate near the inner wall. In addition, the diameter of the needle used for injection is usually smaller than that of the syringe, so the tensile force at the interface between the syringe and the needle increases dramatically. The uneven distribution of these forces exerts huge shear stress on the cells, which may result in the rupture of the cell membrane and the rapid death of some necrotic cells, as well as the induction of the apoptotic process, which may further kill the cells injected into the host. Furthermore, the presence of shear forces during the injection process may also affect the gene expression and phenotype of cells, which has a significant effect on the differentiation of stem cells in particular.

[0004] Shear-thinning biomaterials can liquefy in the presence of shear stress to form a lubricating layer on the inner wall surface of the syringe, reducing the flow resistance of the entire material; furthermore, during the injection process, the material has a similar flow rate at the center and end of the syringe, forming a plug flow, thereby reducing the shear stress. This provides mechanical protection to cells during the injection process and reduces the shear force applied to the cells, thereby improving cell viability and reducing the effect of shear force on intracellular gene expression, etc. Examples of such biomaterials include natural polymers and supramolecular hydrogels prepared based on hyaluronic acid, sodium alginate, etc., and polypeptide-based supramolecular hydrogels. Although these materials have a certain degree of shear-thinning properties, they have drawbacks such as potential biological safety issues and lack of structural permeability, which hinder their further application in cell culture and tissue regeneration.

[0005] In addition to cell damage caused by shear during the injection process, cells injected into a joint may be damaged by the shear environment, such as friction within the joint, which may affect the therapeutic effect. Thus, there is a need to further improve the shear thinning property in order to further reduce cell damage in a shear environment.

[0006] In addition, in conventional cell therapy, there is a problem that cell viability and therapeutic efficacy are significantly reduced due to cell damage caused by shear during the cell injection process using an aqueous solution, and existing shear-thinning materials have potential biological safety issues and problems of insufficient structural permeability. In response to these problems, there is an urgent need to develop a method that can further reduce shear damage to cells in the treatment of osteoarthritis, as well as a cell preparation that reduces shear damage to cells and has desirable properties such as good biocompatibility required for the treatment of osteoarthritis. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Piuzzi, NS; Ng, M.; Chughtai , M. ; Khlopas, A.; Ramkumar , PN ; Harwin, SF; Mont, MA; Bauer , TW ; Muschler, GF, Accelerated Growth of Cellular Therapy Trials in Musculoskeletal Disorders: An Analysis of the NIH Clinical Trials Data Bank. Orthopedics 2019, 42(2), e144–150.

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[0008] The present inventors have found that when a specific nucleic acid supramolecular hydrogel is used as a cell delivery vehicle, the nucleic acid hydrogel protects the treated cells almost 100%, and the cell viability is as high as 99% or more during the cell injection process and in a shear environment simulating the inside of a joint. The present inventors have found that the nucleic acid hydrogel has a unique three-dimensional mesh structure and excellent shear thinning properties, which can significantly reduce cell shear damage, provide anti-shear protection to cells, improve cell viability in a shear environment, and improve the effectiveness of cell therapy. Meanwhile, compared with natural polymers and supramolecular hydrogels prepared based on hyaluronic acid, sodium alginate, etc., the nucleic acid supramolecular hydrogel of the present invention has excellent properties suitable for cartilage tissue engineering, such as fast gelation, good biocompatibility and permeability, and is therefore suitable for the formation of a cell gel preparation for osteoarthritis.

[0009] Based on the above findings, the present invention has been completed.

[0010] Specifically, the present invention relates to a cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells for the treatment and / or prevention of acute or chronic osteoarthritis and / or symptoms thereof via intra-articular injection; a method for treating or preventing acute and chronic osteoarthritis and associated inflammatory symptoms (particularly bone joint pain, reduced mobility or function) using said cell gel formulation; and a method for reducing shear damage to cells in a shear environment using said nucleic acid hydrogel.

[0011] The cell gel preparation of the present invention can reduce the shear damage of cells caused by shear environments such as the injection process and friction in joints, and the cells in the cell gel preparation are hardly damaged even in shear environments. Specifically, the nucleic acid hydrogel of the present invention provides anti-shear protection to cells during cell injection process, and the cell survival rate is improved from 85% to 99% or more compared with conventional injection methods; the nucleic acid hydrogel provides anti-shear protection to cells in a simulated joint shear environment, and the cell survival rate is improved from 75% to 99% or more compared with cells in culture medium; and the therapeutic effect of the cells delivered by the nucleic acid hydrogel of the present invention on knee osteoarthritis in rabbits is significantly better than that of conventional cell therapy. [Brief description of the drawings]

[0012] [Figure 1] 1 is a test graph of the rheological properties (including time sweep and strain sweep) of cell-free hydrogels and mesenchymal stem cell-loaded nucleic acid hydrogels. [Diagram 2] FIG. 1 shows a process diagram of cell injection using a conventional injection method and a nucleic acid hydrogel, and the viability determined by labeling cells with Calcein-AM / PI staining before and after injection for both injection methods. [Diagram 3] 13 is a statistical graph of cell viability before and after injection using both injection methods. [Figure 4] FIG. 1 shows a process diagram for simulating the joint shear environment and the viability measured by labeling cells with Calcein-AM / PI staining before and after friction in medium and in hydrogel. [Diagram 5]13 is a statistical graph of cell viability in media and in hydrogel before and after friction in a simulated joint shear environment. [Figure 6] FIG. 1 shows overall views of knee joints in rabbits 12 and 24 weeks after intra-articular injection of nucleic acid hydrogel carrying mesenchymal stem cells for the treatment of knee osteoarthritis. [Figure 7] FIG. 1 shows tissue staining images of knee joints 12 and 24 weeks after intra-articular injection of nucleic acid hydrogel carrying mesenchymal stem cells for the treatment of knee osteoarthritis in rabbits. [Figure 8] FIG. 1 shows the effect of different nucleic acid solids contents on cell viability after injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Scientific and technical terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art, but in case of conflict, the definitions used herein shall control.

[0014] In a first aspect, the present invention relates to a cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells for the treatment and / or prevention of acute or chronic osteoarthritis and / or symptoms thereof by intra-articular injection.

[0015] In one embodiment, the nucleic acid hydrogel comprises a scaffold unit comprising a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end; a crosslinking unit comprising a crosslinking core and at least two single-stranded nucleic acids bound to the crosslinking core, each single-stranded nucleic acid having at least one crosslinking sticky end; and an aqueous medium, wherein the scaffold unit and the crosslinking unit form a three-dimensional spatial network structure by crosslinking through complementary base pairing of the scaffold sticky ends and the crosslinking sticky ends.

[0016] In one embodiment, the nucleic acid includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), etc., and is preferably deoxyribonucleic acid (i.e., DNA). The nucleic acid may be a natural D-nucleic acid or an L-nucleic acid, and the L-nucleic acid refers to an L-nucleic acid in which L-nucleotides are polymerized. Specifically, the L-DNA refers to DNA in which L-deoxyribonucleotides are polymerized. The L-nucleic acid is preferably used.

[0017] In one embodiment, the scaffold unit includes a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end. Preferably, the scaffold core is a nucleic acid, specifically, it may be a D-nucleic acid or an L-nucleic acid, more specifically, it may be a D-DNA or an L-DNA. In one embodiment, the nucleic acid as the scaffold core has a complementary pairing region, and the length of the complementary pairing region may be 4 to 150 bp, preferably 5 to 50 bp, more preferably 6 to 30 bp, and even more preferably 8 to 20 bp.

[0018] In one embodiment, the scaffold core may be a polypeptide, which is a compound in which two or more amino acids are linked by peptide bonds. The polypeptide as the scaffold core specifically includes dipeptides, tripeptides, tetrapeptides, etc. Furthermore, the polypeptide of the present invention also includes oligopeptides, proteins, and the like.

[0019] In one embodiment, the bridging unit comprises a bridging core and at least two single-stranded L-nucleic acids bound to the bridging core, each single-stranded L-nucleic acid having at least one bridging sticky end. Preferably, the bridging core may be a nucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or an L-DNA. In one embodiment, the nucleic acid as the bridging core has a complementary pairing region, and the length of the complementary pairing region may be 4 to 150 bp, preferably 5 to 50 bp, more preferably 6 to 30 bp, and even more preferably 8 to 20 bp.

[0020] In one embodiment, the scaffold sticky end or crosslinking sticky end has a length of 4 nt or more, which is easily crosslinked under physiological conditions, and preferably has a length of 150 nt or less, preferably 50 nt or less, more preferably 30 nt or less, more preferably 30-50 nt, more preferably 20 nt or less.

[0021] In one embodiment, scaffold core and crosslink core are the same, and the single-stranded nucleic acid that is bound to scaffold core and the single-stranded nucleic acid that is bound to crosslink core are the same, for example, the same L-nucleic acid; when the number of single-stranded nucleic acids that is bound to scaffold core and the number of single-stranded nucleic acids that is bound to crosslink core (both are ≧3) are the same, scaffold unit and crosslink unit are the same.Therefore, in one embodiment, scaffold unit and crosslink unit are the same.In one embodiment, scaffold unit and crosslink unit are different.

[0022] In an embodiment, the molar ratio of the scaffold unit to the crosslinking unit in the nucleic acid hydrogel is 2:1 to 1:3, preferably 1:1 to 1:2, and more preferably 1:1.5.

[0023] In one embodiment, the scaffold unit and the crosslinking unit form a three-dimensional spatial network structure by crosslinking through complementary base pairing between the scaffold sticky end and the crosslinking sticky end. Preferably, the scaffold unit, the crosslinking unit and the three-dimensional spatial network structure are in a stable crosslinked state under physiological conditions (37°C, pH 7.2 to 7.4, 0.9 wt% NaCl, isotonic).

[0024] In one embodiment, the aqueous medium refers to water or an aqueous solution. The aqueous solution is preferably a buffer solution containing a buffer salt. The aqueous solution can preferably form an environment similar to the in vivo microenvironment of stem cells, such as physiological conditions (37°C, pH 7.2 to 7.4, 0.9 wt% NaCl, isotonic).

[0025] In one embodiment, the nucleic acid hydrogel has a suitable mechanical strength, for example, the mechanical strength may be 0.1 Pa or more, preferably 1 Pa or more, more preferably 10 Pa or more, and preferably 10,000 Pa or less, more preferably 1,000 Pa or less.

[0026] In one embodiment, the nucleic acid hydrogel has a storage modulus G' that dominates over the loss modulus G" at all strain values ​​from 0.1 to 100 rad / s as measured by a frequency sweep at 25°C and a strain amplitude of 1% strain. Preferably, the ratio of the storage modulus G' to the loss modulus G" at strain values ​​from 0.1 to 100 rad / s is greater than 2, more preferably greater than 10.

[0027] In one embodiment, the nucleic acid hydrogel of the present invention may have a desirable stability, e.g., may stably maintain its structure in the presence of a restriction endonuclease for 24 hours, preferably 36 hours, more preferably 48 hours or more.

[0028] In one embodiment, the scaffold unit or crosslinking unit of the nucleic acid hydrogel can include a CpG sequence. The CpG sequence is a palindrome with a cytosine-phosphate-guanosine (CpG) dinucleotide core, with two purines at the 5' end and two pyrimidines at the 3' end, i.e., 5'-PurPur-CG-PyrPyr-3'. The CpG sequence is recognized by mammalian cells and can trigger a series of biological defense mechanisms, such as complement activation, phagocytosis, and expression of inflammatory cytokine genes. Currently, examples of CpG sequences known to have strong immune stimulating effects include 5'-TCCATGACGTTCCTGACGTT-3'.

[0029] In one embodiment, the sequence of the scaffold unit is 5'-CGATTGACTCTCCACGCTGTCCTAACCATGACCGTCGAAG-3', 5'-CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3' or 5'-CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3'. In one embodiment, the sequence of the bridge unit is 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' or 5'-GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

[0030] The nucleic acid hydrogel of the present invention can be prepared by the method described in CN201610740754X, for example, a single strand of DNA (i.e., the scaffold unit and the crosslinking unit) can be prepared separately, then mixed and self-assembled to obtain the hydrogel of the present invention; alternatively, the scaffold unit and the crosslinking unit can be mixed separately with the aqueous medium to obtain an aqueous medium solution of the scaffold unit and an aqueous medium solution of the crosslinking unit, and then the two solutions can be mixed and crosslinked to form a three-dimensional spatial network structure, thereby obtaining the hydrogel of the present invention.

[0031] In one embodiment, the mesenchymal stem cells are embedded or dispersed in the nucleic acid hydrogel. Preferably, the mesenchymal stem cells are embedded in the nucleic acid hydrogel.

[0032] In one embodiment, the mesenchymal stem cells are derived from bone marrow, umbilical cord or placenta.The mesenchymal stem cells are derived from any one of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, peripheral blood mesenchymal stem cells, synovial mesenchymal stem cells, umbilical cord mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.The mesenchymal stem cells have desirable anti-inflammatory and regenerative properties.

[0033] In one embodiment, the density of the mesenchymal stem cells is 10 5 ~10 8 cells / mL (per milliliter of cell gel formulation), preferably 2 x 10 7 ~5×10 7 1 x 10 cells / mL, more preferably 1 x 10 7 pieces / mL.

[0034] In one embodiment, the gel has a nucleic acid solid content of 0.7-5.0% (mass / volume ratio, nucleic acid solid content per 1 ml of gel) to protect cells during the injection process. If the nucleic acid solid content is less than 0.7% (mass %), the gel cannot be formed, and if the nucleic acid content in the gel is more than 5.0%, the strength is high and the resistance during the injection process is excessively large, making injection difficult. In a preferred embodiment, the nucleic acid solid content is 1.5-4.0%, 2.0-4.0%, 3.0-4.0%, 3.5-4.0%, or 3.8%.

[0035] In one embodiment, the mesenchymal stem cells are added or mixed into the hydrogel after the nucleic acid hydrogel is formed, thereby forming the cell gel formulation. In one embodiment, the mesenchymal stem cells are mixed with a DNA assembly to form a nucleic acid hydrogel loaded with mesenchymal stem cells according to a conventional method. In one embodiment, the mesenchymal stem cells are mixed with a single strand of DNA (three scaffold units and two crosslinking units) to first form a DNA assembly loaded with cells, and then form a nucleic acid hydrogel.

[0036] In one embodiment, the cell gel preparation further comprises an anti-inflammatory agent, preferably selected from steroidal anti-inflammatory compounds (e.g., prednisolone, dexamethasone, betamethasone and triamcinolone), non-steroidal anti-inflammatory compounds (e.g., ibuprofen, diclofenac, naproxen, etc.), anti-rheumatic drugs (e.g., methotrexate, leflunomide, etc.), anti-CD20 drugs, anti-cytokine drugs (e.g., anti-IL1 drugs, anti-IL6 drugs, anti-IL-17 drugs), anti-TNF drugs (e.g., infliximab, etanercept, adalimumab, rituximab, etc.), or mixtures thereof.

[0037] In a second aspect, the present invention relates to a method for treating or preventing acute and chronic osteoarthritis and associated inflammation-derived symptoms, particularly bone joint pain, reduced mobility or function, using said cell gel formulation. In one embodiment, said disease or condition is selected from, for example, osteoarthritis, degenerative arthritis, knee joint disease, hip joint degenerative joint disease, and other inflammatory systemic conditions involving the joints, such as autoimmune diseases, particularly rheumatoid arthritis and systemic lupus erythematosus (SLE), spondyloarthropathy, polymyalgia rheumatica, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic arthritis (related to inflammatory bowel diseases such as hemorrhagic colitis and Crohn's disease), neuropathic arthropathy, acute rheumatic fever, gout, chondrocalcinosis, calcium hydroxyapatite crystal deposition disease, Lyme disease, and all other degenerative joint diseases.

[0038] In a third aspect, the present invention relates to a method for reducing shear damage to cells in a shear environment using a nucleic acid hydrogel, the method comprising: (i) providing a nucleic acid hydrogel comprising a scaffold unit and a bridging unit, and (ii) loading mesenchymal stem cells onto the nucleic acid hydrogel; or (i) separately preparing the scaffold unit and the bridging unit of the nucleic acid hydrogel, and (ii) mixing mesenchymal stem cells with the scaffold unit and the bridging unit, and obtaining the cell gel formulation of the present invention by self-organization; or measuring the anti-shear protective effect of the cell-loaded nucleic acid hydrogel on cells by a cell injection test or the like.

[0039] In a fourth aspect, the present invention relates to the use of a cell gel formulation for the manufacture of a medicament for the treatment or prevention of acute and chronic osteoarthritis and associated inflammatory conditions.

[0040] In one embodiment, the nucleic acid hydrogel is as described above. In one embodiment, the mesenchymal stem cells are embedded or dispersed in the nucleic acid hydrogel. In one embodiment, the shear environment includes injection processes and internal friction within joints. EXAMPLES

[0041] Experimental materials The nucleic acid hydrogel was prepared using the method disclosed in CN201610740754X. First, the DNA single strands (Y1, Y2, and Y3) and (L1C and L2C) shown in Table 1 were respectively self-assembled in an aqueous medium into DNA assemblies (which are 1 mmol / L of scaffolding units and 1.5 mmol / L of crosslinking units), and then the DNA assemblies were mixed to form the desired DNA hydrogel samples (i.e., nucleic acid hydrogels).

[0042] [Table 1]

[0043] The cell culture medium used was Minimum Essential Medium (MEM) α (Gibco, Thermo Fisher, USA).

[0044] The live / dead cell staining kit used was Calcein-AM / PI (Solarbio, Solarbio, Beijing).

[0045] Rheological properties were measured using a Komexus rheometer (Malvern); cell images were acquired using a LSM 710Meta laser scanning confocal microscope (Carl Zeiss AG, Germany).

[0046] Example 1: Preparation of nucleic acid hydrogel carrying mesenchymal stem cells 1 mmol / L of the Y-scaffold unit in Table 1 above was completely dissolved in a certain amount of stem cell (derived from rabbit bone marrow mesenchymal stem cells extracted in the laboratory) suspension, and 1.5 mmol / L of the crosslinking unit in Table 1 above was completely dissolved in an equal volume of the solution, and the two were mixed to form a gel (the gel could be formed after a few seconds). The final cell concentration of the resulting nucleic acid hydrogel was 1×10 7 cells / ml and the nucleic acid solids content was 3.8% (mass %).

[0047] Example 2: Rheological testing of cell-loaded nucleic acid hydrogels The nucleic acid hydrogel carrying mesenchymal stem cells prepared in Example 1 was taken out with a medicine spoon and placed on the test plate of the rheometer. The cone plate of the rheometer was adjusted to slowly descend so as to contact the hydrogel sample, and finally the distance between the cone plate and the plate was fixed at 150 μm.

[0048] A total of two types of rheological tests were carried out in this section.

[0049] The first one is the time scan test, that is, the samples are continuously sheared in a certain time with a constant sweep frequency, to test the shear resistance ability of the samples, and further to reflect the mechanical strength of different samples by comparing the ratio of storage modulus to loss modulus. The parameters set in this experiment are strain=1%, frequency=1Hz, and temperature is constant at 25℃.

[0050] The second was a strain sweep test, which measured the change trends of the storage modulus and loss modulus of different samples under different strains to reflect whether the samples had shear thinning behavior. The parameters set in this experiment were strain range = 0.1-1000%, frequency = 1Hz, and temperature was constant at 25℃.

[0051] Figures 1A and 1B show the results of rheological tests in time and strain modes for the cell-free hydrogel, and Figures 1C and 1D show the results of rheological tests in time and strain modes for the cell-containing hydrogel. As can be seen from Figure 1, the nucleic acid hydrogel loaded with mesenchymal stem cells has shear thinning properties while maintaining good mechanical strength to support the cells.

[0052] Example 3: Shear protection of cells from cell-loaded nucleic acid hydrogels In this example, the anti-shear protective effect of the nucleic acid hydrogel prepared in Example 1 was investigated during cell injection and in a simulated joint shear environment.

[0053] Using a 29G insulin syringe (needle diameter is only 330 μm), a cell injection test was performed on the cell-loaded nucleic acid hydrogel of Example 1, while cells in the medium were used as a control. The cell viability was characterized by Live / Dead staining after injection. The experimental results are shown in Figures 2 and 3. Figure 2 shows the process diagram of cell injection using the conventional injection method and the nucleic acid hydrogel, and the viability determined by labeling cells with Calcein-AM / PI staining before and after injection for both injection methods. In Figure 2, row A shows the injection method, row B shows the distribution of live cells (live cells are green bright spots (original image) or white spots (black and white image)), row C shows the distribution of dead cells (dead cells are red bright spots (original image) or light gray spots (black and white image)), and row D shows the distribution of live cells / dead cells. For convenience, the distribution of only dead cells is shown in row C. As can be seen from row C of FIG. 2, before injection using the conventional injection method, many dead cells were already observed, for example, in region 1C of FIG. 2, whereas before injection protected with nucleic acid hydrogel, no dead cells were observed, for example, in region 3C of FIG. 2. After injection, the conventional injection method caused a large amount of cell death, for example, in region 2C of FIG. 2, whereas after protection with nucleic acid hydrogel and injection, almost no cell death was observed, for example, in region 4C of FIG. 2. This shows that the nucleic acid hydrogel carrying mesenchymal stem cells itself can provide protection to cells (i.e., protection before injection), and furthermore, it was revealed that the protective effect of the nucleic acid hydrogel carrying mesenchymal stem cells on cells after injection was particularly remarkable.

[0054] In order to simulate the shear environment in a joint, the nucleic acid hydrogel carrying mesenchymal stem cells of Example 1 was placed between two cover slips with a distance of 330 μm from each other, and the two cover slips were slid in both directions to simulate the shear environment in a joint. After two minutes of bidirectional sliding, the cell viability was characterized by Live / Dead staining. The experimental results are shown in Figures 4 and 5. Figure 4 shows a process diagram of simulating the joint shear environment and the viability measured by labeling cells with Calcein-AM / PI staining before and after rubbing the cells in an aqueous medium medium and in a hydrogel. Friction in an aqueous medium caused a large amount of cell death (dead cells are bright red spots (original image) or light gray spots (black and white image)), as shown in region 2A of Figure 4, whereas almost no dead cells were observed in the nucleic acid hydrogel.

[0055] The results of these two experiments demonstrated that, compared with direct injection of a cell suspension, the nucleic acid hydrogel provided anti-shear protection to cells during the injection process and in a simulated joint shear environment, with almost no cell death observed and significantly improved cell viability in a shear environment. Specifically, the nucleic acid hydrogel provided anti-shear protection to cells during the cell injection process, improving cell viability from 85% to more than 99% compared with conventional injection methods, and the nucleic acid hydrogel provided anti-shear protection to cells in a simulated joint shear environment, improving cell viability from 75% to more than 99% compared with that in culture medium.

[0056] Example 4: Treatment of knee osteoarthritis in rabbits by intra-articular injection of nucleic acid hydrogel loaded with mesenchymal stem cells Skeletally mature male New Zealand rabbits (age: 6 months, weight: 3.5 ± 0.5 Kg, n = 60) were purchased from the Animal Experiment Center of Peking University. Osteoarthritis was induced in the rabbits by anterior cruciate ligament transection and medial meniscus resection. All animal experimental procedures were approved by the Peking University Animal Care and Use Committee and followed the Guidelines for the Care and Use of Laboratory Animals (National Academy of Sciences Press, National Institutes of Health Press, No. 85-23, revised 1996). The specific procedures are as follows: After anesthesia and routine aseptic preparation, the joint cavity was entered through a medial patellar incision, the anterior cruciate ligament was transected, and the medial meniscus was completely resected. The decrease in stability of the knee joint was confirmed by an anterior drawer test. After surgery, all animals were returned to the corresponding rabbit cages, allowed to move freely, and necessary infection prevention and pain relief treatments were administered, allowing the knee joint to recover function.

[0057] All animals were randomly divided into five groups, each of which underwent surgery and injection treatments on the left knee: 1. Sham group: simply incised and sutured the skin; 2. Blank group: simply performed knee osteoarthritis model surgery on rabbits; 3. PDS group: only injected nucleic acid hydrogel into the knee of rabbits with osteoarthritis; 4. MSCs group: only injected mesenchymal stem cells into the knee of rabbits with osteoarthritis; 5. Hybrid group: injected mesenchymal stem cell-loaded nucleic acid hydrogel into the knee of rabbits with osteoarthritis. All injections were performed once a week for a total of three times, starting 8 weeks after the rabbit knee osteoarthritis model surgery. The PDS group was injected with 200 μl of nucleic acid hydrogel per knee each time, and the MSCs group was injected with 200 μl of basal medium suspension of cells per knee each time (cell concentration 1 × 10 7 cells / ml), and the Hybrid group received 200 μl of the nucleic acid hydrogel carrying mesenchymal stem cells from Example 1 (cell concentration 1×10 7 At 12 and 24 weeks after the end of the injection treatment, the rabbits in each group were euthanized and knee joint samples were taken for further study.

[0058] As can be seen from the general observations, the Blank and PDS groups showed cartilage erosion and cartilage defects that worsened over time, whereas the cartilage damage in the MSCs-treated and Hybrid-treated groups was significantly improved, where the general observations of the Hybrid group were closer to normal cartilage (Sham group). Sample tissues were further subjected to H&E, Safranin O-Fast Green, Toluidine Blue staining, and collagen type II immunohistochemistry. Compared with the MSCs-treated group, the cartilage morphology and collagen type II expression in the mesenchymal stem cell-loaded nucleic acid hydrogel-treated group (Hybrid group) were closer to those of normal cartilage tissue, especially at 24 weeks. Similarly, the microstructure of the cartilage surface was observed by scanning electron microscope (SEM), and the cartilage surface of the conventional mesenchymal stem cell-loaded aqueous solution-treated group (MSCs group) was not cracked, but still had many hills. Meanwhile, the cartilage surface of the Hybrid group was smooth with only small irregularities. The results are shown in Figures 6 and 7.

[0059] Example 5: Effect of nucleic acid solid content on nucleic acid hydrogels loaded with mesenchymal stem cells As in Example 1, nucleic acid hydrogels carrying mesenchymal stem cells with nucleic acid solid content of 0.95%, 1.9%, and 4.2% were prepared, and the shear protection effect on cells during cell injection was tested for these three types of nucleic acid hydrogels carrying mesenchymal stem cells as in Example 3. 24 hours after injection, no dead cells were observed in the nucleic acid hydrogels carrying mesenchymal stem cells with nucleic acid solid content of 0.95%, 1.9%, and 4.2%, as well as those with nucleic acid solid content of 3.8%. However, when prepared in the same manner, the nucleic acid hydrogel carrying mesenchymal stem cells with nucleic acid solid content of 3.8% had more uniform cell distribution, more suitable nucleic acid strength, and more suitable injectability. The results are shown in FIG. 8.

[0060] Although examples of the present invention have been shown and described above, it is understood that the above examples are illustrative and are not to be construed as limiting the present invention, and those skilled in the art can change, modify, substitute, and change the above embodiments within the scope of the present invention.

Claims

1. 1. A method for reducing shear damage to cells in a shear environment using a cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells, comprising: The method comprises: (i) providing a nucleic acid hydrogel comprising a scaffold unit and a crosslinking unit; and (ii) loading mesenchymal stem cells in the nucleic acid hydrogel; or (i) separately preparing a scaffold unit and a crosslinking unit of a nucleic acid hydrogel; and (ii) mixing mesenchymal stem cells with the scaffold unit and the crosslinking unit to obtain the cell gel preparation of the present invention by self-organization, further comprising subjecting the cell gel formulation to a shear environment; The shear environment consists of the injection process and internal friction within the joint; The nucleic acid hydrogel comprises a scaffold unit comprising a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end; a cross-linking unit comprising a cross-linking core and at least two single-stranded nucleic acids bound to said cross-linking core, each single-stranded nucleic acid having at least one cross-linked sticky end; and Contains an aqueous medium, The method, wherein the scaffold unit and the cross-linking unit form a three-dimensional spatial network structure by cross-linking through complementary base pairing of the scaffold sticky end and the cross-linking sticky end.

2. The method of claim 1, wherein the scaffold unit comprises a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end; the scaffold core is a nucleic acid; the nucleic acid as the scaffold core has a complementary pairing region, and the length of the complementary pairing region is 4 to 150 bp.

3. The method according to claim 1 or 2, wherein the bridging unit comprises a bridging core and at least two single-stranded L-nucleic acids bound to the bridging core, each single-stranded L-nucleic acid having at least one bridging sticky end; the bridging core is a nucleic acid; the nucleic acid as the bridging core has a complementary pairing region, and the length of the complementary pairing region is 4 to 150 bp.

4. The method according to any one of claims 1 to 3, wherein the molar ratio of scaffold units to crosslinking units in the nucleic acid hydrogel is from 2:1 to 1:

3.

5. The method according to any one of claims 1 to 4, wherein the scaffold units, the crosslinking units and the three-dimensional spatial network structure are in a crosslinked state under physiological conditions (37°C, pH 7.2 to 7.4, 0.9 wt% NaCl, isotonic).

6. 6. The method of any one of claims 1 to 5, wherein the nucleic acid hydrogel has a storage modulus G' that dominates over the loss modulus G" at all strain values ​​from 0.1 to 100 rad / s as measured by a frequency sweep at 25°C and a strain amplitude of 1%.

7. The method according to any one of claims 1 to 6, wherein a scaffold unit or a crosslinking unit of the nucleic acid hydrogel comprises a CpG sequence.

8. The method according to any one of claims 1 to 7, wherein the mesenchymal stem cells are embedded or dispersed in the nucleic acid hydrogel.

9. The density of the mesenchymal stem cells is 10 ５ ~10 ８ The method according to any one of claims 1 to 8, wherein the cell concentration is 100% or more and the cell gel concentration is 100% or more.

10. The method of any one of claims 1 to 9, further comprising an anti-inflammatory agent.

11. The method of any one of claims 1 to 10, wherein the cell gel formulation has a nucleic acid solids content of 3.8%.

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