Stem cell composition with knee joint injury repair function and preparation method thereof

Abstract

The present invention provides a stem cell composition with knee joint injury repair function and a preparation method thereof, wherein the stem cell composition comprises mesenchymal stem cells and anti-NGF antibodies, wherein the anti-NGF antibodies comprise a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2; and the mesenchymal stem cells are adipose mesenchymal stem cells that have been activated. The activated adipose mesenchymal stem cells are used in combination with the anti-NGF antibodies to promote knee arthritis repair, inhibit the expression of inflammatory factors, and relieve pain symptoms.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a stem cell composition with knee joint injury repair function and a preparation method thereof. Background Art

[0002] Knee joint injury is a common orthopedic disease, and osteoarthritis (OA) is a degenerative disease. It is the most common and difficult to cure type of knee joint injury. It can be divided into primary OA and secondary OA. The cause of primary OA is unknown, while the cause of secondary OA is clear, including endocrine system disorders, anatomical abnormalities, post-traumatic arthritis and inflammatory arthritis (see Koh YG, Choi YJ Infrapatellar fat pad-derived mesenchymal stem cell therapy for knee osteoarthritis. The Knee, 2012, 19(6): 902–907). OA is characterized by a decrease in articular chondrocytes and destruction of the joint matrix. Specific symptoms include persistent chondrocyte damage, loss of articular chondrocytes, subchondral microfractures, subchondral bone exposure, and hyperplasia of the joint margin and subchondral bone (see Ghosh P., Smith M. Osteoarthritis, genetic and molecular mechanisms. Biogerontology, 2002, 3(1–2): 85–88; Espinoza F, Matas J, Orrego M, et al. Allogeneic mesenchymal stromal cell (MSC) therapy for knee osteoarthritis (OA): a phase I / II randomized controlled trial. Cytotherapy, 2017, 19(5): 24). Clinically, OA patients present with slowly progressive joint pain, joint stiffness, joint swelling, reduced range of joint motion, and joint deformity (see Hootman JM, Helmick CG, Barbour KE, et al. Updated projected prevalence of self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitation among US adults, 2015-2040. Arthritis Rheum, 2016, 68(7): 1582–1587).

[0003] Epidemiological statistics show that the overall prevalence of primary OA in people over 40 years old is 46.3%, including 41.6% in men and 50.4% in women. The incidence rate in the middle-aged and elderly population can reach 40% and 80%. When the condition is mild, it affects the quality of life. When the condition is severe, it can cause disability, and the final disability rate can reach more than 50% (see Zhu Y, Wang Y, Zhao B, et al. Comparison of exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells and synovial membrane-derived mesenchymal stem cells for the treatment of osteoarthritis. Stem Cell Res Ther, 2017, 8(1): 64). Therefore, OA has become a serious health problem.

[0004] In addition, sports activities are directly related to the subsequent development of knee OA. Sports activities performed by professional athletes are a significant risk factor for knee OA. According to statistics (see Driban JB, Hootman JM, Sitler MR, et al. Isparticipation in certain sports associated with knee osteoarthritis? J AthlTrain, 2017, 52(6): 497-506), the prevalence of OA is significantly increased in specific sports, and the prevalence of knee osteoarthritis is football (OR is 3.5), elite long-distance running (OR is 3.3), competitive weightlifting (OR is 6.9) and wrestling (OR is 3.8), respectively. Elite sports (football or orienteering) and non-elite sports participants have a greater prevalence. Therefore, knee OA has also become one of the serious diseases that plague professional athletes.

[0005] Currently, the treatment of OA is very limited, and conventional treatments include physical therapy, nonsteroidal anti-inflammatory drugs, analgesics, hyaluronic acid, platelet-rich plasma (PRP) or corticosteroid intra-articular injections, traditional Chinese medicine, and knee arthroscopy (see Dantas LO, Salvini TF and McAlindonb TE, Knee osteoarthritis: key treatments and implications for physical therapy, Braz J Phys Ther, 2021, 25(2): 135–146). All of the above treatments can only relieve symptoms but cannot repair cartilage. As OA worsens, total knee replacement is required (see Jones I A., Togashi R., Wilson M L. et al. Intra-articular treatment options for knee osteoarthritis. Nat Rev Rheumatol, 2019, 15(2): 77–90).

[0006] Mesenchymal stem cells (MSCs) have the potential for self-renewal and multidirectional differentiation, and can play a therapeutic role in a variety of diseases through directional differentiation, immune regulation, anti-inflammation, angiogenesis, improvement of microenvironment and promotion of regeneration. MSCs have been used to treat a variety of diseases, such as premature ovarian failure, Parkinson's disease, nervous system damage and amyotrophic lateral sclerosis. MSC therapy can be used to treat OA and has shown encouraging results. Stem cell therapy for OA will become a milestone in regenerative medicine.

[0007] Studies have shown that MSCs have the following functions: (1) interact with the immune system and promote immune regulation (see Coulson-Thomas VJ, Coulson-Thomas YM, Gesteira TF, et al. Extrinsic and intrinsic mechanisms by which mesenchymal stem cells suppress the immune system. Ocul Surf, 2016, 14(2): 121–134); (2) migrate to the site of injury, enhance the tolerance of peripheral tissues, inhibit the release of inflammatory factors, promote the repair of damaged tissues, and increase the activity of damaged cells (Ryu JS, Jung YH, ChoM Y, et al. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodiumnitroprusside-stimulated chondrocytes. Biochem Biophys Res Commun, 2014, 447(4): 715–720); (3) have great multidirectional differentiation and proliferation potential; (4) secrete a variety of cytokines, such as transforming growth factor β1, hepatocyte growth factor, fibroblast growth factor and vascular endothelial growth factor, which have anti-inflammatory, anti-apoptotic, anti-fibrotic, pro-angiogenic, pro-mitotic and pro-wound healing effects (see Susanne G., Julia L. Tissue-engineering strategies to repair chondral and osteochondral tissue inosteoarthritis: use of mesenchymal stem cells. Curr Rheumatol Rep, 2014, 16(10): 452).The biggest advantage of MSCs is that they can improve or even repair cartilage (see Im GIT issue engineering inosteoarthritis: current status and prospect of mesenchymal stem cell therapy. BioDrugs, 2018, 32(4): 183–192), and regenerate damaged cartilage. This is also one of the biggest reasons why knee joint injection of MSCs may become a mainstream treatment method in the future. The factors secreted by MSCs can target synovial and articular chondrocytes, regulate anabolic and catabolic factors, and thus induce / mediate tissue regeneration. Other studies have shown that MSCs can stimulate the proliferation and anabolic activity of articular chondrocytes, or promote the recruitment and chondrogenic differentiation of stem / progenitor cells (Saulnier N., Viguier E., Perrier-Groult E, et al. Intra-articular administration of xenogeneic neonatal mesenchymal stromal cells early after meniscal injury downregulates metalloproteinase gene expression in synovium and preventscartilage degradation in arabbit model of osteoarthritis. Osteoarthritis Cartilage, 2015, 23(1): 122–133).

[0008] The above evidences all indicate that MSCs can effectively treat OA. Researchers have summarized and analyzed 15 RCTs and 4 retrospective studies involving a total of 584 patients, comparing the efficacy of MSCs treatment with traditional treatment. The results showed that MSCs treatment is safe and effective, and can significantly reduce postoperative pain (see Song YC, Zhang JH, Xu HL, et al. Mesenchymal stem cells in knee osteoarthritis treatment: A systematic review and meta-analysis J Orthop Translat., 2020, 24: 121–130.).

[0009] However, there are also certain limitations in stem cell treatment of OA, such as doubts about safety, different methods for culturing and preserving MSCs, certain risks in clinical application, and MSCs do not seem to have satisfactory performance in relieving pain, etc. To this end, the present invention provides a stem cell composition with knee joint injury repair function and a preparation method thereof, so as to overcome the above-mentioned defects and provide an effective and process-controllable stem cell treatment method for OA. Summary of the invention

[0010] The first aspect of the present invention provides a stem cell composition, comprising mesenchymal stem cells and anti-NGF antibodies, wherein the anti-NGF antibodies comprise a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2.

[0011] NGF not only plays an important role in the maintenance and development of the sensory nervous system, but is also a major cause of inflammation and pain (see Lewin GR, Mendell L M. Nerve growth factor and nociception. Trends Neurosci, 1993, 16 (9): 353-9). Lewin et al. reported that systemic injection of NGF can induce thermal and mechanical hyperalgesia (see Lewin GR, Rueff A, Mendell L M. Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur. J Neurosci, 1994, 6 (12): 1903-12.). In addition, systemic injection of anti-NGF antibodies can reduce abnormal pain and hyperalgesia in animal models of neuropathic pain (including nerve trunk or spinal nerve ligation) (see Wild KD, Bian D, Zhu D, et al. Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance, J Pharmacol. Exp. Ther, 2007, 322 (1): 282–7). Some researchers have used anti-NGF antibodies to treat OA. In a mouse knee OA pain model, intraperitoneal injection of anti-NGF antibodies inhibited this gait disorder and the upregulation of CGRP in DRG neurons, indicating that anti-NGF therapy may be valuable for the treatment of knee OA pain (see Miyagi M, Ishikawa T, Kamoda H, et al. Efficacy of nerve growth factor antibody in a kneeosteoarthritis pain model in mice, BMC Musculoskelet Disord, 2017, 18: 428). Therefore, in the present invention, a novel anti-NGF antibody is screened and obtained, and it is combined with mesenchymal stem cells to form a therapeutic composition, in order to effectively alleviate OA symptoms and accelerate the treatment process of OA.

[0012] Furthermore, the mesenchymal stem cells are selected from at least one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

[0013] Furthermore, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0014] Furthermore, the preparation method of the adipose mesenchymal stem cells includes: cutting the adipose tissue into pieces, washing the adipose tissue, filtering the adipose tissue through a 100-mesh filter, and digesting with collagenase; collecting the cells by centrifugation, washing the cells again with PBS and collecting the cells by centrifugation; resuspending in culture medium, and subculturing at 37°C and 5% CO2 for a total of 2-5 subcultures.

[0015] Furthermore, the preparation of adipose-derived mesenchymal stem cells also includes an activation step, which includes inoculating ADSCs cells and culturing them until the cell confluence reaches more than 70%, adding BMP-9 with a final concentration of 100 ng / mL and incubating for more than 24 hours.

[0016] Furthermore, the composition is in the form of an injection.

[0017] The second aspect of the present invention provides the use of the stem cell composition in the preparation of a drug for repairing knee joint injuries.

[0018] Furthermore, the knee joint injury is knee arthritis.

[0019] The third aspect of the present invention provides a method for preparing the stem cell composition, comprising: extracting adipose mesenchymal stem cells from adipose tissue and activating them using BMP-9; preparing an anti-NGF antibody, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2.

[0020] Beneficial Effects

[0021] The present invention provides a stem cell composition having the function of repairing knee joint injury and a preparation method thereof, with the following specific beneficial effects:

[0022] (1) Provide a novel anti-NGF antibody that can specifically bind to the target antigen;

[0023] (2) The preparation and activation methods of adipose-derived mesenchymal stem cells were optimized. The use of BMP-9 to activate adipose-derived mesenchymal stem cells can effectively promote the proliferation of chondrocytes.

[0024] (3) The combination of activated adipose-derived mesenchymal stem cells and anti-NGF antibodies can promote the repair of knee arthritis, inhibit the expression of inflammatory factors, and relieve pain symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Transwell co-culture model;

[0026] Figure 2 :ADSCs promote chondrocyte proliferation;

[0027] Figure 3 : Stem cell therapy improves OA pain severity;

[0028] Figure 4 :Observation of tissue morphology in OA animal models;

[0029] Figure 5 :Stem cell therapy inhibits the secretion of inflammatory factors. DETAILED DESCRIPTION

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0031] Example 1 Preparation and induced activation of adipose-derived mesenchymal stem cells

[0032] 1.1 Preparation of adipose-derived mesenchymal stem cells

[0033] Adipose tissue from healthy volunteers was obtained and rinsed three times with phosphate buffered saline (PBS) containing penicillin and streptomycin. The adipose tissue was cut into pieces, repeatedly rinsed with sterile PBS, filtered through a 100-mesh filter, added with 1% type I collagenase, and digested on a shaker at 37°C for 1 hour; the cells were collected by centrifugation at 1000 rpm for 5 min; the cells were washed again with PBS and centrifuged at 1000 rpm for 5 min to collect the cells; the cells were resuspended in DMEM medium containing 10% fetal bovine serum, and cultured at 37°C and 5% CO2. The medium was changed every 48 hours. When the density reached 80%, 0.25% trypsin was used for digestion and passage, and the third generation of adipose-driven stem cells (ADSCs) were taken for subsequent experiments.

[0034] 1.2 Activation of Adipose-Derived Mesenchymal Stem Cells

[0035] The biological activity of mesenchymal stem cells is regulated by a variety of cytokines in the body. Among the numerous cytokines and chemical regulators, the present invention selects bone morphogenetic protein 9 (BMP-9) as an activator, which can effectively regulate the activity of mesenchymal stem cells, promote osteogenic differentiation and subsequent knee injury treatment.

[0036] ADSCs cells were inoculated and cultured at 37°C and 5% CO2 until the cell confluence reached more than 70%. BMP-9 (purchased from RD System) with a final concentration of 100 ng / mL was added and incubated for 24 h. The cells were collected by trypsin digestion to obtain activated ADSCs cells, which were recorded as AC-ADSCs.

[0037] 1.3 Adipose-derived mesenchymal stem cells promote chondrocyte proliferation

[0038] In this section, the effect of ADSCs on chondrocytes was investigated in the Transwell co-culture model. The normal human chondrocyte cell line C28 / I2 (purchased from Millipore, USA) was used as the experimental object. C28 / I2 cells were inoculated and cultured until the logarithmic growth phase, and then digested and centrifuged to collect the cells. C28 / I2 cells were cultured at 5×10 3 The cells were seeded at a density of 5×10 cells / well in the lower layer of the Transwell chamber, and then the cells were divided into three groups, each with 3 replicate wells, namely: ADSCs group, 5×10 cells / well were seeded in the upper layer of the Transwell chamber 3 In the AC-ADSCs group, 5×10 3 AC-ADSCs; in the control group, no cells were seeded on the upper layer. Figure 1 As shown, ADSCs (1) were seeded on the upper layer, and C28 / I2 cells (2) were seeded on the lower layer.

[0039] After inoculation, culture at 37°C and 5% CO2 for 24h. Cell viability was detected by MTT method. The upper chamber of Transwell was removed, and 10μL MTT solution (5mg / mL) was added to the lower chamber in the dark, and the cells were incubated in the incubator in the dark for 4h. After incubation, the old culture medium in the well was discarded, and 150μL DMSO was added to each well in the dark to terminate the reaction. Finally, the absorbance of each well at 490nm was detected by microplate reader. Relative cell viability = (OD value of experimental well - OD value of blank control well) / (OD value of control well - OD value of blank control well), with the well of single culture medium as blank control well.

[0040] The results are as follows Figure 2 Mesenchymal stem cells can significantly promote the proliferation of chondrocytes, and ADSCs activated by BMP-9 have a stronger ability to promote proliferation, which is beneficial to the subsequent treatment of knee injuries and functional recovery.

[0041] Example 2 Preparation of anti-NGF antibody

[0042] After recombinant human NGF (purchased from Beijing Yiqiao Shenzhou Company) was mixed with equal volumes of Freund's complete adjuvant, BALB / c mice were immunized for the first time; the second immunization was performed 21 days later, and booster immunization was performed every 2 weeks thereafter, for a total of 5 immunizations. One week after the last immunization, mouse spleen cells were taken and fused with SP2 / 0 cells to obtain hybridoma cells and identify positive clones. The antibody titer was determined using the indirect ELISA method, and antibodies with higher titers were selected and analyzed using a surface plasmon resonance instrument. 8K (purchased from Cytiva) to identify the affinity of the antibody to the target antigen, and the antibody with the highest affinity (Kd value up to 2.68 nM) was selected for subsequent experiments.

[0043] According to bioinformatics identification, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0044] Example 3 Adipose-derived mesenchymal stem cell composition for treating OA in animal models

[0045] 3.1 OA animal model preparation and drug administration

[0046] Healthy SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution; the right knee joint was depilated and disinfected, the knee joint was flexed 90 degrees, and the patella, femoral condyle and patellar ligament were palpated and located. A microsyringe was used to insert the needle close to the femoral condyle under the patella on both sides of the patellar ligament, and 50 μL of 8% sodium iodoacetate (MIA) solution was injected into it. Then the knee joint was massaged to promote uniform distribution of the solution. After 7 days, obvious swelling and pain in the rat knee joints were observed, and the rats developed lameness, indicating that the knee OA model was successfully established.

[0047] OA animals were randomly divided into 3 groups, 10 in each group: AC-ADSCs group, 1×10 6 AC-ADSCs; AC-ADSCs+antibody group, 1×10 6 AC-ADSCs were injected with 100 mg / kg anti-NGF antibody every 2 days; the control group was injected with an equal volume of saline every 2 days. Each group was intervened for 4 weeks, and follow-up tests were performed after 4 weeks.

[0048] 3.2 Pain assessment

[0049] In this section, Von-Frey fibers were used to measure the mechanical paw withdrawal threshold (PWT) of the rat model to evaluate mechanical and thermal allodynia. The specific method was based on the literature (Fernihough J, Gentry C, Malcangio M, et al. Pain-related behavior in two models of osteoarthritis in the rat knee. Pain, 2004, 112: 83–93). Specifically, von Frey fibers were used to vertically stimulate the center of the rat's hind paw with increasing intensity. If the rat quickly withdrew or licked its paw, it was considered a positive response. Then, adjacent fibers with decreasing intensity were selected for stimulation. If the rat's hind paw had a negative response, adjacent fibers with increasing intensity were selected for stimulation until the rat's hind paw had a positive response. If there were 3 positive responses in 5 consecutive stimulations, the von Frey fiber value was defined as PWT.

[0050] like Figure 3 As shown, although stem cell therapy can relieve pain, the degree of improvement is low; after using anti-NGF antibodies, PWT can be significantly increased and pain can be significantly improved, indicating that the combination of adipose mesenchymal stem cells provided in the present invention and anti-NGF antibodies can effectively reduce pain in OA and relieve OA symptoms.

[0051] 3.3 Observation of knee tissue morphology

[0052] The rats were killed after 4 weeks, and the knee tissues were fixed in paraformaldehyde. 4 μm thick tissue sections were cut, stained with HE, and observed under a microscope. Figure 4 As shown, in the control group, chondrocytes were arranged irregularly, the whole layer was disordered, the cartilage structure was unclear, the cartilage was thinned, a large number of fibrocartilage cells appeared, and the tide line was incomplete; in the AC-ADSCs group, the cartilage structure was improved, the cartilage thickness increased, the arrangement state tended to be regular, and the inflammatory cells decreased; in the AC-ADSCs+antibody group, the cartilage structure was significantly improved, the layers were evenly distributed, there were no cracks, and the chondrocytes were arranged in an orderly manner. This shows that AC-ADSCs+antibodies can effectively treat OA and promote the repair of cartilage tissue after injury.

[0053] 3.4 Detection of IL-1β expression in joint fluid

[0054] IL-1β is the main inflammatory factor in OA. Its increased expression level can mediate multiple tissue and cell damage, making OA more severe. In this section, after 4 weeks of treatment, various rat knee joint fluids were collected, centrifuged at 3000r / min for 15min, and the supernatant was collected. The ELISA kit (purchased from Shanghai Future Industrial Co., Ltd.) was used to detect the IL-1β concentration in the supernatant. The specific steps were carried out according to the kit instructions.

[0055] The results are as follows Figure 5 As shown in the data, stem cell therapy can significantly reduce the expression level of IL-1β. When used in combination with anti-NGF antibodies, the expression of IL-1β is further inhibited, indicating that this treatment method can effectively inhibit the inflammatory response in OA.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-NGF antibody, characterized in that The anti-NGF antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

2.

2. A stem cell composition, characterized in that: The method comprises mesenchymal stem cells and the antibody according to claim 1.

3. The stem cell composition according to claim 2, characterized in that The mesenchymal stem cells are selected from at least one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

4. The stem cell composition according to claim 3, characterized in that The mesenchymal stem cells are adipose-derived mesenchymal stem cells.

5. The stem cell composition according to claim 4, characterized in that The preparation method of adipose mesenchymal stem cells comprises: cutting adipose tissue into pieces, washing the adipose tissue, filtering the adipose tissue through a 100-mesh filter, and digesting the cells with collagenase; collecting the cells by centrifugation, washing the cells again with PBS and collecting the cells by centrifugation; resuspending in a culture medium, and subculturing at 37° C. and 5% CO2 for 2-5 times in total.

6. The stem cell composition according to claim 5, characterized in that The preparation of adipose-derived mesenchymal stem cells also includes an activation step, which includes inoculating ADSCs cells and culturing them until the cell confluence reaches more than 70%, adding BMP-9 with a final concentration of 100 ng / mL and incubating for more than 24 hours.

7. The stem cell composition according to any one of claims 2 to 6, which is in the form of an injection.

8. Use of the stem cell composition according to any one of claims 2 to 7 in the preparation of a drug for repairing knee joint injuries.

9. The use according to claim 8, characterized in that: The knee joint injury is knee arthritis.

10. A method for preparing the stem cell composition according to any one of claims 2 to 7, comprising: Adipose mesenchymal stem cells were extracted from adipose tissue and activated using BMP-9; An anti-NGF antibody is prepared, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 2.

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