Intervertebral disc regeneration model as well as preparation method and application thereof

Abstract

The invention provides an intervertebral disc regeneration model, human nucleus pulposus cells (iHNPCs) and human fibrous ring cells (iHAFCs) reversibly immortalized by hTERT (human telomerase reverse transcriptase) are successfully constructed, and based on the two cells, after stimulation of chondrogenic differentiation factors and osteogenic factors, an iHAFCs / PPCNg mixture and an iHNPCs / GelMA hydrogel mixture are respectively loaded to the periphery and the central area of a 3D intervertebral disc-like structure of a POC support, so that the 3D intervertebral disc-like structure of the POC support is formed. After the intervertebral disc model is implanted under the skin of a nude mouse for 42 days, cartilage-like tissues exist in the central area of the intervertebral disc model, and mature bone tissues exist in the peripheral area of the intervertebral disc model. The intervertebral disc regeneration model simulates the structure of a natural intervertebral disc, provides an important biomedical engineering material for IVD tissue engineering, has potential application value in intervertebral disc degenerative diseases, and also provides an important means for researching intervertebral disc cell functions and developing an effective IVD tissue engineering method.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to an intervertebral disc regeneration model and a preparation method and application thereof. Background Art

[0002] Low back pain (LBP) is a common chronic disease affecting middle-aged and elderly people worldwide. The etiology of most LBP is intervertebral disc (IVD) degeneration (IVDD). The IVD is a highly specialized avascular tissue structure that provides separation and connection between vertebrae. The typical IVD is composed of different related tissues, including a central highly hydrated nucleus pulposus (NP), a surrounding elastic annulus fibrosus (AF), and a cartilaginous endplate (CEP) connecting the vertebral bodies. The NP is a water-rich tissue with a high concentration of proteoglycans that has the function of maintaining IVD swelling pressure and hydraulic shock absorption, while the AF has a layered structure formed by collagen fibers that can resist tensile and compressive strains. A healthy IVD requires a balance between matrix synthesis and degradation. When this balance is disturbed, it leads to intervertebral disc degeneration (IVDD), which is usually characterized by the loss and dysfunction of IVD cells and the exhaustion of IVD progenitor cells. IVD degeneration usually originates from the NP region and is usually manifested as dehydration of the nucleus pulposus with or without rupture of the annulus fibrosus.

[0003] In the early stages of IVDD, the goal of conservative treatment is to relieve pain and delay degenerative progression rather than to restore the structure and function of the IVD. In patients with advanced IVDD, the IVD often decompensates with NP protrusion, compressing the spinal cord and causing symptoms of spinal stenosis such as low back pain, limited lower limb mobility, numbness, incontinence and even paralysis. At this stage, invasive treatments are usually required, such as chemical nucleolysis, disc replacement, NP discectomy, elastic fixation and spinal fusion.

[0004] In recent years, tissue engineering to replace damaged IVDs with scaffolds and appropriate cells has emerged as a potential treatment for IVDD. Successful tissue engineering requires an ideal tripartite combination: differentiation-potential cells, biocompatible scaffolds, and differentiation-promoting factors. In the past few decades, significant progress has been made in the development of biocompatible scaffold materials for tissue engineering. Among them, citric acid-based polymers such as poly (1,8-octanediol-co-citric acid) (POC) and polyethylene glycol citrate-co-N-isopropylacrylamide (PPCN) have attracted much attention due to their excellent biocompatibility and degradability in regenerative medicine. Composite materials of POC and hydroxyapatite have been used to manufacture degradable implantable medical devices approved by the US FDA for musculoskeletal surgery; while temperature-responsive PPCN has been reported for various biomedical applications including biologic delivery. Another commonly used non-toxic biomaterial hydrogel, gelatinmethacryloyl (GelMA), has good mechanical strength, viscoelasticity and swelling capacity, and has been shown to promote cartilage or cartilage-like differentiation under high stiffness conditions.

[0005] Although human IVDs are incapable of self-repair and spontaneous regeneration, the discovery of progenitor cells from NPs, AFs, and CEPs has raised hopes for resurrecting degenerated IVDs by differentiating the respective progenitor cells into corresponding mature IVD cells to promote the production of IVD matrix and fulfill IVD functions. Although human primary NPCs and AFCs are theoretically suitable for IVD tissue engineering, primary AFCs and NPCs are difficult to obtain and have a limited lifespan, further hindering their potential application in preclinical and clinical studies. Therefore, the main challenges for effective IVD regeneration are to obtain healthy NPCs and AFCs for extensive cell phenotyping and to identify the most appropriate biological factors to maintain IVD health by supporting cell survival and promoting the production of a highly hydrated extracellular matrix for the IVD structure. Summary of the invention

[0006] Tissue engineering has become a potential treatment for IVDD. In order to solve the current situation that there is no suitable scaffold and cells to effectively replace the damaged intervertebral disc, the present invention provides an intervertebral disc regeneration model and its preparation method and application. The purpose of the present invention is achieved through the following technical solutions: The present invention provides an intervertebral disc regeneration model, comprising a three-dimensional disc-shaped structure of a mixture of human annulus fibrosus cells (HAFCs) and polyethylene glycol citric acid-poly N-isopropylacrylamide hydrogel (PPCNg), a mixture of human nucleus pulposus cells (HNPCs) and methacryloyl chloride gelatin (GelMA), and a poly-1,8-octanediol-co-citric acid (POC) scaffold material.

[0007] Furthermore, the human annulus fibrosus cells and human nucleus pulposus cells are reversibly immortalized human annulus fibrosus cells (iHAFCs) and human nucleus pulposus cells (iHNPCs).

[0008] Furthermore, the genomes of the above-mentioned reversibly immortalized human annulus fibrosus cells (iHAFCs) and human nucleus pulposus cells (iHNPCs) contain the human reverse transcriptase telomerase gene, and the gene has flippase recognition sites on both sides.

[0009] Furthermore, the human annulus fibrosus cells are stimulated with osteogenic factors, and the human nucleus pulposus cells are stimulated with chondrogenic factors.

[0010] Furthermore, the human annulus fibrosus cells are stimulated with BMP9, and the human nucleus pulposus cells are stimulated with BMP2.

[0011] Furthermore, the three-dimensional disk-shaped structure is a disk-shaped structure of 5 mm×3 mm (diameter×height), and the central gap is 1 mm×1 mm (diameter×height).

[0012] Furthermore, a mixture of human annulus fibrosus cells and PPCNg was added to the periphery of the disc-shaped structure of the above intervertebral disc regeneration model, and a mixture of human nucleus pulposus cells and GelMA was added to the central space.

[0013] Furthermore, the mixture of human annulus fibrosus cells and PPCNg was resuspended in 10% PPCNg to about 5×10 6 The mixture of human nucleus pulposus cells and GelMA, the human nucleus pulposus cells were resuspended to about 5×10 6 Pieces / ml.

[0014] The present invention also provides a method for preparing an intervertebral disc regeneration model, comprising the following steps: Step 1, stimulate reversibly immortalized human NPCs (iHNPCs) and AFCs (iHAFCs) with chondrogenic and osteogenic factors; Step 2: 3D print the POC stent, which is a disc-shaped structure of 5mm×3mm (diameter×height) with a central gap of 1mm×1mm (diameter×height); Step 3, adding a mixture of iHAFCs / PPCNg stimulated by BMP9 to the periphery of the scaffold and a mixture of iHNPCs / GelMA stimulated by BMP2 to the central space to establish an intervertebral disc regeneration model; In step 4, the assembled disc regeneration construct was implanted into the spinal segment and histologically evaluated after 6-8 weeks.

[0015] The present invention also provides application of the above intervertebral disc regeneration model in intervertebral disc degenerative diseases. Beneficial Effects

[0016] The present invention provides an intervertebral disc regeneration model, which successfully constructs reversibly immortalized human nucleus pulposus cells (iHNPCs) and human annulus fibrosus cells (iHAFCs). Based on these two cells, after stimulation by chondrogenic differentiation factors and osteogenic factors, the iHAFCs / PPCNg mixture and the iHNPCs / GelMA hydrogel mixture are loaded into the peripheral and central regions of the 3D intervertebral disc-like structure of the POC scaffold, respectively. After 42 days of subcutaneous implantation in nude mice, cartilage-like tissue exists in the central region of the intervertebral disc model, while mature bone tissue exists in the peripheral region. This intervertebral disc regeneration model simulates the structure of a natural intervertebral disc and provides important biomedical engineering materials for IVD tissue engineering.

[0017] The present invention reversibly immortalizes healthy human nucleus pulposus cells (iHNPCs) and annulus fibrosus cells (iHAFCs) by stably expressing hTERT. The immortalization process can be reversed by FLP recombinase and is not tumorigenic. iHAFCs show osteogenic differentiation under BMP9 stimulation, while iHNPCs show chondrogenic differentiation under BMP2 stimulation, providing valuable cell resources for studying intervertebral disc cell function and developing effective IVD tissue engineering methods.

[0018] The present invention also developed an ex vivo intervertebral fusion model, by replacing the native intervertebral disc with iHAFCs stimulated by BMP9, and subcutaneously implanting these engineered intervertebral fusion structures into athymic nude mice. There was obvious bone fusion in the intervertebral segment injected with iHAFCs, which provided an important means for studying the function of intervertebral disc cells and developing effective IVD tissue engineering methods. iHAFCs showed high osteogenic potential under BMP9 stimulation and had potential and significant application prospects in spinal fusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the intervertebral disc regeneration model process Figure 2Schematic diagram and microscopic morphology of iHAFCs and iHNPCs (A) Schematic diagram of primary cell isolation (B) Vector expression of hTERT and Flippase recognition target (FRT) (C) Cell morphology of iHAFCs and iHNPCs Figure 3 pMLV-hTERT lentiviral vector plasmid map Figure 4 Expression of iHAFC- and iHNPCs-specific markers The expression of AFC-specific genes (indicated by blue letters) and NPC-specific genes (indicated by red letters) was analyzed using TqPCR and compared with the human articular chondrocyte cell line C28 / 12.

[0020] Figure 5 iHAFC and iHNPC cells were infected with Ad-FLP or Ad-GFP (A) Crystal violet staining (a) and quantitative OD reading analysis (b); (B) Analysis of clonal proliferation capacity after removal of hTERT. After 72 hours of infection with Ad-FLP or Ad-GFP, cells were re-plated into 12-well plates at low density (about 50 cells per well), and microscopic images were taken on days 1, 3, 5, and 7 (a), and crystal violet staining was performed on day 7 (b); (C) Expression of hTERT after Ad-FLP infection; (D) Cell proliferation was evaluated by cell cycle analysis. All experiments were repeated three times, and "**" indicates p<0.01 compared with the Ad-GFP group. Figure 6 iHAFCs showed higher osteogenic potential under BMP9 stimulation. (A) BMP9-induced iHAFCs early osteogenic marker alkaline phosphatase (ALP) staining; (B) ALP activity; (C) BMP9-induced iHAFCs mineralized matrix formation, "**" indicates p<0.01 compared with the Ad-GFP group; (D) BMP9-induced iHAFCs osteogenic regulatory factors and marker expression, "**" indicates p<0.01 compared with the Ad-GFP group; (E) BMP9-stimulated iHAFCs ectopic bone formation, Micro CT imaging (a), H&E staining GFP group (b) and BMP9 group (c) Figure 7 The osteogenic potential of iHNPCs and the chondrogenic potential of iHAFCs were tested. (A) to (F) The osteogenic potential of iHNPCs was analyzed. The experimental methods were the same as those in Figure 6Same; (G) TqPCR analysis of chondrogenic markers in BMP2-induced iHAFCs, “*” indicates p < 0.05 compared with the GFP control group Figure 8 (A) Micromass staining of Alcian blue (a) and Safranin O (b) for the chondrogenic differentiation potential of iHNPCs; (B) Expression of chondrogenic differentiation regulatory factors and markers in BMP2-induced iHNPCs, "**" represents p<0.01 compared with Ad-GFP group. (C) In vitro GelMA3D culture, HE staining, Alcian blue staining and Safranin O staining Fig. 9 To detect the tumorigenicity of iHAFC and iHNPCs Fig.10 Biocompatibility of iHAFCs and iHNPCs with POC scaffolds and PPCNg and GelMA hydrogels in intervertebral disc (IVD)-like structures. (A) Experimental design of IVD-like structures loaded with cells; GFP signal levels (B) and (C) and GLuc activity (relative units, RU) (D) were evaluated at the indicated time points, with white arrows indicating newly infected cells under high magnification; (E) POC scaffolds have good biocompatibility, with white arrows indicating cells with different morphologies adhering to the scaffolds; (F) Co-seeding of GFP-labeled iHAFCs (periphery, PPCNg, green) and RFP-labeled iHNPCs (center, GelMA, red) Fig.11 Intervertebral disc (IVD) models were prepared for iHAFCs and iHNPCs. (A) Experimental design; (B) Micro CT imaging (a and b), the height of the removed POC disc was quantitatively calculated based on the 3D reconstruction of the micro CT imaging data (c), "*" indicates p < 0.05; (C) HE staining, Safranin O / Fast Green staining and Masson's trichrome staining, NB indicates new bone, SC indicates scaffold, NDC indicates NPC-derived cartilage or chondrocytes Fig.12(A) Experimental design of the cell- and gene-mediated in vitro model of intervertebral disc fusion; (B) Micro CT imaging of the implanted intervertebral segment (a), followed by quantitative analysis of the new bone density (HU) (b) and the relative volume of new bone (c) in the projected IVD area, with the newly formed bone structure indicated by arrows. "**" indicates p < 0.01, which is statistically significant compared with the control group. (C) Histological and special staining analysis: HE staining (a), Masson's trichrome staining (b), and modified PAS staining (c); the magnified area is shown in the right panel; NB indicates new bone; AF indicates annulus fibrosus; NP indicates nucleus pulposus; EP indicates endplate. DETAILED DESCRIPTION

[0021] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art in this field can make some non-essential improvements and adjustments to the present invention based on the above invention content.

[0022] Example 1 Construction of immortalized cell lines of human AFCs and NPCs 1. Primary cell culture: Primary cells were provided by the research team of the Department of Surgery and Rehabilitation (Philadelphia, PA) of the University of Pennsylvania Medical Center. The donor was a healthy young male who was injured in a car accident. The donor was informed and agreed to the research. The specimen was obtained with the approval of the Ethics Committee of the University of Pennsylvania Medical Center. Primary AFCs and NPCs were isolated from the annulus fibrosus and nucleus pulposus of the intervertebral disc ( Figure 2 -A), using DMEM medium containing 10% fetal bovine serum (FBS, Gemini Bio-Products), 100U / ml penicillin and 100U / ml streptomycin, and the cell culture environment was 37℃ and 5% carbon dioxide. Cells within 3 generations were used to prepare immortalized cell lines.

[0023] 2. Lentivirus construction: The pMLV-hTERT lentiviral vector plasmid was constructed by the Molecular Oncology Laboratory at the University of Chicago (https: / / boneandcancer.org / researchtools / ), as shown in the figure. Figure 3pMLV-hTERT expresses hTERT (NM_198253.3) and a hygromycin resistance gene linked to a self-cleaving E2A flanked by flippase recognition target sites (FRT) for removal of hTERT and reversal of immortalization using the recombinase flippase (FLP) ( Figure 2 -B), pMLV-hTERT (10 μg), packaging plasmid pCMV-VSVG (1 μg, Addgene) and psPAX2 (10 μg, Addgene) were co-transfected into HEK-293 cells using liposomes, and the culture supernatant containing hTERT lentiviral particles was collected 48 h and 72 h after transfection.

[0024] 3. Obtaining reversibly immortalized cell lines: Sub-confluent primary human AFCs and NPCs cells were infected with the collected lentiviral supernatant and screened with hygromycin B (0.3 mg / mL, Invitrogen) for 7-10 days. The immortalized human AFCs and NPCs cell pools obtained were named iHAFCs and iHNPCs, respectively. Both cell lines were passaged for more than 30 generations. From a morphological point of view, iHAFCs are fibroblast-like and grow faster than iHNPCs, while iHNPCs are more round in shape ( Figure 2 -C).

[0025] 4. Identification of iHAFCs and iHNPCs AFCs and NPCs express different marker genes, AFCs are osteoblast-like, while NPCs are more chondrogenic. Using the established human articular chondrocyte cell line C28 / 12, a comprehensive qPCR analysis of specific genes of AFCs and NPCs was performed. Primer primers were designed using the platform, and the specific sequences are shown in Table 1.

[0026] Compared with C28 / 12 cells, 7 of 10 AF-specific genes were significantly elevated in iHAFCs, while 16 of 25 NP-specific genes were significantly elevated in iHNPCs. When comparing iHAFCs and iHNPCs, 5 AF-specific genes were significantly elevated in iHAFCs, while 11 NP-specific genes were significantly elevated in iHNPCs ( Figure 4 ). The results showed that the established iHAFCs and iHNPCs represented different cell types, expressing AF- and NP-specific marker genes, respectively.

[0027] Table 1. PCR primer sequences for specific genes related to AFCs and NPCs

[0028]

[0029] 5. Introduction of FLP recombinase can reverse the immortalized phenotype: The immortalized system has two FRT sites that can be recombined by FLP to remove the immortalized hTERT gene, this process is called reverse immortalization. In order to identify the proliferation capacity of cell immortalization and the effect of reverse immortalization, the following experiments were carried out: (1) Construction, amplification and use of recombinant adenovirus Ad-GFP, Ad-RFP, Ad-GFP-Gluc, Ad-FLP, Ad-BMP2 and Ad-BMP9 used in this study all adopt AdEasy technology or Gibson DNA Assembly-based OSCA system. Ad-GFP and Ad-RFP are green fluorescence and red fluorescence as controls; Ad-GFP-Gluc co-expresses GFP fluorescence and secreted luciferase (GaussiaLuciferase, GLuc) for cell activity determination; Ad-FLP is an adenovirus expressing flippase (FLP) for reverse immortalization; Ad-BMP2 Ad-BMP9 can infect target cells to overexpress BMP2 and BMP9.

[0030] Construction process: The coding region of each gene is amplified by PCR. The adenovirus shuttle plasmid pAdTrack-CMV was cloned and then combined with the adenovirus backbone plasmid pAdEasy1. Homologous recombination was performed in BJ5183 competent cells. The recombinant plasmid was used as an adenovirus plasmid for adenovirus Packaging and amplification of adenovirus. Adenovirus packaging is to use PEI to transfect 293pTP or 293GP cells with RNA-free adenovirus plasmids after Pac I digestion. After waiting for 7-10 days, the packaged adenovirus is collected. Adenovirus collection adopts three freeze-thaw and three thaws, that is, 293pTP or 293GP cells are blown, centrifuged at 800rpm for 3 minutes after collection, and about 0.5-1ml supernatant is removed and retained. Then, it is repeatedly frozen and thawed three times by dry ice and 37℃ water bath, and centrifuged at 3000rpm for 5 minutes to collect the supernatant containing adenovirus. When amplifying, add the packaged adenovirus supernatant to HEK-293 or RAPA cells in appropriate amount, and collect high titer adenovirus after amplification for about 48 hours. When using adenovirus to infect target cells, add Polybrene to a final concentration of 6μg / ml to improve the infection efficiency. The determination of virus titer is determined by target cells, that is, the target cells are plated into a 24-well plate, gradient amounts of adenovirus and Polybrene are added, and the fluorescence is observed after 24 or 48 hours. It is generally considered that 50-70% of the target cells are infected as the appropriate virus titer range, and subsequent studies will make appropriate adjustments based on actual usage.

[0031] (2) Crystal violet cell proliferation assay: iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-FLP and Ad-GFP, respectively. After 24 hours, the cells were resuspended and plated in a 24-well plate at a cell density of about 30%. At the preset time point, the cells were resuspended again and plated in a new 24-well plate. This step was to remove unhealthy After the cells adhere to the wall for 4-6 hours, remove the supernatant, rinse with PBS, and then use Stain with 0.5% crystal violet-formalin solution for 10 minutes, remove the crystal violet, and rinse with water until there is no residue After that, let it dry and observe. Scan and take photos ( Figure 5 -Aa), the dye was dissolved with 10% acetic acid, and the absorbance of the obtained solution at 592nm was measured using a spectrophotometer (Cary 60 UV-Vis Spectrophotometer, Agilent, USA) as quantitative data. The results are shown in Figure 5 -Ab, the cell viability of the FLP group was weaker than that of the GFP control group.

[0032] (3) Single-cell colony proliferation assay. iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-FLP and Ad-GFP, respectively. After 72 hours, the cells were resuspended to a very low density and plated in a 12-well plate (approximately 50 cells / well). Cells / well). Cell morphology was recorded and observed at preset time points, and the results were analyzed on the last day. Crystal violet staining. Figure 5 -B, The proliferation rate of cells treated with FLP was significantly slower than that of the GFP control group.

[0033] (4) hTERT gene expression TqPCR: iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-FLP and Ad-GFP. After 36 hours, RNA was extracted and subjected to reverse transcription and TqPCR. Figure 5 -C, compared with the GFP control group, hTERT expression was significantly reduced after Ad-FLP infection, and the removal rate could reach 70-80%.

[0034] (5) Flow cytometric cell cycle analysis. iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-FLP and Ad-GFP, respectively. After 72 hours, the cells were resuspended in PBS and rinsed twice. Propidium iodide (PI, BD Bioscience, USA) was used to stain the cells for 20 minutes at 4°C in the dark. The cells were analyzed using a BD LSR II Flow Cytometer (USA) and FlowJo software (V3.05478). Cell cycle analysis was performed. Figure 5 -D, compared with the GFP control group, the proportion of cells in the G1 phase of Ad-FLP-infected cells increased significantly (iHAFCs increased from 33.60% to 52.49%, iHNPCs increased from 38.37% to 65.83%), while the proportion in the S phase decreased (iHAFCs decreased from 46.93% to 35.84%, iHNPCs decreased from 41.92% to 24.43%), and the proportion in the G2 phase also showed a downward trend (iHAFCs decreased from 18.20% to 10.83%, iHNPCs decreased from 18.95% to 9.33%).

[0035] Taken together, these results indicate that iHAFCs and iHNPCs have long-term proliferation capacity, which can be effectively reversed by FLP recombinase-mediated hTERT removal.

[0036] Example 2 Detection of biological properties of immortalized cells 1. iHAFCs showed higher osteogenic differentiation properties under BMP9 stimulation: (1) Alkaline Phosphatase (ALP) qualitative staining and quantitative reading test: iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-BMP9 and Ad-GFP, respectively. After 24 hours, the cells were resuspended and plated in 24-well plates at a cell density of about 30%. The cells were qualitatively stained for ALP and quantitatively read at the predetermined time points. For staining, the culture medium was aspirated, rinsed with PBS, and then stained with 0.1 mg / ml napthol AS-MX phosphate mixed with 0.6 mg / ml Fast Blue BB salt for 20 minutes in the dark. The stained cells were observed and recorded by a microscope or scanner. For reading, the culture medium was aspirated, the cells were lysed after PBS rinse, and the SEAP fluorescence kit Great EscapeSEAP Chemiluminescence assay kit (BD Clontech) was used for quantitative analysis.

[0037] The results showed that compared with the Ad-GFP group, the activity of the early osteogenic marker alkaline phosphatase (ALP) in the Ad-BMP9 group was induced on the first day and increased significantly on the seventh day ( Figure 6 -A). Quantitative ALP assay further confirmed these results, showing that the ALP activity of BMP9-stimulated iHAFCs was 7- to 10-fold higher than that of the GFP group on days 1, 3, 5, and 7 ( Figure 6 -B).

[0038] (2) Matrix mineralization test (Alizarin Red S staining): iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-BMP9 and Ad-GFP, respectively. After 24 hours, the cells were resuspended and plated in 24-well plates at a cell density of about 30%. After the cells adhered to the wall, the culture medium was replaced with matrix mineralization medium, that is, 0.5 mg / ml L-Ascorbic acid and 0.1 M Sodium β-glycerophosphate pentahydrate were added to the ordinary complete culture medium. At the predetermined time point, the culture medium was aspirated, and the cells were fixed at room temperature for 10 minutes by adding 2.5% glutaraldehyde, and then stained with 0.4% Alizarin red S solution for 5 minutes, and the residue was washed with water. The stained cells were observed and recorded by a microscope or scanner. The stain was then dissolved with 20% methanol + 10% acetic acid aqueous solution, and the absorbance of the obtained solution at 450 nm was measured by a spectrophotometer as quantitative data.

[0039] After 14 days of culture in mineralization medium, a large number of mineralized nodules were observed in the BMP9 group ( Figure 6-Ca, b). Quantitative analysis showed that BMP9-induced mineralized matrix increased by at least 6-fold compared with the GFP control group ( Figure 6 -Cc).

[0040] (3) TqPCR of osteogenic regulatory factors and markers. iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-BMP9 and Ad-GFP, respectively. After 36 hours, RNA was extracted and reverse transcribed and TqPCR was performed. Primers were designed using the Primer3 platform, and the specific sequences are shown in Table 2.

[0041] Table 2. PCR primer sequences of osteogenic regulatory factors and markers

[0042] The results showed that compared with the GFP control, the expressions of RUNX2, OSX, OCN, OPN and COL1A1 in the BMP9 stimulation group were significantly upregulated ( Figure 6 D).

[0043] (4) Detection of iHAFCs’ osteogenic capacity in vivo: Subcutaneous injection experiments were performed in skeletally mature thymic nude mice (Envigo, Indianapolis, IN; 6 weeks old, weighing 15-20 g, regardless of gender). The thermosensitive polymer PPCNg was synthesized and characterized. iHAFCs in the logarithmic growth phase were infected with Ad-BMP9 and Ad-GFP, respectively. After 36 hours, the cells were digested and resuspended with PPCNg to approximately 5 × 10 6 The cell suspension was injected into the subcutaneous tissues of both sides of the abdomen of nude mice using a 0.5 ml syringe (2-3 × 10 cells / 100 µl at each injection site). 6 Each nude mouse was injected with 6-8 masses. After 28 days of injection, the nude mice were sacrificed and the subcutaneous masses were collected for Micro-CT and histological analysis.

[0044] Micro CT imaging analysis of tumors removed from the Ad-BMP9 group showed significant bone formation, whereas no bone formation was observed in the Ad-GFP group ( Figure 6 -Ea). Histological analysis of the removed tumors showed that BMP9 induced significant trabecular bone formation ( Figure 6 -Eb), while the GFP group showed only residual PPCNg polymers and injected cells ( Figure 6 The above results indicate that iHAFCs exhibit high osteogenic potential stimulated by BMP9 both in vitro and in vivo.

[0045] It is worth mentioning that we conducted similar osteogenesis experiments in iHNPCs and found that they showed some but limited osteogenesis potential in vitro after BMP9 stimulation and also failed to induce significant new bone formation in vivo ( Figure 7 -AF); TqPCR analysis of chondrogenic markers in BMP2-induced iHAFCs ( Figure 7 -G), the results showed that its chondrogenic potential was limited.

[0046] 2. iHNPCs showed high chondrogenic differentiation properties under BMP2 stimulation.

[0047] (1) Micromass cell 3D culture: iHNPCs and C28 / 12 cells in the logarithmic growth phase were infected with Ad-BMP2 and Ad-GFP, respectively. After 36 hours, the cells were resuspended to approximately 5×10 6 / ml concentration. In a 48-well plate, 10μl of cell suspension was dropped into a hemispherical shape, and then placed in an incubator for 2 hours to allow the cells to sink to the bottom to form a micromass. Gently add 0.5ml of normal medium (NM) to the GFP-iHNPCs group, and chondrogenic differentiation medium (CDM) to the BMP2-iHNPCs group and BMP2-C28 / 12 group. CDM is based on NM, with 10% ITS + Premix Tissue Culture Supplement (Becton Dickinson), 0.1 μM dexamethasone (Sigma) and 1 μM ascorbate-2-phosphate (Sigma). GFP-NM-iHNPCs was the negative control group, BMP2-CDM-C28 / 12 was the positive control group, and BMP2-CDM-iHNPCs was the experimental group. Micromass was stained on culture days 1, 7, 14, and 21. For staining, the culture medium was aspirated, and the cells were fixed with 2.5% glutaraldehyde, followed by staining with Alcian blue (30 minutes) and Safranin O (15 minutes), and then the residual dye was rinsed with water until clear.

[0048] The results showed that compared with the positive control C28 / 12 cells under the same conditions, iHNPCs were able to effectively undergo chondrogenic differentiation when cultured in chondrogenic differentiation medium and stimulated with BMP2, as shown by Alcian blue staining ( Figure 8 -Aa) and Safranin O staining ( Figure 8In fact, BMP2-induced chondrogenic differentiation of iHNPCs was similar to that of C28 / 12 cells, showing a time-dependent manner, whereas no significant chondrogenic differentiation was observed in the GFP-stimulated iHNPCs group.

[0049] (2) TqPCR of chondrogenic regulatory factors and markers: iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-BMP2 (CDM culture) and Ad-GFP (NM culture), respectively. After 36 hours, RNA was extracted and reverse transcribed and TqPCR was performed. Primers were designed using the Primer3 platform, and the specific sequences are shown in Table 3.

[0050] Table 3. PCR primer sequences of cartilage regulatory factors and markers

[0051] The results showed that compared with the GFP control, the expression levels of SOX9, ACAN and COL2A1 in the BMP2 stimulation group were significantly upregulated ( Figure 8 -B).

[0052] (3) GelMA hydrogel cell 3D culture: GelMA hydrogel is a photocurable hydrogel. The specific synthesis is described below. iHNPCs and C28 / 12 cells in the logarithmic growth phase were infected with Ad-BMP2 and Ad-GFP, respectively. After 36 hours, the cells were resuspended in 10% GelMA to a volume of approximately 5×10 6 Pipette 100μl GelMA-cell suspension, add it into a sterilized ring mold, and cure it with UV light for 30 seconds to form a circular GelMA cell disk with a diameter of about 5mm and a thickness of 3mm. The cell disk was immersed in culture medium, and the GFP group was cultured with NM, and the BMP2 group was cultured with CDM. After 21 days of culture, the GelMA cell disk was removed, fixed, dehydrated, paraffin sectioned, and H&E staining, Alcian blue staining, and Safranin O staining were performed.

[0053] H & E staining showed that compared with the Ad-GFP group, the Ad-BMP2 group had more chondrocytes ( Figure 8 -C, left column). Alcian blue staining ( Figure 8 -C, middle column) and Safranin O staining ( Figure 8 -C, right column) showed that the signal of the Ad-BMP2 group was stronger than that of the Ad-GFP group, indicating that iHNPCs could effectively undergo chondrogenic differentiation and produce a large amount of extracellular matrix.

[0054] Interestingly, when the same experiment was performed using iHAFCs, no obvious chondrogenic differentiation was observed. Collectively, these results suggest that iHNPCs can undergo efficient chondrogenic differentiation under BMP2 stimulation, whereas iHAFCs do not possess this property.

[0055] 3. Identification of the tumorigenic properties of immortalized cells in vivo: A small number of hTERT-immortalized non-tumor cells may proliferate indefinitely in the body and lead to tumor formation due to their greatly enhanced proliferation ability. It is necessary to verify the tumorigenic potential of iHAFCs and iHNPCs in vivo and use in vivo imaging technology to evaluate the proliferation of cell clusters in vivo.

[0056] The firefly luciferase (Fluc) gene was transferred into The retrovirus was transfected into iHAFCs and iHNPCs to form stable cell lines iHAFCs-Fluc and iHNPCs-Fluc. Fluc is a luciferase expressed by cells, which can generate bioluminescence under the action of substrates. The packaging and infection process of retrovirus is basically similar to the immortalized lentivirus process described above. In brief, the retroviral vector pSEPI-Fluc, which was made in our laboratory, was co-transfected with packaging plasmids pCMV-VSVG and pCL-Ampho, and fluorescent plasmid pMPB-BiFP into HEK-293 cells to package retrovirus91,92. After the collected retroviral supernatant was infected with iHAFCs and iHNPCs cells, Puromycin (0.3-0.4 µg / mL, Invitrogen) was used for selection for 7-10 days to obtain stable cell lines iHAFCs-Fluc and iHNPCs-Fluc.

[0057] The fluorescence in vitro identification of iHAFCs-Fluc and iHNPCs-Fluc was completed by Pierce™ FireflyLuciferase Glow Assay Kit (Thermo Fisher Scientific, USA). iHAFCs and iHNPCs were used as negative controls, and the human osteosarcoma cell line 143B-Fluc previously established in the laboratory was used as a positive control.

[0058] After confirming the stable expression of Fluc gene in vitro, we performed subcutaneous injection of thymic nude mice and in vivo imaging monitoring. We also identified the tumorigenic properties of de-immortalized and non-de-immortalized cells. Specifically, iHAFCs-Fluc and iHNPCs-Fluc were infected with Ad-GFP and Ad-FLP for 48 hours, then resuspended in about 80μl PBS and injected subcutaneously into thymic nude mice (6-8 weeks old, 15-20g, regardless of gender; Envigo, Indianapolis, IN) using a 0.5ml syringe. Live imaging was performed on nude mice on days 3, 7, 10, and 14 after injection. The fluorescent substrate was VivoGlo™ Luciferin In Vivo Grade (Promega, USA) injected intraperitoneally at 100mg / kg body weight, and scanned using IVIS Spectrum (PerkinElmer, Waltham, MA). During the whole process, the nude mice were fully anesthetized with 2% isoflurane.

[0059] To track the proliferation status of iHAFCs and iHNPCs in vivo, iHAFCs and iHNPCs were stably labeled with firefly luciferase (Fluc) using retroviral vectors to generate iHAFCs-Fluc and iHNPCs-Fluc cell lines. Subconfluent fluc-labeled cells were infected with Ad-FLP or Ad-GFP (MOI: 40) for 16 h, and then the cells were collected and injected subcutaneously into the flanks of thymic nude mice (5 × 10 6 cells / injection site, 9 injections per group). Human osteosarcoma cell line 143b-Fluc was used as a positive control. On days 3, 7, and 14, nude mice were anesthetized with 2% isoflurane, intraperitoneally injected with D-Luciferin, and placed in a Lago X imaging system (Spectral Instruments Imaging, Tucson, AZ) for bioluminescence imaging. The imaging data were analyzed using the accompanying Aura image analysis software.

[0060] Bioluminescence imaging results ( Fig. 9 ) showed that on the 7th day, the signals of the iHAFCs-Fluc and iHNPCs-Fluc groups were weaker than those of the 143B-Fluc group. On the 14th day, the masses and signals of the iHAFCs-Fluc and iHNPCs-Fluc groups disappeared, while the 143B-Fluc group formed significant masses and produced strong signals ( Figure 1-4 B). We monitored the animals for 4 weeks and did not find any detectable tumors in the iHAFCs-Fluc and iHNPCs-Fluc groups. The results indicate that hTERT-immortalized iHAFCs and iHNPCs, with or without hTERT gene ablation by FLP, have no obvious tumorigenicity.

[0061] Example 3 Biocompatibility determination of scaffold materials POC, PPCNg and GelMA

[0062] This study used three scaffold materials as composite materials to conduct intervertebral disc related tissue engineering research and explore the feasibility of in vitro intervertebral fusion gene therapy. Poly (1,8-octanediol-co-citric acid) (POC) is translated into Chinese as poly 1,8-octanediol-co-citric acid, which is a citrate-based polymer material with good biocompatibility and absorbability. Polyethylene glycol citrate-co-Nisopropylacrylamide pre-mixed withgelatin (PPCNg), translated into Chinese as polyethylene glycol citrate-co-N-isopropylacrylamide hydrogel, PPCN is a citrate-based thermosensitive polymer. After mixing with hydrogel, it can make the hydrogel change from liquid to semi-solid at 37°C. Gelatin methacryloyl (GelMA), translated into Chinese as methacryloyl chloride gelatin, is also a type of hydrogel that can quickly change from liquid to gel under ultraviolet irradiation.

[0063] In this part of the experiment, the POC polymer was made into a cylindrical scaffold with a diameter of 5mm and a thickness of 3mm (diameter × height = 5mm × 3mm) by 3D printing, with a cavity with a diameter of 1mm and a thickness of 1mm in the center. The disc-shaped scaffold was used for intervertebral disc tissue engineering. All scaffolds need to complete biocompatibility tests in vitro for different cells. The main test methods are as follows: iHAFCs and iHNPCs in the logarithmic growth phase were infected with Ad-GFP-Gluc to express GFP fluorescence and Gluc secretory luciferase at the same time. After 24 hours, the cells were digested, and iHAFCs were resuspended with PPCNg and iHNPCs were resuspended with GelMA. The two cell suspensions were carefully added to the pre-sterilized POC scaffolds and the cells were allowed to attach to their surfaces. PPCNg was properly heated by infrared lamps to solidify, while GelMA was properly irradiated by ultraviolet lamps to solidify. After solidification, the scaffolds with attached cells were immersed in the culture medium. In the co-culture experiment of iHAFCs and iHNPCs, GelMA-iHNPCs suspension was added to the central cavity of the POC scaffold and cured by UV light, and then PPCNg-iHAFCs suspension was added to the surrounding part, properly heated and cured by infrared, and immersed in the culture medium. At the predetermined time point, GFP fluorescence was used to observe the cell morphology and growth status, and Gluc was used to quantitatively determine the biological activity of the cells ( Fig.10 -A).

[0064] Taking advantage of the qualitative (GFP signal) and quantitative (secreted Gluc activity) properties of the Ad-GFP-Gluc vector, iHAFCs and iHNPCs infected with Ad-GFP-Glu were mixed with PPCNG and seeded onto GelMA 3D structures submerged in culture medium. GFP signal levels and Gluc activity were used as surrogate markers of cell survival. On day 6 of infection, the cell-bearing scaffolds were reinfected with Ad-GFP-Gluc. We found that both iHAFCs and iHNPCs were able to tolerate the POC scaffolds and could survive for more than 10 days. After reinfection with Ad-GFP-Gluc on day 6, the reappearance of the GFP signal was observed overnight ( Fig.10 -BC), while Gluc activity was partially restored ( Fig.10 -D), indicating that the adherent cells in the scaffold are still alive. Under a high-power microscope, it was observed that the adherent cells were attached to the POC scaffold in different structures, shapes and dimensions ( Fig.10 -E). Co-culture experiments showed that cells were well attached to different regions of the POC disc-shaped three-dimensional structure, with iHNPCs-GelMA concentrated in the central part and iHAFCs-PPCNg distributed in the peripheral part ( Fig.10 -F). Overall, these results indicate that POC, PPCNG, and Gelma scaffolds exhibit good biocompatibility for both cell types and are suitable for cell-based tissue engineering research.

[0065] Example 4 Establishment of an effective intervertebral disc regeneration model

[0066] The POC scaffold was 3D printed into a 5 mm × 3 mm disc-shaped structure with a gap in the middle (1 mm × 1 mm) (Figure S3). iHAFCs in the logarithmic growth phase were infected with Ad-BMP9, and iHNPCs were infected with Ad-BMP2. After 48 hours, the cells were digested and the supernatant was removed by centrifugation. BMP9-iHAFCs were resuspended with 10% PPCNg (mass percentage) to obtain 5×10 6 / ml cell suspension, and resuspend BMP2-iHNPCs with 10% GelMA (mass percentage) to obtain 5×10 6 / ml cell suspension. 100 μl of GelMA-iHNPCs suspension was first added into the central cavity of the disc-shaped POC, and irradiated with UV light for 30 seconds to solidify it. Then the assembled POC was placed into the PPCNg-iHAFCs suspension to allow it to fully absorb the cell suspension. The intervertebral disc scaffold absorbed about 500 μl of PPCNg-iHAFCs ( Fig.11 A).

[0067] After nude mice were anesthetized by inhalation of isoflurane, ketamine (50–75 mg / kg) plus xylazine (7-10 mg / kg) were injected intraperitoneally to completely anesthetize them. The mice were placed in a supine position. After routine disinfection, a longitudinal incision of about 1 cm was made in the middle of the abdomen on one side of the nude mice. The subcutaneous tissue and fascia were bluntly dissected with vascular clamps and nerve strippers. Then, the POC soaked in the PPCNg-iHAFCs suspension was carefully placed under the skin of the nude mice, and the remaining suspension was dripped on the placed POC. After the placement was completed, an infrared lamp was quickly used to properly heat the surgical site to solidify the PPCNg and adhere cells. The contralateral side was similar, and 4 POC models were implanted in one nude mouse. POC without any treatment and cell adhesion was implanted subcutaneously in the same way as a negative control. After 42 days of implantation, the nude mice were killed and the subcutaneous masses were collected for Micro-CT and histological analysis.

[0068] Compared with the GFP-treated cell group or the POC scaffold-only group, the 3D reconstruction results of the implanted IVD model suggested that the BMP9 / iHAFCs and BMP2 / iHNPCs co-seeded group (IVD model group) exhibited significant bone formation in the surrounding area ( Fig.11 -B-ab). At the same threshold, the total bone volume of the IVD model in the peripheral area was much higher than that of the POC scaffold-only group. In addition, the average height of the IVD model was significantly higher than that of the POC scaffold-only group ( Fig.11 -Bc), suggesting that BMP9-stimulated iHAFCs may effectively form new bone, thereby improving the mechanical strength of the IVD model.

[0069] The removed IVD model samples showed that the shape was well maintained. HE staining showed that new bone nodules were formed in the peripheral area of ​​iHAFCs infected with Ad-BMP9, while cartilage-like tissue was found in the central area of ​​iHNPCs infected with Ad-BMP2 ( Fig.11 -C, left). Safranin O / Fast Green staining and Masson's trichrome staining further confirmed the presence of cartilage-like tissue in the central region of the IVD model and mature bone tissue in the peripheral region ( Fig.11 -C, middle and right). These results indicate the feasibility of establishing an IVD regeneration model using POC scaffolds loaded with iHNPCs stimulated by chondrogenic differentiation factors and iHAFCs stimulated by osteogenic differentiation factors.

[0070] Example 5 Ex vivo intervertebral fusion model

[0071] Since iHAFCs showed high osteogenic potential under BMP9 stimulation, we further investigated Potential application of iHAFCs in spinal fusion.

[0072] Freshly killed rats (Sprague Dawley, 8 weeks old, weighing 200–350 g, regardless of sex; Envigo, Indianapolis, IN) made an incision of about 5 cm in length at the lumbosacral position of the midline of the back, separated the subcutaneous tissue and fascia, and removed the entire thoracic and lumbar vertebrae of the spine. The entire spine was cut from the middle of the vertebral body, and each two intervertebral discs contained the upper and lower halves of the vertebral body as one section. The posterior structure of the vertebral body was removed from the pedicle position, leaving only the intervertebral disc and vertebral body. Use appropriate tools to hollow out the intervertebral disc and grind until the upper and lower end plates are exposed. The processed disc-free spinal segments were stored in sterile gauze moistened with PBS. iHAFCs infected with Ad-BMP9 or Ad-GFP were collected, mixed with the thermosensitive polymer PPCNg, and injected into the cavity of the lumbar segment. These segments were transplanted into the backs of athymic nude mice. Intervertebral segments with intact intervertebral discs or with discs removed but not loaded with cells were used as negative controls ( Fig.12 -A).

[0073] Six weeks after transplantation, the rats were killed and the implanted intervertebral segments were removed for Micro CT imaging analysis. The results showed that compared with the original disc segment, new bone structure was formed between the end plates ( Fig.12 -Ba). Quantitative analysis of the disc area showed that the mean bone density (Hounsfield units, HU) and relative bone volume of the fusion group were 2 to 3 times higher than those of the original disc ( Fig.12 -B-bc). The removed samples were also evaluated histologically. HE staining showed that in the disc fusion model, mature bone structure and bony connection between the end plates were visible, while the discs in the control group maintained the histological characteristics of the normal annulus fibrosus and nucleus pulposus between the end plates ( Fig.12 -Ca). Masson trichrome staining showed that mature bone was formed in the intervertebral disc fusion model ( Fig.12 -Cb). In contrast, modified PAS staining showed that cartilage tissue was significantly reduced in the disc fusion model compared with the control disc model ( Fig.12 -Cc).

[0074] Overall, these results suggest that iHAFCs could serve as an ideal cell source for ex vivo cell-based gene therapy for spinal intervertebral fusion.

[0075] In summary, the present invention has the potential of intervertebral disc tissue engineering based on cells, factors and scaffolds. Human healthy nucleus pulposus cells (iHNPCs) and annulus fibrosus cells (iHAFCs) are reversibly immortalized by stably expressing hTERT. The immortalization process can be reversed by FLP recombinase and is not tumorigenic. iHAFCs show osteogenic differentiation under BMP9 stimulation, while iHNPCs show chondrogenic differentiation under BMP2 stimulation. The present invention has developed an IVD regenerative tissue engineering model, using a 3D-printed cylindrical POC scaffold, loading a BMP9-stimulated iHAFCs / PPCNg mixture in the peripheral area, and a BMP2-stimulated iHNPCs / GelMA hydrogel mixture in the central area, simulating the structure of a natural intervertebral disc, and subcutaneously transplanted into athymic nude mice. The IVD model has significant bone nodule formation in the peripheral area, and chondrogenic differentiation in the central area. The present invention also developed an ex vivo intervertebral fusion model, by replacing the native intervertebral disc with iHAFCs stimulated by BMP9, and implanting these engineered intervertebral fusion structures subcutaneously into athymic nude mice, and there was obvious bone fusion in the intervertebral segment injected with iHAFCs. The intervertebral disc regeneration model and the ex vivo gene therapy model provide important means for studying the function of intervertebral disc cells and developing effective IVD tissue engineering methods.

Claims

1. A disc regeneration model comprising a three-dimensional disc-like structure of a mixture of human annulus fibrosus cells (HAFCs) and polyethylene glycol citric acid-poly (N-isopropylacrylamide) hydrogel (PPCNg), a mixture of human nucleus pulposus cells (HNPCs) and methacryloyl chloride gelatin (GelMA), and a poly (1,8-octanediol-co-citric acid) (POC) scaffold material.

2. The intervertebral disc regeneration model according to claim 1, further characterized by: The human annulus fibrosus cells and human nucleus pulposus cells are reversibly immortalized human annulus fibrosus cells (iHAFCs) and human nucleus pulposus cells (iHNPCs).

3. The intervertebral disc regeneration model according to claim 2, further characterized by: The genomes of the reversibly immortalized human annulus fibrosus cells (iHAFCs) and human nucleus pulposus cells (iHNPCs) contain a human reverse transcriptase telomerase gene, and both sides of the gene have flippase recognition sites.

4. The intervertebral disc regeneration model according to claim 3, further characterized by: The human annulus fibrosus cells are stimulated with osteogenic factors, and the human nucleus pulposus cells are stimulated with chondrogenic factors.

5. The intervertebral disc regeneration model according to claim 4, further characterized by: The human annulus fibrosus cells are stimulated with BMP9, and the human nucleus pulposus cells are stimulated with BMP2.

6. The intervertebral disc regeneration model according to claims 1-5, further characterized by: The three-dimensional disk-shaped structure is a disk-shaped structure of 5 mm×3 mm (diameter×height), and the central gap is 1 mm×1 mm (diameter×height).

7. The intervertebral disc regeneration model according to claim 6, further characterized by: A mixture of human annulus fibrosus cells and PPCNg was added to the periphery of the disc-shaped structure, and a mixture of human nucleus pulposus cells and GelMA was added to the central space.

8. The intervertebral disc regeneration model according to claim 6, further characterized by: The mixture of human annulus fibrosus cells and PPCNg is prepared by resuspending the human annulus fibrosus cells to about 5×106 cells / ml with 10% PPCNg; the mixture of human nucleus pulposus cells and GelMA is prepared by resuspending the human nucleus pulposus cells to about 5×106 cells / ml with 10% GelMA.

9. A method for preparing an intervertebral disc regeneration model, comprising the following steps: Step 1, stimulate reversibly immortalized human NPCs (iHNPCs) and AFCs (iHAFCs) with chondrogenic and osteogenic factors; Step 2: 3D print the POC stent, which is a disc-shaped structure of 5mm×3mm (diameter×height) with a central gap of 1mm×1mm (diameter×height); Step 3, adding a mixture of iHAFCs / PPCNg stimulated by BMP9 to the periphery of the scaffold and a mixture of iHNPCs / GelMA stimulated by BMP2 to the central space to establish an intervertebral disc regeneration model; In step 4, the assembled disc regeneration construct was implanted into the spinal segment and histologically evaluated after 6-8 weeks.

10. Use of the intervertebral disc regeneration model as described in claims 1 to 8 in intervertebral disc degenerative diseases.

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