Core-shell structure biological scaffold for treating bone defect as well as preparation method and application of core-shell structure biological scaffold

Abstract

The invention provides a core-shell structure biological scaffold for treating bone defects as well as a preparation method and application of the core-shell structure biological scaffold. The preparation method comprises the following steps: (1) adding a fibroblast protein inhibitor (FAPI) into a polycaprolactone solution, and preparing a drug-loaded polycaprolactone fiber membrane by adopting an electrostatic spinning technology; (2) preparing a nano polymer by adopting plasmids of which the SHN-3 is knocked out and G5-GBA, then adding the nano polymer and phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate into a hydrogel solution composed of GelMA and HAMA, and carrying out ultraviolet irradiation; and (3) winding the obtained drug-loaded polycaprolactone fiber membrane on the surface of a mold, placing the product obtained in the step (2) in the mold, and curing under ultraviolet light to form a core-shell structure. The obtained biological scaffold with the core-shell structure can prevent excessive fibrosis of calluses, promote formation of H-type blood vessels in new calluses, effectively avoid bone nonunion and accelerate the repair process of key bone defects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a core-shell structure biological scaffold for treating bone defects, and a preparation method and application thereof. Background Art

[0002] Critical bone defects refer to the smallest bone defects that cannot be repaired by their own repair mechanisms, and are often caused by sequelae of trauma, tumors, infection, or congenital diseases. Currently, autologous bone transplantation is the main treatment method for repairing critical bone defects in clinical practice, but its clinical application is limited due to insufficient bone graft donors, large surgical trauma, postoperative donor site pain, and high treatment costs. In recent years, tissue engineering strategies have been adopted on an international scale to treat this defect, with the goal of creating artificial bone grafts / bone growth factors to meet the growing clinical needs.

[0003] However, current bone tissue regeneration strategies usually focus on the bone tissue itself, while ignoring the problem of excessive growth of fibrous tissue in the critical bone defect area in the early stage, which hinders bone regeneration. During the bone regeneration process in the critical bone defect area, the fibrous tissue in the bone defect area grows faster and the bone tissue grows slower; the growth rates of new bone tissue and new fibers should maintain a relatively balanced state to effectively promote normal bone tissue regeneration. If this balance is broken, it will lead to a corresponding pathological state, that is, if the growth of fibroblasts is very robust, it will lead to a large amount of fibrous tissue filling the bone defect, which will eventually manifest as delayed fracture healing or even nonunion.

[0004] During bone repair and remodeling, H-type angiogenesis is closely associated with osteogenesis. H-type vessels are a special subtype of blood vessels in bone that play an important role in bone regeneration and are characterized by strong CD31 and endomucin (Emcn) positivity in endothelial cells.

[0005] Therefore, how to construct a solution that has both anti-fibrotic activity and promotes H-type vascularization and bone regeneration is urgently needed in this field. Summary of the invention

[0006] In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a method for preparing a core-shell structure biological scaffold for treating bone defects, the preparation method comprising the following steps:

[0007] (1) adding fibroblast inhibitor FAPI to a polycaprolactone solution and using electrospinning technology to prepare a drug-loaded polycaprolactone fiber membrane;

[0008] (2) using a SHN-3 knockout plasmid and G5-GBA to prepare a nanopolymer, then adding the nanopolymer and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate to a hydrogel solution composed of GelMA and HAMA, and irradiating with ultraviolet light; the SHN-3 knockout plasmid is a mixed plasmid of plasmids whose gene knockout target sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 respectively;

[0009] (3) rolling the obtained drug-loaded polycaprolactone fiber membrane on the surface of a mold, placing the obtained substance in step (2) on the mold, and curing it under ultraviolet light to form a core-shell structure.

[0010] Preferably, the solvent in the polycaprolactone solution includes chloroform. More preferably, the polycaprolactone solution is prepared by dissolving polycaprolactone in chloroform at a weight to volume ratio of 1 g:9 mL, and then adding dimethylformamide in an amount of one fifth of the weight of the polycaprolactone.

[0011] Preferably, the volume weight ratio of the fibroblast inhibitor FAPI to the sum of the weights of polycaprolactone and the fibroblast inhibitor FAPI is 0.5-2‰ v / w; the N / P ratio of the SHN-3 knockout plasmid to G5-GBA is 1-20. In the SHN-3 knockout plasmid, the number ratio of each plasmid is 1:1:1:1

[0012] Preferably, during electrospinning, the voltage is 18 kV, the collection distance is 20 cm, and the flow rate is 2.5 mL / h.

[0013] Preferably, the weight ratio of GelMA to HAMA is 1:1.

[0014] Preferably, the added amount of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.5% w / v.

[0015] Preferably, the wavelength of the ultraviolet light is 405 nm.

[0016] Another object of the present invention is to provide a core-shell structure biological scaffold for treating bone defects prepared by the above preparation method.

[0017] Another object of the present invention is to provide an application of the obtained core-shell structure biological scaffold for treating bone defects in preparing products for treating bone defects.

[0018] Beneficial effects of the present invention:

[0019] The core-shell structure biological scaffold obtained by the present invention can prevent excessive fibrosis of callus tissue, promote the formation of H-type blood vessels in new callus tissue, effectively avoid bone nonunion, and accelerate the repair process of key bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results are the characterization results of the core-shell structure of the biological scaffold of the present invention; wherein, Part A is the scanning electron microscope observation of polycaprolactone electrospinning, GelMA hydrogel, GELMA-Hama hydrogel and core-shell scaffold; Part B is the element mapping of the surface of PCL electrospinning film; Part C is the frequency distribution table and physical diagram of the diameter of electrospinning fibers; Part D is the frequency distribution table and physical diagram of the pore size of hydrogels; Part E is the H-NMR characterization result diagram of GelMA, HAMA and Gelma-HAMA; Part F is the XPS analysis of the elements on the surface of PCL electrospinning membrane; Part G is the Fourier transform infrared spectrum of the surface of PCL electrospinning film; Part H is the statistical data of the water contact angle on the surface of PCL electrospinning membrane; Part I is the drug release curve of PCL electrospinning membrane; the experiment was repeated three times; p<0.05 has statistical significance, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0021] Figure 2 The results of the biocompatibility experiment of the product obtained in the present invention are as follows; Part A shows the live and dead staining after co-culture of PCL electrospinning membranes with different drug loading ratios and fibroblasts (electrospinning drug loading ratio: blank, 0.5‰, 1‰, 2‰); Part B shows the observation results of stereoscopic imaging of live and dead cells on PCL electrospinning membranes; Part C shows the quantitative analysis results of PCL electrospinning membranes and the ratio of live and dead fibroblasts; Part D shows the proliferation activity of fibroblasts on PCL electrospinning membranes; Part E shows the results of co-culture of osteoblast precursor cells with PCL electrospinning membranes. After culture, live and dead staining of nanopolymers with different N / P ratios (N / P: 1, 2, 4, 8, 12, 16, 18, 20); Part F shows the quantitative localization of the ratio of live and dead cells in nanopolymers and osteoblast precursor cells; Part G shows the proliferation inhibition rate of osteoblast precursor cells co-cultured with nanopolymers with different N / P ratios; The experiment was repeated three times; p<0.05 showed statistical significance, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0022] Figure 3The figure is the experimental result of the anti-fibrosis effect of the product of the present invention on fibroblasts; Part A is a schematic diagram of the blocking effect of the core-shell scaffold on fibroblasts in bone defects; Part B is a schematic diagram of the separation of cells by PCL membrane in a transwell plate; Part C is the observation of cell migration on the PC membrane under a light microscope and quantitative analysis; Part D is the expression result of adhesion plaques in different groups; Part E is the expression result of TGF-β1 protein in different groups; Part F is the WB band of TGF-β1 protein after treatment in different groups; Part G is the expression result of related fibroblast genes (TGF-β1, S-mad 1); Part H is the fluorescence area and intensity analysis results of Vinculin protein; Part I is the fluorescence area and intensity analysis results of TGF-β1 protein; The experiment was repeated three times; p<0.05 is statistically significant, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0023] Figure 4 The figure is a diagram of the formation and function experiment results of GBA@pkSHN-3 of the present invention; wherein, part A is a schematic diagram of GBA-G5 synthesis and binding plasmid; part B is the agarose electrophoresis experiment result of GBA@pkSHN-3; part C is the expression of GFP protein in osteoblasts 48 hours after transfection; part D is the particle size and Zeta potential measurement of GBA@pkSHN-3; part E is the transfection efficiency of different reagents detected by flow cytometry; part F is the quantitative analysis of the transfection efficiency of osteoblast precursor cells by flow cytometry; part G is the co-localization result of plasmid and osteoblast precursor cells; part H is the test tube formation experiment result of HUVECS; part I is the scratch experiment result of HUVECS; part J is the migration experiment result of HUVECS; part K is the expression of MC3T3E1SHN-3 gene before and after transfection; part L is the expression of MC 3T3 before and after transfection. Changes in slit3 protein secreted by E1 (ELISA); Part M is the number of duct nodes; Part N is the scratch healing area of ​​different groups; Part O is the number of HUVECS migrating cells; The experiment was repeated three times. p<0.05 is statistically significant, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0024] Figure 5Figure 1 is the result of osteogenesis and H-type angiogenesis experiment; Part A shows the changes of differentially expressed genes related to osteogenic mineralization after knocking out the SHN-3 gene in osteogenic precursor cells; Part B shows the TOP up-regulated signaling pathway of MC 3T3 E1 after gene knockout; Part C shows the changes of angiogenesis-promoting genes in gene knockout cells; Part D shows the type I collagen staining results of gene knockout cells; Part E shows the CD31 and EMCN fluorescence staining analysis results of HUVECS after slit3 factor intervention; Part F shows the fluorescence intensity analysis results of type I collagen; Part G shows the fluorescence intensity analysis of EMCN; Part H shows the fluorescence intensity analysis results of CD31; Part I is a schematic diagram of pathways related to cell transfection, gene changes and protein secretion; The experiment was repeated three times; p<0.05 shows statistically significant differences, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0025] Figure 6 It is the result of the in vivo experiment of the present invention; Part A is a schematic diagram of the in vivo experimental procedure; Part B is the X-ray images of different groups; Part C is the 2D-Mirco CT image of the bone defect area; Part D is the HE staining result of the regenerated bone in the defect area, the red arrow indicates the bone regeneration in the defect area, and the white arrow indicates the fibroblast tissue; Part E is the Masson staining result of the regenerated bone in the defect area; Part F is the HE and Sirian red staining results of the regenerated soft tissue at the bone defect (blank group on the left, PCL group on the right); Part H is the new bone density analysis of each treatment group; Part I is the proportion of Masson staining positive tissue; The experiment was repeated three times; p<0.05 is statistically significant, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0026] Figure 7 The results of tissue immunofluorescence detection of bone defect sites in SD rats; Part A shows that the 4-week and 8-week tissue sections of different treatment groups were stained with CD31 and Emcn target proteins (green represents CD31, red represents Emcn, and H-type vessels are double-expressing tissues); Part B shows the fluorescence area analysis results of CD31, Emcn, and H-type vessels; the experiment was repeated three times; p<0.05 showed statistically significant differences, *: p<0.05; **: p<0.01; ***: p<0.001; ns: not statistically significant;

[0027] Figure 8 TEM image of GBA@pkSHN-3 nanopolymer;

[0028] Fig. 9The morphological observation results of fibroblasts grown on PCL electrospun membranes (blue represents DAPI, and red represents phalloidin);

[0029] Fig.10 This is a diagram of the plasmid construction used in the present invention;

[0030] Fig.11 The diagrams show the changes in the osteogenic mineralization ability of knockout cells, which are Alizarin red and ALP staining experiments respectively; Fig.12 The results of the biocompatibility validation experiment in rats; liver and kidney sections were embedded in the subcutaneous core-shell scaffolds for 2 weeks, and there were no signs of inflammation and necrosis in the patients in each group;

[0031] Fig.13 Models of various groups of animals of the present invention and photos of some specimens;

[0032] Fig.14 The figure is a schematic diagram of the technical concept of the present invention; wherein, part A is the manufacturing process of the core-shell structure scaffold; part B shows that the core-shell structure scaffold is implanted into a rat femoral severe bone defect model and plays an anti-tissue space-occupying role; part C shows that the nanopolymer induces osteoblast gene changes, enhances paracrine effects, and promotes vascularization;

[0033] Fig.15 Schematic diagram of the construction of pGPU6 / GFP / Neo vector;

[0034] Fig.16 This is the linearization result diagram of pGPU6 / GFP / Neo vector and the marker map;

[0035] Figures 17 to 19 It is the identification map of the recombinant vector plasmid cut with PstI and the identification map of the recombinant vector plasmid cut with BamHI Marker map (1Kb DNA Marker);

[0036] Fig. 20 The co-localization fluorescence images were obtained by transfection with different plasmids. DETAILED DESCRIPTION

[0037] 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. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Materials: Polycaprolactone (molecular weight: 80,000) was purchased from Sora Biosciences (Beijing, China); GelMA and HAMA were purchased from China Eastern Airlines (Suzhou, China); G5-PAMAM was purchased from Shunna (Shanghai, China); the circular plasmid (GFP) construct was customized by GemmaBio (Chengdu, China); FAPI-4 reagent was purchased from Abmole (Shanghai, China). Cell culture reagents such as DMEM, fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin 100 U / mm-streptomycin 0.1 mg / mL (penicillin-streptomycin) were purchased from GibcoBRL (Rockville, MD, USA); DAPI, DAPI glycoside, alkaline phosphatase (ALP) and corresponding kits were purchased from Sora Biosciences; polymerase chain reaction (PCR) and western blotting (WB) related reagents were purchased from Baiyun Company (Shanghai, China); Lipofectamine TM3000 transfection reagent was purchased from Thermo Fisher Scientific (MA, USA); rat homolog slit3 protein ELISA kit was purchased from Enfi Biotechnology (Wuhan, China); Label IT nucleic acid labeling kit and cy3 kit were purchased from Mirusbio (WI, US); focal adhesion protein, TGF-β1, Col-I and Emcn antibodies were purchased from Affinity Industries (Jiangsu, China); CD31 antibody and fluorescent secondary antibody were purchased from Proteintech (IL, USA).

[0040] 2. Preparation of SHN-3 knockout plasmid

[0041] 1. Experimental Materials

[0042] ①DNA oligo was designed using Designer3.0 (GenePharma) software, and the synthetic primers were completed by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0043] ②List of instruments used in the experiment:

[0044] Table 1

[0045]

[0046] ③List of reagents used in the experiment:

[0047] Table 2

[0048]

[0049]

[0050] 2. Construction process

[0051] Selection of shRNA targets (or provided by customers): Based on the target gene sequence of the target gene, multiple RNA interference target sequences are designed using GenePharma Designer 3.0 according to the RNA interference sequence design principles provided on the public website, and the target with the best kinetic parameters is selected to enter the subsequent experimental process.

[0052] (1) shRNA DNA template design and synthesis

[0053] The loop structure in the shRNA DNA template uses TTCAAGAGA to avoid the formation of a termination signal, and the transcription termination sequence of shRNA uses a T6 structure. CACC is added to the 5' end of the sense strand template to complement the sticky end formed after BbsI digestion; GATC is added to the 5' end of the antisense strand template to complement the sticky end formed after BamHI digestion; if the first base of the siRNA is not G, a G is added after CACC.

[0054] The relevant sequence information is shown in Table 3.

[0055] Table 3

[0056]

[0057]

[0058] (2) Annealing of shRNA DNA template

[0059] Dissolve the DNA oligo in TE (pH 8.0) to a concentration of 100 μM. Take the corresponding sense chain and antisense chain oligo solutions and prepare the annealing reaction system according to the following ratio.

[0060] Table 4

[0061]

[0062] Annealing was performed on a PCR instrument according to the following procedure: 95°C for 5 min; 85°C for 5 min; 75°C for 5 min; 70°C for 5 min; and stored at 4°C. After annealing, a shRNA template with a concentration of 10 μM was obtained. The obtained template solution was diluted 50 times to a final concentration of 200 nM for ligation reaction.

[0063] (3) Linearization of pGPU6 / GFP / Neo vector

[0064] Take 10 μg of pGPU6 / GFP / Neo vector and perform enzyme digestion according to the following system:

[0065] Table 5

[0066]

[0067] Digest at 37℃ for 1 hour, perform agarose electrophoresis, recover using Agarose Gel DNA Purification Kit Ver2.0, estimate the concentration by electrophoresis, and dilute to 50ng / μl.

[0068] Schematic diagram of the construction method and results, see Fig.15 and 16 .

[0069] (4) Construction of pGPU6 / GFP / Neo-shRNA vector

[0070] Carry out the vector ligation reaction according to the following system:

[0071] Table 6

[0072]

[0073] 22℃ for 1 hr, and then transferred to Top10 competent cells.

[0074] (5) Preparation of competent cells: (Calcium chloride method)

[0075] 5.1 Pick a single colony from a fresh plate cultured at 37°C for 16 hours and transfer it to a 1L flask containing 100ml LB medium. Culture at 37°C for 3 hours with vigorous shaking (rotating shaker, 300 rpm).

[0076] 5.2 Aseptically transfer the bacteria into a sterile, disposable, ice-cold 50 ml polypropylene tube and place on ice for 10 minutes to cool the culture to 0°C.

[0077] 5.3 Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells.

[0078] 5.4 Pour out the culture medium and invert the tube for 1 minute to allow the last trace of culture medium to flow out.

[0079] 5.5 Resuspend each pellet with 10 ml of ice-cold 0.1 mol / L CaCl2 and place on an ice bath.

[0080] 5.6 Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells.

[0081] 5.7 Pour out the culture medium and invert the tube for 1 minute to allow the last trace of culture medium to flow out.

[0082] 5.8 Resuspend each cell pellet with 2 ml of ice-cold 0.1 mol / L CaCl2 (containing 20% ​​glycerol) for every 50 ml of initial culture.

[0083] 5.9 Divide the cells into small portions (100 μl / vial) and freeze them at -70°C.

[0084] (For the preparation of competent cells, please refer to: Molecular Cloning Experiment Guide, 2nd edition, page 55)

[0085] (6) Transformation of ligation products

[0086] 6.1 Take out the competent cells from -70℃ and place the centrifuge tube containing the competent cells on ice for 4 minutes. After the competent cells are thawed, add 10μl of the ligation product, gently mix the contents, and place it on ice for 30 minutes.

[0087] 6.2 Place the centrifuge tube on a test tube rack in a water bath preheated to 42°C and leave it there for 90 seconds without shaking the centrifuge tube.

[0088] 6.3 Quickly transfer the centrifuge tube to an ice bath and allow the cells to cool for 3 minutes.

[0089] 6.4 Add 800 μl of LB medium (without antibiotics) to each centrifuge tube, then transfer the centrifuge tube to a 37°C shaker at 250 rpm and incubate for 45 minutes to allow the bacteria to recover.

[0090] 6.5 Take 200 μl of the cultured cells and spread them evenly on an LB plate containing 50 μg / ml Kanamycin.

[0091] 6.6 After the liquid on the plate is absorbed, place the plate upside down in a 37℃ incubator and incubate for 16 hours.

[0092] (7) Identification and sequencing of positive clones

[0093] 7.1 Pick 5 colonies from each plate, inoculate them into LB medium containing 50 μg / ml Kanamycin, and culture at 37℃ for 16 hours.

[0094] 7.2 Extract plasmid using alkaline lysis method:

[0095] 7.2.1 Take 1.5 ml of the culture into a microcentrifuge tube, centrifuge at room temperature at 12000g for 30 seconds, discard the supernatant, and invert the centrifuge tube to allow the liquid to drain out as much as possible.

[0096] 7.2.2 Resuspend the bacterial pellet in 100 μl of pre-cooled solution I and shake vigorously to disperse and mix the bacteria.

[0097] 7.2.3 Add 200 μl of freshly prepared Solution II, invert several times to mix (do not shake violently), and place the centrifuge tube at room temperature for 2-3 minutes to lyse the cells (Solution II is the lysis solution, so the bacterial solution in the centrifuge tube gradually becomes viscous and clear).

[0098] 7.2.4 Add 150μl pre-cooled solution III, gently invert the tube several times to mix, and white flocculent precipitate will appear. It can be placed on ice for 3-5 minutes. Solution III is a neutralizing solution. At this time, plasmid DNA renatures, chromosomes and proteins irreversibly denature, forming insoluble complexes. At the same time, K + The SDS-protein complexes were precipitated.

[0099] 7.2.5 Add 450 μl of phenol / chloroform / isoamyl alcohol, vortex to mix, and centrifuge at 12,000 g for 5 min at 4°C.

[0100] 7.2.6 Carefully transfer about 400 μl of the supernatant to a new microcentrifuge tube, add 2 volumes of pre-cooled anhydrous ethanol, mix well, place at -20°C for 20 min, centrifuge at 12000 g for 15 min at 4°C, and discard the supernatant.

[0101] 7.2.7 Wash the precipitate 1-2 times with 1 ml of pre-cooled 70% ethanol, centrifuge at 8000g for 7 min at 4°C, discard the supernatant, and dry the precipitate at room temperature.

[0102] 7.2.8 Dissolve the precipitate in 50 μl TE (containing 20 μg / ml RNaseA), place at room temperature for 10 min to degrade RNA, and store at -20°C for later use.

[0103] 7.3 The obtained plasmid was digested with BamHI and PstI respectively for identification. The positive recombinant vector should be digested by BamHI but not by PstI. The digestion results were correct and sent to sequencing (Qingke). The sequencing results can be found in the sequencing folder.

[0104] The results are as follows Figures 17 to 19 As shown. Among them, Fig.17 and 18 The gene names corresponding to the lanes are as follows:

[0105] Table 7

[0106] Lane number Gene name 1 Hivep3-Rat-1801 2 Hivep3-Rat-2892 3 Hivep3-Rat-4270 4 Hivep3-Rat-5370

[0107] 7.4 The strains with correct sequencing are extracted using a high-purity plasmid midi extraction kit, and the resulting plasmids can be used for conventional molecular biology experiments and cytology experiments. If the cell toxicity is high when used for cell transfection, please re-transform into E. coli Top10, and then use a kit or CsCl ultracentrifugation method to prepare a higher purity plasmid.

[0108] 7.5 Mix the four plasmids in equal numbers (1:1:1:1).

[0109] Preparation of core-shell bioscaffolds for treating bone defects:

[0110] (1) 0.5 g polycaprolactone (PCL) was dissolved in 4.5 mL chloroform solvent to prepare PCL electrospinning solution, and 0.1 g dimethylformamide equilibrium solution was added to prepare PCL fiber by electrospinning. FAPI was directly added during the PCL dissolution process, and the drug loading was set to 1‰ v / w according to the weight fraction of FAPI / (FAPI+PCL). The suspension was transferred to a 10 mL syringe needle. The high voltage electrode and receiver were set to 18 kV voltage, the collection distance was 20 cm, and the flow rate was controlled at 2.5 mL / h; after 2 hours, the prepared fiber membrane was collected on aluminum foil, and the prepared fiber membrane was vacuum dried overnight.

[0111] (2) Synthesis of G5-GBA was prepared by referring to the paper (Cai, C., et al., MMP-2 Responsive Unidirectional Hydrogel-Electrospun Patch Loading TGF-β1 siRNA Polyplexes for Peritendinous Anti-Adhesion. Advanced Functional Materials, 2020.31(6)). Fig.10 ) and G5-GBA have an N / P ratio of 12. Commercial GelMA and HAMA hydrogels were added to a pure water bath at a ratio of 1:1 to dissolve (37°C, 20% w / v); after complete dissolution, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (0.5% w / v) and nanopolymers were added and fixed in the gel under ultraviolet light at a wavelength of 405 nm.

[0112] (3) The PCL film was rolled into a cylinder and placed in a specific cylindrical mold. The hydrogel was cured under ultraviolet light (405 nm, 30 s) to form a core-shell structure.

[0113] Example 2

[0114] The difference from Example 1 is that the N / P ratio is 1.

[0115] Example 3

[0116] The difference from Example 1 is that the N / P ratio is 2.

[0117] Example 4

[0118] The difference from Example 1 is that the N / P ratio is 4.

[0119] Example 5

[0120] The difference from Example 1 is that the N / P ratio is 8.

[0121] Example 6

[0122] The difference from Example 1 is that the N / P ratio is 16.

[0123] Example 7

[0124] The difference from Example 1 is that the N / P ratio is 20.

[0125] Example 8

[0126] The difference from Example 1 is that the drug loading amount of FAPI is 0.

[0127] Example 9

[0128] The difference from Example 1 is that the drug loading amount of FAPI is 0.5‰ v / w.

[0129] Example 10

[0130] The difference from Example 1 is that the drug loading amount of FAPI is 2‰ v / w.

[0131] Comparative Example 1

[0132] When constructing a plasmid for knocking out SHN-3, only the plasmid with the gene knockout target sequence as SEQ ID NO.1 (named Hivep3-Rat-1801) was used, and the rest was consistent with Example 1.

[0133] Comparative Example 2

[0134] When constructing a plasmid for knocking out SHN-3, only the plasmid with the gene knockout target sequence as SEQ ID NO.2 (named Hivep3-Rat-2892) was used, and the rest was consistent with Example 1.

[0135] Comparative Example 3

[0136] When constructing a plasmid for knocking out SHN-3, only the plasmid with the gene knockout target sequence as SEQ ID NO.3 (named Hivep3-Rat-4270) was used, and the rest was consistent with Example 1.

[0137] Comparative Example 4

[0138] When constructing a plasmid for knocking out SHN-3, only the plasmid with the gene knockout target sequence such as SEQ ID NO.4 (named Hivep3-Rat-5370) was used, and the rest was consistent with Example 1.

[0139] Experimental example

[0140] The following tests were performed on the products obtained in the above examples:

[0141] 1. Detection method

[0142] 1. Sample preparation, gold spraying, and observation of the micromorphology of PCL electrospun fibers and hydrogels under a scanning electron microscope (Zeiss Sigma 300, Germany) and analysis of elemental composition (mapping / EDS). More precise elemental composition was quantified by energy dispersive X-ray spectroscopy (Thermo Scientific K-Alpha, USA). The Fourier transform infrared (ATR-FTIR) spectrum of the material was recorded using an Avatar 380 infrared spectrometer (USA) to analyze the chemical composition and structure of PCL electrospun fibers. The change in water contact angle of PCL electrospun fibers was measured using a water contact angle meter (Sunglass, Suzhou). The chemical modification of hydrogels and G5-GBA was evaluated by 1H NMR (BrukerAvance 400, Germany). The morphology of the GBA@pk SHN-3 complex was observed by transmission electron microscopy (TEM; TALOSF200X). The particle size and zeta potential of GBA@pk SHN-3 polymers were measured at 25 °C using a Malvern Nano ZS90 (Malvern, UK).

[0143] 2. Drug release experiment

[0144] A standard solution containing FAPI was prepared, and its absorbance and peak area were determined by high performance liquid chromatography (HPLC), and a curve function of drug concentration was plotted. Each drug-loaded stent was cut into 50-100 mg diaphragms, and the cut diaphragms were placed in a 2 ml microinfusion tube. 1 mL of PBS solution was added to each test tube and placed in a shaking incubator at 37 ° C. At a certain time point (1-14 days), 100 μL of solution was taken out and an equal volume of PBS solution was added. The drug concentration was calculated based on the peak area of ​​each sample.

[0145] 3. In vitro experiments

[0146] 3.1 Cell culture

[0147] The cells used in the present invention include osteoblast precursor cells (MC3T3-E1), rat fibroblasts (RAT2) and human umbilical vein endothelial cells (HUVECs); all cells were purchased from Procell Biotech Co., Ltd. (Suzhou, China). The cells were cultured to the 3rd to 5th generations for subsequent experiments. The cell culture medium contained cell-based medium (88%), fetal bovine serum (10%) and penicillin-streptomycin (1%). The cells were cultured in an incubator at 37°C, 5% carbon dioxide and 95% relative humidity. The culture medium was changed every 3 days. When the cell adhesion rate reached 80%, it was treated with trypsin for further use.

[0148] 3.2 Cell counting kit-8 (CCK-8) detection

[0149] To evaluate the effect of core-shell scaffolds on cell proliferation, we performed cytotoxicity tests using the CCK-8 method. Fibroblasts and osteoblast precursor cells were co-cultured with PCL electrospun fibers and GBA@pk SHN-3, respectively, at a density of 5 × 104 cells / well on a 12-well plate. Cell proliferation was then detected using CCK-8 after 1, 3, 5, and 7 days using an enzyme labeling assay (Thermo Fisher Scientific, USA). The absorbance at 450 nm was measured, and the cell proliferation rate was calculated. This experiment was repeated three times.

[0150] 3.3 Live and dead staining experiment

[0151] The cytotoxicity of the core-shell scaffolds was evaluated by live and dead cell staining (Beijing, China). Fibroblasts and osteoblast precursor cells were co-cultured with PCL electrospun fibers and GBA@pk SHN-3 at a density of 5 × 104 cells / well, respectively. The cells were then stained with a live or dead staining kit and observed under a fluorescence microscope. The measurements were analyzed using ImageJ software, and the experiments were repeated three times.

[0152] 3.4 Anti-fibroblast adhesion assay

[0153] PCL electrospun fibers were cut to the appropriate size and placed in the upper chamber of the transwell plate to completely cover the polycarbonate film. Rat fibroblasts (RAT2) were seeded at a density of 5×104 well / well in different subgroups. After 48 h of culture, the wells were washed three times with PBS solution, fixed, stained with crystal violet, and the polycarbonate (PC) membrane was removed to observe the delivery effect of PCL electrospinning on fibroblasts. This experiment was repeated three times.

[0154] The cells on the nanofiber membrane were fixed and incubated with rhodamine and DAPI. The cell morphology and spreading were observed by cytoskeletal and nuclear staining. The cells were observed under a confocal laser scanning microscope (Olympus, Japan).

[0155] 3.5 Cell Immunofluorescence

[0156] In order to observe the expression of cell-related proteins, RAT2 and MC3T3-E1 cells were seeded into different treatment groups at a density of 5×104 cells / well. RAT2 cells were treated with PBS, FAPI-4 (2.2μg / well), PCL and PCL-FAPI to observe the expression of focal adhesion protein and TGF-β1 protein; HUVECs were treated with PBS, DFO (50ug / well) and half culture medium to observe the expression of CD31 protein and Emcn protein. MC3T3-E1 cells were treated with dexamethasone (10ug / well), PBS, terribatide (10ug / well) and Pasimd (1ug / well) to observe the expression of collagen-1 protein. After 48h of culture, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 30min, lysed with 0.5% TritonX-100 for 15min, and blocked with rapid blocking solution at room temperature for 1h. Primary antibodies (Vinculin, TGF-β1, CD31, Emcn, Collagen-1) were added and incubated at 4°C overnight, then fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 hour, and then washed with PBS. The cytoskeleton was stained with phagophorin for 30 minutes, and the nucleus was stained with DAPI for 10 minutes. The expression of target proteins was observed using a laser confocal microscope (Japan). Fluorescence intensity and fluorescence area were measured using ImageJ software. This experiment was repeated three times.

[0157] 3.6 RT-qPCR experiments

[0158] Total RNA was extracted from fibroblasts 3 days after FAPI intervention to detect the mRNA expression levels of fibrosis-related genes (Triazole, China). RNA concentration was determined, and total RNA was reverse transcribed into mRNA using Biosharp Universal Reverse Transcription Kit (Shanghai, China). Then, the expression of target genes was amplified and quantified using Gene Amplification Kit (Takara, Japan) according to the pre-designed target gene primers (Table 1). Data were analyzed using GraphPad software. This experiment was repeated three times.

[0159] 3.7 Western blotting detection

[0160] The third generation RAT2 cells were seeded on 6-well plates at a density of 5×105 cells / well and co-cultured under different interventions. After 10 days of induction, the protein concentration of each group was determined using a total protein extraction kit (Solarbio, China), followed by centrifugation for 10 min (4°C, 12000g). The proteins were then separated by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking the membrane for 1 h, it was then incubated with the primary antibody (TGF-β1 and Gapdh) at 4°C overnight. After washing 6 times with PBS containing 0.1% Tween 20 (PBST) for 30 min, and after further incubation for 1 h, the immunoreactive bands were detected using an ultrasensitive enhanced chemiluminescence kit. This experiment was repeated three times.

[0161] 3.8 Agarose gel electrophoresis experiment

[0162] The binding capacity of GBA@pkSHN-3 plasmid was detected by agarose gel electrophoresis (1%, v / w). GBA@pkSHN-3 nanopolymers with different N / P ratios (1-20) were prepared in water treated with diethyl pyrocarbonate. The group, time, and voltage (80 kV) were set, and the nucleic acid development bands of different N / P ratio groups were analyzed using an automatic gel imaging system (QuantumCX5, France).

[0163] 3.9 Cell transfection and flow cytometry experiments

[0164] In order to study the transfection efficiency of GBA@pkSHN-3 on MC3T3-E1 cells, the density of MC3T3-E1 cells before transfection was 5×10 5 Cells / well were seeded in 6-well plates. After reaching 70% confluency, G5-PAMAM, G5-GBA and commercial reagents were used. Osteoblast precursor cells were transfected for 24 hours. Then, the expression of green fluorescent protein (GFP) in each group of cells was observed under a bright field microscope. Flow cytometry was used to detect the proportion of GFP fluorescent cells in the whole cells and analyze the transfection efficiency. The data were analyzed using FlowJo software. This experiment was repeated three times.

[0165] 3.10 Plasmid / cell fluorescence confocal experiment

[0166] In order to study the micromorphology of MC3T3-E1 uptake by different transfection reagents, the plasmid was labeled with fluorescein using the IT nucleic acid labeling kit cy3 (Mirus, USA), fixed 4h after transfection, and then double stained with DAPI and FITC-fluorescein. Cells were observed under a laser confocal microscope (Olympus, Japan) and measured using ImageJ. This experiment was repeated three times.

[0167] 3.11ELISA experiment

[0168] To analyze the changes in Slit3 protein secretion after transfection of MC3T3-E1 plasmid, the culture medium of osteoblast precursor cells after plasmid intervention was collected, the supernatant was collected after centrifugation, and frozen for further detection. About 100 μl of standard or sample was added to each well, the culture plate was sealed, and incubated at 37°C for 90 min. After washing twice in wash buffer, 100 μL of biotin-labeled antibody working solution was added to each well, the plate was sealed, and incubated at 37°C for 60 min. The cells were washed three times, soaking for 1 min each time. Then, 100 μL of HRP-streptavidin conjugate (SABC) working solution was added to each well, the plate was sealed, and incubated at 37°C for 30 min. The cells were washed 5 times, soaking for 1 min each time. Then, 90 μL of TMB substrate solution was added, the plate was sealed, and incubated at 37°C for 10-20 min. After adding 50 μL of stop solution, the absorbance was measured at 450 nm using a UV spectrophotometer. This experiment was repeated three times.

[0169] 3.12 HUVECs scratch test, migration test, and tube formation test

[0170] To analyze the effect of slit3 protein on the migration ability of endothelial cells, HUVECs were seeded in 6-well plates at a density of 5 × 105 cells / well. When the cells reached 100% confluence, a straight scratch was made on the base of each well using a sterile 1000-μL pipette. Each well was washed twice with PBS, and then the cells were treated with different interventions. Pictures were taken after 0, 24, and 48 h. HUVECs were seeded in Transwell-24 well plates at a density of 2 × 105 cells / well. The migration ability of endothelial cells treated with different intervention factors was analyzed. After removing the PC membrane, the cells were observed under a microscope and photographed. Quantitative analysis was performed using ImageJ software. This experiment was repeated three times.

[0171] HUVECs were cultured in 48-well plates and the matrix TM (BD Biosciences, USA) coating, density of 2 × 104 cells / well, with base and fresh medium ratio of 1:1. After 24 h of culture, the tubules formed in each group were stained with Calcein-AM, and the images were observed under a fluorescence microscope. Quantitative analysis was performed using ImageJ software.

[0172] 3.13 Alizarin red and ALP staining experiments

[0173] To analyze the osteogenic expression and mineralization capacity of osteoprogenitor cells after gene intervention, MC3T3-E1 was seeded on 6-well plates at a density of 5×105 cells per well. After different intervention treatments, cells were fixed in 4% paraformaldehyde at specific times and stained with Alizarin Red Kit and ALP Kit. After repeated washing, cells were observed under a light microscope.

[0174] 3.14 Transcriptome sequencing (RNA-seq) and bioinformatics analysis

[0175] Total RNA was extracted from transfected MC3T3-E1 cells using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Purity and quantification were assessed, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were constructed using the VAHTS Universal V6 RNA-seq Library Preparation Kit according to the manufacturer's instructions. Libraries were sequenced on a Dabaihe Novasquer 6000 sequencing platform at Shanghai Oyi Biotechnology Co., Ltd. (Shanghai, China). Raw reads in FASTQ format were processed using Fastp software. Clean reads were obtained after removing low-quality reads in subsequent data analysis. Differentially expressed gene analysis was performed using DESeq2 software, and genes that met the thresholds of q-value < 0.05 and fold change > 2 or fold < 0.5 were defined as differentially expressed genes (DEGs). Subsequently, the hypergeometric distribution algorithm was used to enrich the DEGs of GO and KEGG pathes and other databases to screen for projects with significant enrichment functions. R (v 3.2.0) was used to draw column charts, chord diagrams, or enrichment analysis circle diagrams to identify projects with significant enrichment functions (SAMtools, http: / / samtools.sourceforge.net / mpileup.shtml).

[0176] 4. In vivo experiments

[0177] 4.1 Experimental methods

[0178] All animal experiments involved in the present invention were approved by the Ethics Committee of the Animal Experiment Center of Sichuan North Medical College (Ethics Approval No.: 2021-26). Sprague-Dawley (SD) rats aged 6 to 8 weeks were randomly divided into 4 groups (n = 9 / group): sham operation group, blank control group, PCL group, and core-shell scaffold group. Under sodium pentobarbital anesthesia (20 mg / kg), the skin was prepared and disinfected, and a towel was placed in the surgical area. The skin was cut, the soft tissue was separated, and the femur was exposed. A 5 mm bone defect was formed on the femoral shaft using a wire saw, and the bone defect was stabilized with a titanium alloy steel plate and screws. Different scaffolds were implanted in each group according to the experimental grouping. There was no bone defect in the sham operation group. No biological scaffold was implanted in the blank group. In the PCL group, the bone defect area was covered with a 7-8 mm PCL membrane and fixed at the edge with surgical sutures. In the core-shell group, the bone defect area was filled with a core-shell scaffold. The diameter of the complete core-shell scaffold is about 3 mm, and the length of the internal hydrogel core is about 5 mm. The PCL membrane on the outer layer of the scaffold extended 1-2 mm to the edge of the hydrogel to cover the broken end of the defect and the area connected to the hydrogel. Each core-shell scaffold contained 2.2 ug of FAPI and 1 ug of plasmid. At 4, 8, and 12 weeks after surgery, SD rats were anesthetized with an overdose of sodium pentobarbital. The femur, liver, and kidney were quickly removed under sterile conditions and fixed with 4% paraformaldehyde solution for further examination.

[0179] 4.2 Micro-CT

[0180] Rats were killed 4, 8, and 12 weeks after surgery. The collected femurs were fixed with 4% paraformaldehyde and scanned using a Micro-CT system (SkyScan1276, Bruker, Billerica, USA). Each sample was scanned at 18-μm pixel resolution, 100 kV voltage, and 80 μA current. Using the 3D-CT model generated by Datavewer and CTAn software, the cortical bone defect area was selected as the region of interest (ROI), and the new bone-related indices (BV, BMD) of each group were analyzed and compared.

[0181] 4.3 Immunofluorescence experiments on tissue sections

[0182] Immunofluorescence staining showed the morphology of H-type vessels. The whole femur specimens, including the defect area, were collected, fixed and decalcified, and the decalcified bones were embedded in a mixture of 8% (w / v) gelatin, 20% sucrose and 2% polyvinylpyrrolidone for tissue fixation. Cryosections of 70 μm thickness were obtained using a cryostat. Before incubation, the bone sections were infiltrated with 0.3% (v / v) Triton X-100 (Solarbio) for 10 min, and nonspecific antigens were blocked with 5% (vol / vol) fetal bovine serum albumin solution for 60 min. The sections were incubated with primary antibodies diluted in 3% BSA overnight at 4°C. PBS was washed three times for 5 min each time, and the secondary antibodies diluted in 3% BSA were incubated at 25°C for 75 min. Finally, the sections were placed in mounting medium and covered with cover slides. Fluorescence microscope (Olympus) was used for observation and image acquisition. ImageJ software was used to analyze the positive area of ​​H-type vessels in each specimen.

[0183] 4.4 Statistical analysis

[0184] All statistical analyses were performed using SPSS23.0 (IBM Corporation, Armonk, New York, USA) and GraphPad Prism9 (GraphPad Software, USA). Independent sample t-test was used to evaluate the statistical differences between the two groups, and one-way analysis of variance (ANOVA) was used for multiple data sets. Data are expressed as mean ± standard deviation (SD). When P < 0.05, the difference was considered statistically significant.

[0185] 2. Test results

[0186] 1. Physical properties of core-shell scaffolds and their components

[0187] like Figure 1 As shown in part A, the diameter of PCL electrospun nanofibers is concentrated in the range of 200-500nm, with good anisotropy, which can well prevent the growth of cells and tissues. In the core-shell scaffold, electrospinning and hydrogel are well combined through physical penetration, and there is no separation at the interface between electrospinning and hydrogel. Its structure and composition were further analyzed by X-ray diffraction spectroscopy, mapping and FTIR. The content of different elements on the nanofiber membrane was analyzed by mapping, scanning and XPS. The F element was visible on the surface of the nanofiber membrane, proving that the drug FAPI-4 was successfully loaded on the electrospun nanofiber membrane ( Figure 1 Part B and F in the figure). Using Fourier transform infrared spectroscopy, it was found that different drug loading ratios had no effect on the functional groups on the surface of electrospinning fibers ( Figure 1The data from the water contact angle measuring instrument showed that the maximum drug loading rate of the water contact angle of the PCL membrane (110±0.8°) was lower than that of the simple PCL membrane scaffold (120±0.4°), but the overall change was not significant and still maintained good hydrophobicity ( Figure 1 The release curve of FAPI-4 in the electrospun membrane was determined by HPLC. The cumulative release of FAPI-4 reached >90% within 2 weeks ( Figure 1 Part I).

[0188] The microstructure of the hydrogel was observed using a scanning electron microscope. The hydrogel is composed of GelMA, HAMA, and GelMA-HAMA. The hydrogels of different components have different pores. The pores of GelMA hydrogel are larger, while the pores of GelMA-HAMA hydrogel are smaller. Through measurement and analysis, the pore size of GelMA-HAMA hydrogel is concentrated to about 50μm ( Figure 1 The results of the study show that the composite hydrogel has a relatively rigid microstructure. Subsequently, 1H NMR spectroscopy was used to determine whether GelMA-HAMA was successfully prepared and retained the methacrylate functional molecules. The methacrylate group showed double bond peaks at 5.38ppm and 5.61ppm, and chemical shift peaks were also observed at 5.7ppm and 6.13ppm, which is consistent with the 1H NMR spectra of GelMA and HAMA ( Figure 1 These chemical shift peaks confirm that -Ch=CH-CH3 or -CH=CH2 groups are successfully grafted onto the product.

[0189] The microstructure of GBA@pkSHN-3 polymers was observed by transmission electron microscopy. When the N / P ratio was greater than 4, stable and uniform polymers began to appear under TEM, and the diameters of nano-homopolymers with different N / P ratios were between 200 and 300 nm ( Figure 8 ).

[0190] 2. The core-shell scaffold has good biocompatibility

[0191] In order to study the effect of the hydrophobic surface of PCL membrane on cell viability, we seeded fibroblasts on the surface of PCL membrane for co-culture. Figure 2 As shown in part A, when fibroblasts were co-cultured on electrospun membranes with different drug loading ratios, the number of viable cells increased and there was almost no cell death, indicating that the PCL electrospun membrane had good biocompatibility ( Figure 2 A, C, and D in the middle). Three-dimensional layer scanning images of cells on the nanofiber membrane were observed under a confocal laser scanning microscope (live cells were labeled with green fluorescence and dead cells were labeled with red fluorescence) ( Figure 2Part B), fibroblasts grow into the electrospinning and the cell morphology changes. As the drug loading increases during the electrospinning process, the proliferation rate of fibroblasts is inhibited. In order to balance the biological effect and biocompatibility of the scaffold, we selected an electrospinning membrane with a drug loading of 1‰ for subsequent experiments.

[0192] Polymers with different N / P ratios were co-cultured with osteogenic precursors. After co-culture, the cells were stained with live cells and observed under a fluorescence microscope (live cells were labeled with green fluorescence and dead cells were labeled with red fluorescence). The results showed that as the N / P ratio increased, the toxicity of the polymers increased ( Figure 2 Meanwhile, polymers with higher N / P ratios had a greater effect on the proliferation of osteoblast precursor cells ( Figure 2 In order to strike a balance between the biological effect and biocompatibility of the scaffold, we selected a polymer with an N / P ratio of 12 for subsequent experiments.

[0193] 3. Cell anti-fibrosis experiment

[0194] 3.1 Physical barrier effect of PCL electrospun membrane

[0195] In order to simulate the blocking effect of PCL membrane on fibroblasts, we established an in vitro migration model of fibroblasts. Figure 3 As shown in parts A and B, in the Transwell chambers with PCL electrospun membrane barriers, almost no cells can pass through the membrane barrier. This is because the nanofibers have micro-nano pores and dense structures. In those Transwell compartments without PCL nanofiber membrane barriers, fibroblasts can easily pass through the PC membrane. The number of cells on the PC membrane in each group was statistically analyzed, and the difference was statistically significant ( Figure 3 (Part C).

[0196] Fibroblasts were seeded on the electrospun membrane for co-culture. After 2 days of co-culture, the electrospun membrane was stained with live cells and observed under a confocal laser scanning microscope (nuclear staining: DAPI, cytoskeletal element staining: rhodamine cyclotide). These cells seemed to be concentrated on the surface of the PCL nanofiber membrane, making it difficult to enter deeper into the fiber membrane ( Fig. 9 ).

[0197] 3.2 PCL electrospun membrane inhibits the expression of genes and proteins related to fibrosis

[0198] Each treatment group was stained for intravenous protein and TGF-β1 protein. The results showed that the blank group had the highest level of fibroblast proliferation, the best cell morphology, and the richest fibroblast expression. In the PCL-FAPI group, the cell morphology was irregular, the cell number was suppressed, and the expression of adhesion plaques and TGF-β1 antibody was the weakest ( Figure 3 The differences in fluorescence area and intensity among the groups were statistically significant ( Figure 3 H and I in the middle). The blank group and PCL-FAPI group were subjected to RT-PCR and Western blot detection, respectively. The expression of fibroblast-related genes or proteins such as TGF-β1 and Smad1 in the PCL-FAPI group was significantly reduced. This difference in expression was statistically significant ( Figure 3 F and G in the figure).

[0199] 4. Transfection process and composite cell osteogenesis and angiogenesis

[0200] 4.1GBA-G5 combined plasmid and transfected cells

[0201] Figure 4 Part A is a schematic diagram of the modified cationic polymer G5-PAMAM combined with plasmid. When the N / P ratio is >4, uniform cyclic polymers can be observed under transmission electron microscopy ( Figure 8 ). In addition, 1% agarose gel electrophoresis showed that after channel 4, the cationic polymer had almost no nucleic acid development bands in the channel due to good binding with DNA fragments. Few nucleic acid fragments escaped ( Figure 4 Dynamic light scattering measurements confirmed the particle size measurement and zeta potential changes of the polymers ( Figure 4 (section D).

[0202] Different transfection reagents (liposome tm3000, G5-PAMAM, GBA-G5) were used to co-transfect osteoblast precursor cells with plasmids. After 2 days, the expression of GFP protein was recorded under a fluorescence microscope, and the expression ratio of GFP-positive cells was detected by flow cytometry. It can be seen that the MC3T3-E1 cells transfected with modified GBA-G5 had the highest GFP fluorescent protein density and the highest GFP-positive cell ratio. Statistical analysis showed that the average transfection efficiency of liposomes, G5-PAMAM and GBA-G5 was 10%, 15% and 20%, respectively. GBA-G5 had the strongest transfection efficiency, which is due to the strong membrane-breaking effect of the modified guanidine ( Figure 4 In order to explore the microscopic appearance of plasmids delivered by different transfection reagents, the plasmids were labeled with Cy3 fluorescein at the end. After 4 h of transfection, the plasmids were concentrated around the cells. The plasmids transfected with GBA-G5 had a good membrane permeabilization effect and were effectively absorbed by the cells ( Figure 4(see Section G in the Appendix).

[0203] Since the target gene SHN-3 (Hivep3) has a large base pair and CDs of 7000+BP, multiple targeted knockdown plasmids were designed for the target gene. The designed sites are: Hivep3-Rat-1801, Hivep3-Rat-2892, Hivep3-Rat-4270, Hivep3-Rat-5370. The use of 4-site plasmids mixed in the same proportion has a higher success rate and transfection rate than using a single-site plasmid. Fig. 20 As shown, the left part of the figure is the transfection of 4-site plasmids mixed in the same proportion, and the right part of the figure is the co-localization fluorescence image of single-site plasmid (Hivep3-Rat-1801) transfection.

[0204] 3.4.2 Genetic alterations in SHN-3 knockdown cells

[0205] The expression of Slit3 protein in the culture medium of transfected cells was detected using a rat homologous Slit3 protein ELISA kit (Enfeldt, Wuhan). It can be seen that the knockout cells secreted more Slit3 protein ( Figure 4 The expression of SHN-3 gene in gene knockout cells was detected by RT-qPCR, and the results showed that it was significantly reduced ( Figure 4 (see Section K in the figure).

[0206] 4.3Slit3 protein promotes the growth of “H” type blood vessels

[0207] In order to analyze the effect of Slit3 protein on the growth of vascular endothelial cells, we used transfected cell culture medium (half-exchange solution) to intervene in HUVECs and analyzed its effect on vascular endothelial cell migration and tubule formation. After intervention with Slit3 protein, the migration and tubule formation ability of vascular endothelial cells were enhanced, and the difference was statistically significant compared with the blank group ( Figure 4 h, I, J, M, N, O). To analyze the characteristic expression of H-type angiogenesis in endothelial cells, these cells after intervention were stained with CD31 and Emcn antibodies. The expression of these antibodies was weak in cells without intervention. CD31 expression was stronger in cells treated with deferoxamine (DFO). CD31 and Emcn ( Figure 5 In the analysis of fluorescence area and fluorescence intensity, the difference was statistically significant ( Figure 5 F, G, and H parts).

[0208] 4.4 Gene knockout cells have enhanced bone formation and mineralization capabilities

[0209] Type I collagen was stained in osteoblast precursor cells of all treatment groups, while the expression of type I collagen was inhibited in dexamethasone-treated cells. The expression of type I collagen was enhanced in cells treated with teriparatide and cells knocked down ( Figure 5 The blank group cells and knockout gene cells were stained with Alizarin red and ALP, and the results showed that calcium deposition and ALP expression were enhanced in knockout gene cells ( Fig.11 ).

[0210] 4.5 RNA Sequence

[0211] We analyzed the genetic changes in the secretion of osteogenesis, mineralization, and angiogenic factors in gene knockdown cells. By sequencing the genes of knockout osteoblast precursor cells, GO cell function analysis found that this is related to the development of the skeletal system, ossification involved in bone remodeling, vascular development, and Wnt signaling. There were significant differences in the up-regulation levels of pathway-related genes. Further analysis of the enrichment of differentially expressed genes for KEGG pathways revealed that the Wnt signaling pathway was in the top 10 of the differentially expressed gene enriched pathways. Detection of the expression of angiogenic factors showed that only the Slit3 gene showed higher and more stable levels in knockout cells. The above data indicate that the knockout osteoblast progenitor cells have enhanced the osteogenesis and mineralization capabilities associated with the Wnt signaling pathway, and enhanced the slit3 secretion capacity of osteoblast progenitor cells ( Figure 5 AC part).

[0212] 4.6 Osteogenesis ability of scaffolds in vivo

[0213] All SD rats were randomly divided into 4 groups: sham operation group, blank control group, PCL membrane group, and core-shell group. After 1 week of adaptive feeding, all four groups of rats underwent surgery to induce a 5 mm critical bone defect in the femur ( Fig.13 ). After scaffold implantation, rats were euthanized at 4, 8, and 12 weeks. X-ray and micro-CT results of the femurs of rats showed that the volume of new bone tissue in the core-shell group increased significantly at each time point. In addition, the bone volume (BV) and bone mineral density (BMD) of the new bone increased. These results are consistent with the results of the in vitro experiments.

[0214] Histological sections were used to further determine its anti-fibrosis and bone regeneration-promoting effects in vivo. In the gross tissue samples of the two groups implanted with solid scaffolds, some membrane tissue remained, but no hydrogel residue was found. The hematoxylin and eosin (H&E) staining results of femoral defects in SD rats showed that the femoral fracture ends of the blank group and PCL group closed after 8 weeks, suspending the bone regeneration process. The new fibrous tissue produced in the bone defects of the blank group and PCL group was more than that of the core-shell group ( Figure 6 This was confirmed by Masson trichome staining ( Figure 6(Part E). The severe femoral defects in the core-shell group achieved bone reunion at 8 weeks.

[0215] Tissue immunofluorescence was used to observe the regeneration of "H"-type blood vessels in the new femur of rats. At the 4th week, scattered h-type blood vessels were distributed in the bone regeneration tissue around the bone defect in the blank group and PCL group. At the 8th week, the expression of h-type blood vessels in the bone regeneration tissue decreased. At the 4th and 8th weeks, the number of h-type blood vessels in the core-shell group was higher than that in the control group, blank group and PCL group ( Figure 7 Part A and B).

[0216] H&E and Masson trichome staining were used to observe the main organs (liver and kidney) of the four groups of SD rats to further verify the safety of PCL composite scaffold implantation. At the end of the study period, no obvious pathological changes such as necrosis and / or inflammation were found in any organ ( Fig.12 ). Based on these results, the present invention concludes that the core-shell scaffold has good biocompatibility in vivo.

[0217] Bone repair is a highly dynamic process. During the development, modeling, and repair of bone, bone formation is closely related to fibrosis and vascularization. During the repair of critical bone defects, due to the large size of bone defects and slow osteogenesis, fibrous tissue invades and fills the bone defect area, hindering osteogenesis. At present, the clinical treatment of critical bone defects mainly includes filling the bone defect area with autologous bone, allogeneic bone, or other bioactive materials, combined with the application of growth factors such as bone morphogenetic protein or vascular endothelial growth factor. The excessive growth of fibrotic tissue at the bone defect site has not received serious attention. The regeneration of bone and blood vessels after bone injury is closely related to para-osseous tissue (such as fat and connective tissue). Para-osseous tissue participates in the osteogenesis process, which accelerates the fibrosis process on the one hand and reduces the quality of osteogenesis on the other hand. Therefore, isolating the fibrotic microenvironment around the bone defect and creating a regenerative microenvironment that is conducive to bone formation is expected to become a powerful strategy to promote the repair of critical bone defects. On the other hand, due to the large size and slow ossification of critical bone defects, increasing the rate of bone formation is also a core factor in achieving effective repair of critical bone defects. H-type vessels are a special subtype of blood vessels in bone. H-type angiogenesis is closely related to osteogenesis during bone development, formation and repair. Therefore, the combined strategy of anti-fibrosis and promotion of H-type angiogenesis is expected to become a new direction for the treatment of critical bone defects in the clinical treatment of new callus.

[0218] In this invention, we designed a core-shell bionic scaffold PCL-FAPI / GH-GBA@pkSHN-3. This new functional bioscaffold system can significantly promote bone regeneration in a critical bone defect rat model by balancing fibrosis and ossification in callus tissue, inhibiting excessive fibrosis, and promoting bone formation at the defect site. In vivo and in vitro experiments have shown that the bioscaffold system can effectively act on the tissues surrounding the bone defect and has safe and reliable biocompatibility ( Figure 2 , Fig.12 ). Polycaprolactone is an FDA-approved polymer material that is widely used in the medical field due to its rich plasticity and safe biocompatibility. The shell structure uses electrospun PCL nanofibers containing FAPI-4. The PCL electrospun fibers prepared by electrospinning technology have hydrophobicity ( Figure 1 This can effectively block the growth of exogenous fibrous tissue at the physical level ( Figure 3 The released FAPI-4 can inhibit the growth and adhesion of fibroblasts around bone defects and effectively reduce the expression of fibroblast-related proteins ( Figure 3 Meanwhile, FAPI is an osteogenic promoter that promotes osteoblast growth.

[0219] Slit3 protein is a pro-angiogenic factor derived from osteoblasts and is regulated by large zinc finger protein 3 (SHN-3). Downregulating the expression of SHN-3 can enhance the ability of osteoblasts to secrete slit3. The inner core structure is a plasmid / polyamidoamine supported by GelMA / HAMA hydrogel (GBA@plasmid knockout SHN-3). The fifth-generation polyamide modified by GBA (GBA-PAMAM) has strong nucleic acid binding and cell disruption ability, and combines to form a stable nano-scale polymer GBA@pkSHN-3 ( Figure 4 , Figure 8 ). Using Cy3 fluorescent markers to label the plasmid ends and staining the plasmid and granulocytes co-localized, we found that GBA@pk SHN-3 aggregated around the cell nucleus, effectively delivering shRNA to the cell nucleus for gene intervention ( Figure 4 Through quantitative evaluation, the present invention found that GBA@pk SHN-3 had the highest transfection efficiency for osteoblast precursor cells, which was better than G5-PAMAM and commercial reagent liposome ineTM3000 ( Figure 4 To explore the intervention effect of Slit3 protein on vascular endothelial cells, HUVECs were treated with culture medium containing Slit3 protein, which enhanced the migration and tube formation ability of HUVECs ( Figure 4 H, L, and J parts of the middle part), and strongly expressed the characteristic protein of h-type blood vessels ( Figure 5We used transcriptome sequencing (Seq-RNA) to analyze the protein and genomic changes in osteoblast progenitor cells with SHN-3 knockout (SHN-3- / -), and we observed significant differences in the expression of genes related to osteogenesis and mineralization in SHN-3- / - cells ( Figure 5 This was also confirmed by staining for osteogenic proteins and detection of calcium deposition and ALP ( Fig.11 ). Further enrichment of KEGG pathways on differentially expressed genes showed that genes related to the WNT signaling pathway in SHN-3- / - cells were significantly upregulated ( Figure 5 In addition, Slit3 showed higher and more stable levels in osteoblasts knocked out by gene knockout ( Figure 5 In summary, we believe that the strategies of GBA@pk SHN-3 to enhance new callus tissue may be as follows: (1) enhancing the osteogenic and mineralization abilities of knockdown cells; (2) knocking out the SHN-3 gene in osteoblast precursor cells to enhance the secretion of Slit3 protein, thereby promoting the formation of H-type vessels in new callus tissue.

[0220] In the SD rat model of critical femoral defect, the bone defect in the blank control group was not effectively repaired, and a large amount of fibrous tissue was filled in the bone defect ( Figure 6 (Part D in the figure). Bone regeneration was enhanced in the PCL electrospinning membrane coating treatment group, but the critical bone defect was still not effectively repaired. In the PCL-FAPI / GH-GBA@pkSHN-3 treatment group, the critical bone defect was effectively repaired. Immunofluorescence detection results showed that the expression of h-type vessels in the regenerated bone tissue was low in the blank group and PCL group. However, in the PCL-FAPI / GH-GBA@pkSHN-3 scaffold group, the expression of h-type vessels in the regenerated bone tissue was significantly increased (Part 7 in the figure), which was due to the continuous secretion of slit3 protein by knockdown gene cells. This suggests that the successful repair of the critical bone defect is due to the osteogenic environment generated by PCL fibers and the osteogenic and angiogenic effects of Slit3 factors, rather than simply due to the ability of PCL fibers to block the outer fibrous tissue. In addition, as the bone defect repair time increases, the mineralization ability of the regenerated bone tissue is enhanced and the vascularization ability is weakened. However, the expression of H-type vessels in the PCL-FAPI / GH-GBA@pkSHN-3 scaffold-treated group was still higher than that in the blank group and PCL group. Therefore, in the repair of critical bone defects in rats, the core-shell scaffold can achieve anti-fibrosis effects, promote H-type vascularization, and promote bone repair.

Claims

1. A method for preparing a core-shell structure biological scaffold for treating bone defects, characterized in that: The preparation method comprises the following steps: (1) adding fibroblast inhibitor FAPI to a polycaprolactone solution and using electrospinning technology to prepare a drug-loaded polycaprolactone fiber membrane; (2) using a SHN-3 knockout plasmid and G5-GBA to prepare a nanopolymer, then adding the nanopolymer and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate to a hydrogel solution composed of GelMA and HAMA, and irradiating with ultraviolet light; the SHN-3 knockout plasmid is a mixed plasmid of plasmids whose gene knockout target sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 respectively; (3) rolling the obtained drug-loaded polycaprolactone fiber membrane on the surface of a mold, placing the obtained substance in step (2) on the mold, and curing it under ultraviolet light to form a core-shell structure.

2. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 1, characterized in that: The solvent in the polycaprolactone solution includes chloroform.

3. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 2, characterized in that: The polycaprolactone solution is prepared by dissolving polycaprolactone in chloroform at a weight to volume ratio of 1 g:9 mL, and then adding dimethylformamide in an amount that is one fifth of the weight of the polycaprolactone.

4. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 1 or 3, characterized in that: The volume-to-weight ratio of the volume of the fibroblast inhibitor FAPI to the sum of the weights of polycaprolactone and the fibroblast inhibitor FAPI is 0.5-2‰ v / w; the N / P ratio of the SHN-3 knockout plasmid to G5-GBA is 1-20; in the SHN-3 knockout plasmid, the number ratio of each plasmid is 1:1:1:

1.

5. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 4, characterized in that: During electrospinning, the voltage was 18 kV, the collection distance was 20 cm, and the flow rate was 2.5 mL / h.

6. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 1 or 5, characterized in that: The weight ratio of GelMA to HAMA was 1:

1.

7. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 6, characterized in that: The added amount of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.5% w / v.

8. The method for preparing a core-shell structure biological scaffold for treating bone defects according to claim 1 or 7, characterized in that: The wavelength of the ultraviolet light is 405 nm.

9. A core-shell structure biological scaffold for treating bone defects, characterized in that: The core-shell structure biological scaffold is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the core-shell structure biological scaffold for treating bone defects as claimed in claim 9 in preparing products for treating bone defects.

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