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

Abstract

This invention relates to a cell gel formulation for treating and / or preventing acute or chronic osteoarthritis and / or symptoms by intra-articular injection, said cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells; methods for treating or preventing acute and chronic osteoarthritis and related symptoms (especially osteoarthritis pain, loss of mobility or function) using said cell gel formulation; and methods for reducing cellular shear damage in a shear environment using a nucleic acid hydrogel.

Description

Technical Field

[0001] This invention relates to a cell gel formulation for treating and / or preventing acute or chronic osteoarthritis and / or symptoms by intra-articular injection, said cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells; methods for treating or preventing acute and chronic osteoarthritis and related symptoms (especially osteoarthritis pain, loss of mobility or function) using said cell gel formulation; and methods for reducing cellular shear damage in a shear environment using a nucleic acid hydrogel. Background Technology

[0002] Osteoarthritis (OA) is a chronic disease characterized by degenerative changes in articular cartilage and secondary osteophyte formation. Chondrocytes have limited regenerative capacity, therefore, cartilage degeneration is irreversible. Mesenchymal stem cells (MSCs) have been shown to have certain therapeutic effects in repairing cartilage and delaying degenerative changes, and are of great significance in the treatment of osteoarthritis and cartilage defects.

[0003] Cell therapy is one of the promising treatment methods in clinical practice. Traditional cell therapy methods use aqueous solutions as cell delivery media. For example, in traditional cell therapy for osteoarthritis, cell suspensions are injected into the joint cavity via syringe to exert their biotherapeutic effect. However, during the process of transporting cells with aqueous solutions, a large number of cells die several hours to days after injection. Three widely accepted mechanisms explain the massive cell death during transfer: shear forces experienced by cells during injection causing cell death; the lack of growth factors leading to anchorage-dependent cell death; and the inability of injected exogenous cells to reach blood vessels in the host tissue, resulting in cell death due to insufficient nutrient support from the host tissue. Among these, mechanical shear forces during injection are a significant cause of cell death. When cells are injected into a Newtonian fluid like saline, they are affected by shear and tensile forces within the syringe. This is due to flow resistance at the syringe wall and fluid interface, resulting in a higher flow velocity at the center of the syringe than near the inner wall. Furthermore, the diameter of the needle used for injection is usually smaller than the syringe diameter, leading to a sharp increase in tensile forces at the syringe / needle interface. The uneven distribution of these forces can subject cells to extreme shear stress, leading to cell membrane rupture and rapid death of some necrotic cells, and potentially triggering apoptosis, thus causing further cell death after injection into the host. Furthermore, the presence of shear forces during injection can affect gene expression and phenotype, particularly having a significant impact on stem cell differentiation.

[0004] Shear-thinning biomaterials can liquefy on the inner surface of a syringe due to shear stress, forming a lubricating layer that reduces overall flow resistance. During injection, this material exhibits similar flow velocities at the center and edges of the syringe, creating a piston-like flow that further reduces shear stress. Therefore, it can provide mechanical protection for cells during injection, reducing the shear force experienced by cells, thereby improving cell viability and mitigating the impact of shear force on gene expression. Such biomaterials include natural macromolecular supramolecular hydrogels based on hyaluronic acid and sodium alginate, as well as peptide-based supramolecular hydrogels. Despite their shear-thinning properties, these materials suffer from potential biosafety concerns and structural permeability limitations, hindering their further application in cell culture and tissue regeneration.

[0005] Besides the shearing damage during injection, cells injected into the joint are also damaged by the shearing environment of joint friction, thus affecting the therapeutic effect. Therefore, further improvements in shear-thinning properties are needed to further reduce cell damage in shearing environments.

[0006] In response to the problems of cell shear damage during cell injection using aqueous solutions in traditional cell therapy, which significantly reduces cell survival rate and therapeutic efficacy, and the biosafety risks and insufficient structural permeability of existing shear-thinning materials, there is an urgent need for a method to further reduce cell shear damage in the treatment of osteoarthritis, as well as a cell preparation that reduces cell shear damage, while possessing good biocompatibility and other desired properties required for osteoarthritis treatment.

[0007] References:

[0008] 1.Piuzzi, NS; Ng, M.; Chughtai, M.; Khlopas, A.; Ramkumar, PN; Harwin, SF; Mont, MA; Bauer, TW; Muschler, GF. Accelerated Growth of Cellular TherapyTrials in Musculoskeletal Disorders: An Analysis of the NIH Clinical TrialsData Bank.Orthopedics2019,42(2),e144-e150.

[0009] 2. Jones, I.A.; Togashi, R.; Wilson, M.L.; Heckmann, N.; Vangsness, C.T., Jr., Intra-articular treatment options for knee osteoarthritis. Nat Rev Rheumatol 2019, 15(2), 77-90.

[0010] 3. Ng, J.; Little, C.B.; Woods, S.; Whittle, S.; Lee, F.Y.; Gronthos, S.; Mukherjee, S.; Hunter, D.J.; Worthley, D.L., Stem cell directed therapies for osteoarthritis: The promise and the practice: Concise review. Stem Cells 2019.

[0011] 4. Mitrousis, N.; Fokina, A. & Shoichet, M.S. Biomaterials for cell transplantation. Nat Rev Mater 2018, 3, 441–456.

[0012] 5. Yan, C.; Mackay M.E.; Czymmek K.; Nagarkar R.P.; Schneider J.P.; and Pochan D.J., Injectable Solid peptide hydrogel as a cell carrier: effects of shear flow on hydrogels and cell payload. Langmuir 2012 28(14), 6076-6087 Summary of the Invention

[0013] The inventors of this invention have discovered that when a specific nucleic acid supramolecular hydrogel is used as a cell delivery medium, the nucleic acid hydrogel can provide almost 100% protection for therapeutic cells, with cell survival rates exceeding 99% during cell injection and in simulated joint shear environments. The inventors found that due to the unique three-dimensional network structure and excellent shear-thinning properties of this nucleic acid hydrogel, it can significantly reduce cell shear damage, provide shear protection for cells, improve cell survival rates under shear conditions, and enhance the efficacy of cell therapy. Furthermore, compared to natural macromolecular supramolecular hydrogels prepared from hyaluronic acid, sodium alginate, etc., the nucleic acid supramolecular hydrogel of this invention possesses excellent properties suitable for cartilage tissue engineering, such as rapid gelation, good biocompatibility, and permeability, making it suitable for forming cell gel formulations for the treatment of arthritis.

[0014] Based on the above findings, the present invention has been realized.

[0015] Specifically, the present invention relates to a cell gel formulation for treating and / or preventing acute or chronic osteoarthritis and / or symptoms by intra-articular injection, said cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells; methods for treating or preventing acute and chronic osteoarthritis and related symptoms of inflammatory origin (especially osteoarthritis pain, loss of mobility or function) using said cell gel formulation; and methods for reducing cellular shear damage in a shear environment using a nucleic acid hydrogel.

[0016] The cell gel formulation of the present invention can reduce cell shear damage caused by shear environments, including the injection process and intra-articular friction, and the cells in the cell gel formulation suffer almost no damage in the shear environment. Specifically, the nucleic acid hydrogel of the present invention provides shear protection for cells during cell injection, increasing cell survival rate from 85% to over 99% compared to traditional injection methods; the nucleic acid hydrogel provides shear protection for cells in a simulated joint shear environment, increasing cell survival rate from 75% to over 99% compared to cells in culture medium; and the nucleic acid hydrogel-carried cell therapy for knee osteoarthritis in rabbits is significantly more effective than traditional cell therapy. Attached Figure Description

[0017] Figure 1 These are rheological property (including time-scan and strain-scan) test images of cell-free hydrogels and nucleic acid hydrogels loaded with mesenchymal stem cells.

[0018] Figure 2 The diagram shows the process of traditional injection and nucleic acid hydrogel injection of cells, as well as the cell viability before and after injection using Calcein-AM / PI staining.

[0019] Figure 3This is a statistical chart showing the cell survival rate before and after cell injection using two different injection methods.

[0020] Figure 4 This is a process diagram simulating a joint shearing environment, and the cell viability is marked by Calcein-AM / PI staining before and after friction in the culture medium and hydrogel.

[0021] Figure 5 This is a statistical graph showing the cell survival rate before and after friction in a simulated joint shearing environment in culture medium and hydrogel.

[0022] Figure 6 The images show the gross knee joint after 12 and 24 weeks of intra-articular injection of a nucleic acid hydrogel loaded with mesenchymal stem cells into a rabbit for the treatment of knee osteoarthritis.

[0023] Figure 7 This is a staining image of the knee joint tissue 12 and 24 weeks after intra-articular injection of a nucleic acid hydrogel loaded with mesenchymal stem cells into a rabbit for the treatment of knee osteoarthritis.

[0024] Figure 8 This relates to the effect of different nucleic acid solid contents on cell survival after injection. Detailed Implementation

[0025] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0026] In a first aspect, the present invention relates to a cell gel formulation for treating and / or preventing acute or chronic osteoarthritis and / or symptoms by intra-articular injection, said cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells.

[0027] 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 adhesive 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 crosslink adhesive end; and an aqueous medium; wherein the scaffold unit and the crosslinking unit are crosslinked by the scaffold adhesive end and the crosslink adhesive end in a base-complementary pairing manner to form a three-dimensional spatial network structure.

[0028] In one embodiment, the nucleic acid includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), and locked nucleic acid (LNA), preferably deoxyribonucleic acid (i.e., DNA). The nucleic acid can be a natural D-nucleic acid or L-nucleic acid, where L-nucleic acid refers to L-nucleic acid polymerized from L-nucleotides. Specifically, L-DNA refers to DNA polymerized from L-deoxyribonucleotides. L-nucleic acid is preferred.

[0029] In one embodiment, 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 adhesive end. Preferably, the scaffold core is a nucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or L-DNA. In one embodiment, the nucleic acid serving as the scaffold core has a complementary pairing region, the length of which may be 4–150 bp, preferably 5–50 bp, more preferably 6–30 bp, and even more preferably 8–20 bp.

[0030] In one embodiment, the scaffold core may be a polypeptide, which is a compound formed by two or more amino acids linked together by peptide bonds. Specifically, polypeptides serving as the scaffold core include dipeptides, tripeptides, tetrapeptides, etc. Furthermore, the polypeptides of this invention also include oligopeptides, proteins, and so on.

[0031] In one embodiment, the crosslinking unit comprises a crosslinking core and at least two single-stranded L-nucleotides bound to the crosslinking core, each single-stranded L-nucleotide having at least one crosslinking sticky end. Preferably, the crosslinking core may be a nucleic acid, specifically a D-nucleotide or an L-nucleotide, more specifically D-DNA or L-DNA. In one embodiment, the nucleic acid serving as the crosslinking core has a complementary pairing region, the length of which may be 4–150 bp, preferably 5–50 bp, more preferably 6–30 bp, and even more preferably 8–20 bp.

[0032] In one embodiment, the length of the adhesive end or cross-linked adhesive end of the stent is 4 nt or more, which is beneficial for it to be in a stable cross-linked state under physiological conditions. Preferably, the length of the adhesive end or cross-linked adhesive end of the stent is 150 nt or less, more preferably 50 nt or less, more preferably 30 nt or less, more preferably 30-50 nt, and more preferably 20 nt or less.

[0033] In one embodiment, when the scaffold core and the crosslinking core are the same, and the single-stranded nucleic acids bound to the scaffold core and the single-stranded nucleic acids bound to the crosslinking core are the same (e.g., the same L-nucleic acid), and the number of single-stranded nucleic acids bound to the scaffold core and the number of single-stranded nucleic acids bound to the crosslinking core (both ≥ 3) are the same, the scaffold unit and the crosslinking unit are the same. Therefore, in one embodiment, the scaffold unit and the crosslinking unit are the same. In another embodiment, the scaffold unit and the crosslinking unit are different.

[0034] In one embodiment, the molar ratio of scaffold units to crosslinking units in the nucleic acid hydrogel is 2:1-1:3, preferably 1:1-1:2, and more preferably 1:1.5.

[0035] In one embodiment, the scaffold unit and the crosslinking unit are crosslinked through the scaffold adhesive ends and the crosslinking adhesive ends in a base-complementary pairing manner to form a three-dimensional spatial network structure. 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 7.2–7.4, 0.9 wt% NaCl, isotonic).

[0036] In one embodiment, the aqueous medium refers to water or an aqueous solution. Preferably, the aqueous solution is a buffer solution containing buffer salts. The aqueous solution is preferably capable of creating an environment similar to the in vivo microenvironment of stem cells, such as physiological conditions (37°C, pH 7.2–7.4, 0.9 wt% NaCl, isotonic).

[0037] In one embodiment, the nucleic acid hydrogel has suitable mechanical strength, for example, its mechanical strength can be 0.1 Pa or more, preferably 1 Pa or more, more preferably 10 Pa or more, preferably 10000 Pa or less, and more preferably 1000 Pa or less.

[0038] In one embodiment, the nucleic acid hydrogel has a dominant storage modulus G' relative to the loss modulus G'', as measured by frequency scanning at 25°C and a strain amplitude of 1% strain, over all strain values ​​from 0.1 to 100 rad / s. Preferably, the ratio of storage modulus G' to loss modulus G'' is greater than 2 over strain values ​​from 0.1 to 100 rad / s; more preferably, it is greater than 10.

[0039] In one embodiment, the hydrogel of the present invention can have ideal stability, for example, it can maintain its structure stably for 24 hours, preferably 36 hours, preferably 48 hours, or longer in the presence of restriction endonuclease.

[0040] In one embodiment, the scaffold units or cross-linking units of the nucleic acid hydrogel may contain a CpG sequence. A CpG sequence is a palindromic sequence centered on cytosine-guanine dinucleotide (CpG), with two purines at the 5' end and two pyrimidines at the 3' end, i.e., 5'-PurPur-CG-PyrPyr-3'. CpG sequences can be recognized by mammalian cells, thereby triggering a series of immune defense mechanisms, including complement activation, phagocytosis, and the expression of pro-inflammatory cytokine genes. Currently known CpG sequences with strong immunostimulatory effects include, for example, 5'-TCCATGACGTTCCTGACGTT-3'.

[0041] In one embodiment, the sequence of the stent unit is 5'-CGATTGACTCTCCACGCTGTCCTAACCATGACCGTCGAAG-3', 5'-CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3', and 5'-CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3'. In one embodiment, the sequence of the crosslinking unit is 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' and 5'-GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

[0042] The nucleic acid hydrogel of the present invention can be prepared using the method described in CN201610740754X. For example, DNA single strands (i.e., the scaffold unit and the crosslinking unit) can be prepared separately, and then mixed to obtain the hydrogel of the present invention through self-assembly. Alternatively, the scaffold unit and the crosslinking unit can be mixed with the aqueous medium to obtain an aqueous medium solution for the scaffold unit and an aqueous medium solution for the crosslinking unit, and then the two solutions can be mixed to crosslink and form a three-dimensional spatial network structure to obtain the hydrogel of the present invention.

[0043] 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.

[0044] In one embodiment, the mesenchymal stem cells are derived from bone marrow, umbilical cord, or placenta: the source of the mesenchymal stem cells is 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 possess desired anti-inflammatory and regenerative properties.

[0045] In one embodiment, the density of the mesenchymal stem cells is 10-1.5 Up to 10 8 Cells / mL (per milliliter of cell gel formulation), preferably 2 × 10⁶ cells / mL. 7 Up to 5×10 7 Cells / mL; more preferably 1×10⁻⁶ cells / mL; 7 per mL.

[0046] In one embodiment, the nucleic acid solids content in the gel is 0.7%-5.0% (mass / volume ratio, the amount of nucleic acid solids per milliliter of gel volume) to provide protection for cells during injection. When the nucleic acid solids content is below 0.7% (mass percentage), gel formation is impossible; while when the nucleic acid content in the gel is greater than 5.0%, its high strength results in excessive resistance during injection, making injection difficult. In a preferred embodiment, the nucleic acid solids content is 1.5-4.0%, 2.0-4.0%, 3.0-4.0%, 3.5-4.0%, or 3.8%.

[0047] In one embodiment, the mesenchymal stem cells are added to or mixed into the hydrogel after the formation of the nucleic acid hydrogel, thereby forming the cell gel formulation. In one embodiment, the mesenchymal stem cells are mixed together with a DNA assembly to form a nucleic acid hydrogel loaded with mesenchymal stem cells using conventional methods. In one embodiment, the mesenchymal stem cells are mixed with single-stranded DNA (3 scaffold units and 2 cross-linking units) to first form a cell-loaded DNA assembly, and then form a nucleic acid hydrogel.

[0048] In one embodiment, the cell gel formulation further comprises an anti-inflammatory agent, preferably selected from steroidal anti-inflammatory compounds (e.g., prednisolone, dexamethasone, betamethasone, triamcinolone), nonsteroidal anti-inflammatory compounds (e.g., ibuprofen, diclofenac, naproxen, etc.), antirheumatic drugs (e.g., methotrexate, leflunomide, etc.), anti-CD20 drugs, anti-cytokine drugs (e.g., anti-IL1, anti-IL6, anti-IL-17), anti-TNF drugs (e.g., infzumab, etanercept, adalimumab, rituximab, etc.) or mixtures thereof.

[0049] Secondly, the present invention relates to methods for treating or preventing acute and chronic osteoarthritis and related symptoms (especially osteoarthritis pain, mobility, or loss of function) using the aforementioned cell gel formulation. In one embodiment, the disease or symptom is selected from, for example, osteoarthritis, degenerative arthritis, knee joint disease, hip joint degeneration, and other general inflammatory conditions involving the joints, such as autoimmune diseases (especially rheumatoid arthritis and systemic lupus erythematosus (SLE), spondyloarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, enteropathic arthritis (associated with inflammatory bowel diseases such as hemorrhagic colitis and Crohn's disease), neuropathic arthritis, acute rheumatic fever, gout, chondrocalcinosis, hydroxyapatite crystal deposition disease, Lyme disease, and all other degenerative joint diseases.

[0050] Thirdly, the present invention relates to a method for reducing cell shear damage in a shear environment using a nucleic acid hydrogel, the method comprising: (i) providing a nucleic acid hydrogel comprising scaffold units and cross-linking units, and (ii) loading mesenchymal stem cells into the nucleic acid hydrogel; or the method comprising: (i) preparing scaffold units and cross-linking units of the nucleic acid hydrogel respectively, and (ii) mixing mesenchymal stem cells with the scaffold units and cross-linking units to obtain the cell gel formulation of the present invention through self-assembly; or the method comprising determining the shear protection effect of the cell-loaded nucleic acid hydrogel on cells by means of cell injection experiments, etc.

[0051] Fourthly, the present invention relates to the use of cell gel formulations in the preparation of medicaments for the treatment or prevention of acute and chronic osteoarthritis and related symptoms of inflammatory origin.

[0052] In one embodiment, the nucleic acid hydrogel is as described above. In one embodiment, the mesenchymal cells are embedded or dispersed in the nucleic acid hydrogel. In one embodiment, the shearing environment includes injection processes and intra-articular friction.

[0053] Example

[0054] Experimental materials

[0055] The nucleic acid hydrogel was prepared using the method disclosed in CN201610740754X: First, the DNA single strands (Y1, Y2, Y3) and (L1C and L2C) shown in Table 1 were self-assembled into DNA assemblies (which consisted of 1 mmol / L scaffold units and 1.5 mmol / L crosslinking units) in an aqueous medium; then, they were mixed to form the desired DNA hydrogel sample (i.e., nucleic acid hydrogel).

[0056] Table 1. DNA sequence information used to construct the DNA hydrogel

[0057]

[0058] ε represents the absorption coefficient, L / mol·cm. Sequences Y1, Y2 and Y3 form Y-scaffold units; sequences L1C and L2C form crosslinking units.

[0059] The cell culture medium was Minimum Essential Medium (MEM)α (Gibco, Thermo Fisher Scientific, USA).

[0060] The cell viability / death dye kit was Calcein-AM / PI (Solarbio, Beijing).

[0061] Rheological tests were performed using a Malvern (Komexus) rheometer; cell imaging was performed using a Zeiss (LSM 710Meta) laser scanning confocal microscope.

[0062] Example 1: Preparation of nucleic acid hydrogel loaded with mesenchymal stem cells

[0063] A certain volume of stem cell suspension (derived from rabbit bone marrow mesenchymal stem cells, extracted in the laboratory) was used to fully dissolve the 1 mmol / L Y-scaffold unit in Table 1 above, and an equal volume of solution was used to fully dissolve the 1.5 mmol / L crosslinking unit in Table 1 above. The two were then mixed to form a gel (which gelled within seconds). The final cell concentration in the resulting nucleic acid hydrogel was 1 × 10⁻⁶. 7 / ml, and the nucleic acid solid content is 3.8% (mass percentage).

[0064] Example 2: Rheological testing of cell-loaded nucleic acid hydrogels

[0065] The nucleic acid hydrogel loaded with mesenchymal stem cells prepared in Example 1 was removed using a spatula and placed on the test stage of the rheometer. The conical plate of the rheometer was adjusted to slowly descend until it contacted the hydrogel sample, and the final distance between the conical plate and the flat plate was fixed at 150 μm. This section involves two rheological tests:

[0066] The first method is the time-scan test, which involves continuously shearing the sample over a certain period of time while maintaining a constant scanning frequency. This tests the sample's shear resistance, and the mechanical strength of different samples is further reflected by comparing the ratio of the two moduli. The parameters set for this experiment were: Strain = 1%, frequency 1Hz, and constant temperature 25℃.

[0067] The second method is strain sweep testing, which measures the changes in the two moduli of different samples under different strains to reflect the shear-thinning behavior of the samples. The parameters set for this experiment are: strain range of 0.1% to 1000%, frequency of 1 Hz, and constant temperature of 25℃.

[0068] Figure 1 A and Figure 1 B represents the rheological test results of the cell-free hydrogel in time-mode and strain-mode tests. Figure 1 C and Figure 1 D represents the rheological results of the cell-containing hydrogel in time-mode and strain-mode tests. From Figure 1 It can be seen that the nucleic acid hydrogel loaded with mesenchymal stem cells still has good mechanical strength to support the cells, and at the same time still has shear-thinning properties.

[0069] Example 3: Cell shear protection by nucleic acid hydrogel loaded with cells

[0070] This example investigated the shear protection effect of the nucleic acid hydrogel prepared in Example 1 on cells during cell injection and in a simulated joint shearing environment.

[0071] Cell injection experiments were performed on the nucleic acid hydrogels containing cells from Example 1 using a 29G insulin syringe (needle diameter only 330 μm), with cells in culture medium serving as a control. Cell viability was characterized by live / dead staining after injection. Experimental results are as follows: Figure 2 and Figure 3 As shown. Figure 2 The diagram shows the process of traditional injection and nucleic acid hydrogel injection of cells, as well as the cell viability before and after injection using Calcein-AM / PI staining. Figure 2 Row A shows the injection method, row B shows the distribution of live cells (live cells are green highlights (original image) or white dots (black and white image)), row C shows the distribution of dead cells (dead cells are red highlights (original image) or light gray dots (black and white image)), and row D shows the distribution of live / dead cells. For convenience, row C lists the distribution of dead cells separately. Figure 2 As shown in line C, before injection, traditional injection methods revealed a significant number of dead cells, for example... Figure 2 In region 1C, no dead cells were found prior to injection, even with nucleic acid hydrogel protection. Figure 2 In the 3C region; after injection, traditional injection methods lead to a large number of cell deaths, for example... Figure 2 In the 2C region; however, after injection protected with nucleic acid hydrogel, almost no cell death occurred, for example... Figure 2 The 4C region indicates that the nucleic acid hydrogel loaded with mesenchymal stem cells can provide cell protection (i.e., pre-injection protection), and the protective effect of the nucleic acid hydrogel loaded with mesenchymal stem cells is particularly pronounced after injection.

[0072] To simulate the shearing environment of a joint, the nucleic acid hydrogel loaded with mesenchymal stem cells from Example 1 was placed between two coverslips spaced 330 μm apart. The shearing environment of a joint was simulated by bidirectional sliding of the two coverslips. After 2 minutes of bidirectional sliding, cell viability was characterized by live / dead staining. Experimental results are as follows: Figure 4 and Figure 5 As shown. Figure 4 This diagram simulates a joint shearing environment and shows the cell viability before and after friction in aqueous culture medium and hydrogel, marked with Calcein-AM / PI staining. Friction in aqueous medium leads to significant cell death, for example... Figure 4 In region 2A (dead cells are shown as red bright spots (original image) or light gray dots (black and white image)), almost no dead cells were observed when using nucleic acid hydrogel.

[0073] These two experimental results demonstrate that, compared to direct injection of cell suspension, this nucleic acid hydrogel provides shear protection for cells during injection and in a simulated joint shear environment, making it virtually impossible to observe cell death and significantly improving cell survival in shear environments. Specifically, the nucleic acid hydrogel provides shear protection during cell injection, increasing cell survival from 85% to over 99% compared to traditional injection methods; and in a simulated joint shear environment, the nucleic acid hydrogel provides shear protection, increasing cell survival from 75% to over 99% compared to cells in culture medium.

[0074] Example 4: Nucleic acid hydrogel loaded with mesenchymal stem cells was injected intra-articularly into the knee joint for the treatment of knee osteoarthritis in rabbits. Arthritis

[0075] Male, skeletally mature New Zealand rabbits (age: 6 months, weight: 3.5±0.5 kg, n=60) were purchased from the Peking University Animal Management Center. Osteoarthritis in the rabbits was induced by anterior cruciate ligament transection and medial meniscectomy. All animal experimental procedures were approved by the Peking University Animal Protection and Use Committee and complied with the "Guidelines for the Protection and Use of Laboratory Animals" (National Academy of Sciences Press, National Institutes of Health, No. 85-23, revised in 1996). The specific steps were as follows: After anesthesia and routine aseptic preparation, the joint cavity was accessed through a medial patellar incision, the anterior cruciate ligament was transected, and the medial meniscus was completely removed. The anterior drawer test was used to confirm the loss of knee joint stability. Postoperatively, all animals were returned to their respective rabbit cages, allowed free movement, and given necessary infection prevention and pain relief treatment, and knee joint function rehabilitation was initiated.

[0076] All animals were randomly divided into five groups, with surgical and injection interventions performed on the left knee, respectively. These included: 1. Sham surgery group: simple skin incision and suturing; 2. Blank group: simple rabbit knee osteoarthritis modeling surgery; 3. PDS group: rabbit knee osteoarthritis treated with pure nucleic acid hydrogel injection; 4. MSCs group: rabbit knee osteoarthritis treated with pure mesenchymal stem cell injection; 5. Hybrid group: rabbit knee osteoarthritis treated with nucleic acid hydrogel loaded with mesenchymal stem cells. All injections were performed at week 8 post-osteoarthritis modeling, once a week for a total of 3 weeks. The PDS group received 200 μl of nucleic acid hydrogel per knee per injection, while the MSCs group received 200 μl of basal culture medium cell suspension per knee per injection, with a cell concentration of 1 × 10⁻⁶ cells. 7 / ml, the Hybrid group received 200μl of the nucleic acid hydrogel loaded with mesenchymal stem cells from Example 1 per knee injection, with a cell concentration of 1×10⁻⁶ cells / ml. 7 / ml. At 12 and 24 weeks after the completion of injection treatment, rabbits in each group were euthanized, and knee joint samples were collected for further research.

[0077] Gross observations revealed that the blank control group and PDS group exhibited progressively worsening cartilage erosion and defects over time, while the MSCs treatment group and Hybrid treatment group showed significant improvement in cartilage damage. The Hybrid group, in particular, showed gross improvement closer to normal cartilage (sham group). Further analysis of the tissue samples using HE, safranin-O-green, toluidine blue staining, and type II collagen immunohistochemical staining revealed that, compared to the MSCs treatment group, the cartilage morphology and type II collagen expression in the MSC-loaded nucleic acid hydrogel treatment group (Hybrid group) were closer to normal cartilage tissue, especially at week 24. Similarly, scanning electron microscopy (SEM) observation of the cartilage surface microstructure revealed that while the cartilage surface of the conventional aqueous solution-loaded MSCs treatment group (MSCs) lacked cracks, it still exhibited numerous small hill-like features. In contrast, the Hybrid group's cartilage surface was smoother, with only minor irregularities. The results are as follows: Figure 6 and 7 As shown.

[0078] Example 5: Effect of nucleic acid solid content on nucleic acid hydrogels loaded with mesenchymal stem cells

[0079] Nucleic acid hydrogels loaded with mesenchymal stem cells (MSCs) at nucleic acid solid contents of 0.95%, 1.9%, and 4.2% were prepared using a method similar to that in Example 1. The shear protection effect of these three types of MSC-loaded nucleic acid hydrogels on cells during injection was then tested in a manner similar to that in Example 3. At 24 hours after injection, similar to the 3.8% nucleic acid solid content, no dead cells were found in the MSC-loaded nucleic acid hydrogels with nucleic acid solid contents of 0.95%, 1.9%, and 4.2%. However, under the same preparation method, the MSC-loaded nucleic acid hydrogel with a nucleic acid solid content of 3.8% provided a more uniform cell distribution, more suitable nucleic acid strength, and favorable injection results, as shown below. Figure 8 As shown.

[0080] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cell gel formulation for treating osteoarthritis, the cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells, wherein 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 adhesive 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 crosslink adhesive end; and an aqueous medium; wherein the scaffold unit and the crosslinking unit are crosslinked through the scaffold adhesive ends and the crosslink adhesive ends in a base-complementary pairing manner to form a three-dimensional spatial network structure. The cell gel formulation has a nucleic acid solid content of 3.8%. The sequence of the support units is 5'- The sequences of the crosslinking units are 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3', 5'-CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3', and 5'-CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3', wherein the sequences of the crosslinking units are 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' and 5'-GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

2. The cell gel formulation of claim 1, wherein the molar ratio of scaffold units to crosslinking units in the nucleic acid hydrogel is 2:1 to 1:

3.

3. The cell gel formulation of claim 1, wherein the scaffold unit, the crosslinking unit, and the three-dimensional spatial network structure are in a stable crosslinked state under physiological conditions.

4. The cell gel formulation of claim 1, wherein the nucleic acid hydrogel has a dominant storage modulus G' relative to the loss modulus G" as measured by frequency scanning at 25°C and at a strain amplitude of 1% strain, for all strain values ​​from 0.1 to 100 rad / s.

5. The cell gel formulation of claim 1, wherein the scaffold unit or crosslinking unit of the nucleic acid hydrogel comprises a CpG sequence.

6. The cell gel formulation of any one of claims 1-5, wherein the mesenchymal stem cells are embedded or dispersed in the nucleic acid hydrogel.

7. The cell gel formulation of any one of claims 1-5, wherein the density of the mesenchymal stem cells is 10. 5 Up to 10 8 Cells per milliliter of cell gel formulation.

8. The cell gel formulation of any one of claims 1-5, wherein the cell gel formulation further comprises an anti-inflammatory agent.

9. Use of a cell gel formulation in the preparation of a medicament for a method of reducing cell shear damage in a shear environment, said cell gel formulation comprising a nucleic acid hydrogel loaded with mesenchymal stem cells, said nucleic acid hydrogel comprising: a scaffold unit comprising a scaffold core and at least three single-stranded nucleic acids bound to said scaffold core, each single-stranded nucleic acid having at least one scaffold adhesive end; and a crosslinking unit comprising a crosslinking core and at least two single-stranded nucleic acids bound to said crosslinking core, each single-stranded nucleic acid having at least one crosslink adhesive end; And an aqueous medium; wherein the scaffold unit and the crosslinking unit are crosslinked through the adhesive ends of the scaffold and the adhesive ends of the crosslinking in a base-complementary pairing manner to form a three-dimensional spatial network structure. The method includes (i) providing a nucleic acid hydrogel comprising a scaffold unit and a crosslinking unit, and (ii) loading mesenchymal stem cells into the nucleic acid hydrogel; or (i) Scaffold units and cross-linking units of nucleic acid hydrogels were prepared separately, and (ii) mesenchymal stem cells were mixed with the scaffold units and cross-linking units to obtain nucleic acid hydrogels through self-assembly. The method further includes subjecting the cell gel formulation to a shear environment. The shearing environment mentioned above includes the injection process and intra-articular friction. The drug is used to treat osteoarthritis. The sequence of the support units is 5'- CGATTGACTCTCCACGCTGTCCTAACCATGACCGTCGAAG-3', 5'- CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3' and 5'- CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3', wherein the sequence of the crosslinking unit is 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' and 5'- GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

10. The use of claim 9, wherein the molar ratio of scaffold units to crosslinking units in the nucleic acid hydrogel is 2:1 to 1:

3.

11. The use of claim 9, wherein the scaffold unit, the crosslinking unit, and the three-dimensional spatial network structure are in a stable crosslinking state under physiological conditions.

12. The use of claim 9, wherein the nucleic acid hydrogel has a dominant storage modulus G' relative to the loss modulus G', as measured by frequency scanning at 25°C and at a strain amplitude of 1% strain, for all strain values ​​from 0.1 to 100 rad / s.

13. The use of claim 9, wherein the scaffold unit or crosslinking unit of the nucleic acid hydrogel comprises a CpG sequence.

14. Use according to any one of claims 9-13, wherein the mesenchymal stem cells are embedded or dispersed in the nucleic acid hydrogel.

15. The use according to any one of claims 9-13, wherein the density of said mesenchymal stem cells is 10-1. 5 Up to 10 8 Cells per milliliter of cell gel formulation.

16. Use according to any one of claims 9-13, wherein the cell gel formulation further comprises an anti-inflammatory agent.

17. The use according to any one of claims 11-13, wherein the nucleic acid solid content of the cell gel formulation is 3.8%.

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