Macrophage hybrid scaffold as well as construction method and application thereof

Abstract

The invention discloses a macrophage hybrid scaffold as well as a construction method and application thereof, and belongs to the technical field of biological materials. The construction method comprises the following steps: extracting peripheral blood mononuclear cells, inducing the peripheral blood mononuclear cells into mature macrophages, loading the mature macrophages on a tissue engineering scaffold, and adding an immunoregulation factor to regulate the phenotype of the mature macrophages to obtain the M2 type macrophage hybrid scaffold. According to the invention, the macrophages with an immunoregulation effect are introduced into the tissue engineering scaffold, the macrophage hybrid scaffold is constructed together, and the macrophage hybrid scaffold can be used for repairing tissue defects, such as defect tissues such as skin, mucous membrane, viscera, cartilage or hard bone and the like; the macrophage hybrid scaffold can meet various requirements of providing mechanical support and bioremediation functions for tissue defect areas.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a macrophage hybrid scaffold and a construction method and application thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease closely related to the imbalance of the microbiota, which is mainly manifested by the irreversible destruction of the tissues around the teeth and their supporting tissues and the formation of periodontal pockets. When periodontitis develops to the late stage, it can cause loosening, displacement and even loss of teeth. At present, guided tissue / bone regeneration (GTR / GBR) with a barrier membrane is mainly used clinically. It has achieved significant improvements in the regeneration of mild periodontal defects, but it still has certain limitations for the regeneration of large irregular defects. In particular, for large periodontal defects caused by severe periodontitis, due to their complex immune microenvironment and limited residual periodontal supporting tissues, their regeneration and repair remains a major clinical challenge.

[0003] In recent years, tissue engineering technology has attracted much attention in the field of periodontal tissue regeneration and repair, especially the application of biomaterial implants. The traditional view is that ideal biomaterials should promote tissue repair without stimulating the immune system and avoiding foreign body reactions. However, it is often difficult to avoid the occurrence of inflammatory reactions after the implantation of tissue engineering biomaterials, which is a key factor affecting the tissue regeneration and tissue integration effects after the implantation of biomaterials. More and more studies have shown that immunoregulation based on biomaterials has a positive role in promoting tissue regeneration. The repair of tissue defects caused by periodontitis faces more complex challenges than ordinary tissue defects due to its special immune microenvironment, and the efficiency of tissue repair will be affected by the long-term inflammatory environment. Therefore, the immunoregulatory effect of biomaterials has received widespread attention.

[0004] The monocyte-macrophage system is the first line of defense of the immune system, actively participating in pro-inflammatory, anti-inflammatory, phagocytic elimination of bacteria and foreign bodies, and a series of tissue repair functions. In periodontal tissues, the monocyte-macrophage system plays a key role in maintaining environmental homeostasis. Under different stimuli, macrophages can be activated into different phenotypes and divided into two phenotypes, M1 and M2, according to their functions. M1 macrophages mainly play an immune clearance role and can secrete proinflammatory factors and chemokines (such as TNF-α, IL-1β, IL-6, etc.), thereby aggravating the inflammatory response; M2 macrophages are a macrophage phenotype that promotes tissue repair and can release a large number of anti-inflammatory factors (such as IL-10, TGF-β, CCL1, etc.) to promote tissue repair and regeneration. In the periodontitis environment, M1 macrophages are dominant, and the continued existence of this state will affect periodontal healing. Therefore, it is of great significance to endow tissue engineering materials with immunomodulatory functions and promote the transition of tissue microenvironment from an inflammatory state to a pro-repair state, so as to reverse the adverse microenvironment caused by periodontitis and achieve effective regeneration of periodontal tissue. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a macrophage hybrid scaffold and a construction method and application thereof, so as to provide a new strategy for the treatment of late-stage periodontitis by synergizing immune regulation with biomaterials.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: a method for constructing a macrophage hybrid scaffold is provided, comprising the following steps:

[0007] Peripheral blood monocytes were extracted and induced into mature macrophages, the mature macrophages were loaded on tissue engineering scaffolds, and immune regulatory factors were added to regulate the phenotype of mature macrophages to obtain M2 macrophage hybrid scaffolds.

[0008] The beneficial effects of the present invention are as follows: M2 macrophages are a macrophage phenotype that promotes tissue repair, and can release a large amount of anti-inflammatory factors to promote tissue repair and regeneration. The present invention introduces macrophages with immunomodulatory effects on tissue engineering scaffolds to jointly construct macrophage hybrid scaffolds, which can be used to repair tissue defects, such as defective tissues such as skin, mucosa, internal organs, cartilage or bone, and the macrophage hybrid scaffold provides mechanical support and biological repair functions for tissue defect areas.

[0009] Based on the research on regulating macrophage phenotypes by biomaterials to promote tissue repair and regeneration, it is usually through the immune regulatory components in the biomaterials that induce the recruitment of endogenous macrophages in the host in vivo and form M2 macrophages. However, there are many types and numbers of cells in the body, and the efficiency of the immune regulatory components to accurately act on macrophages and regulate their polarization to M2 may be limited and cannot play a role effectively. The macrophage hybrid scaffold constructed in this application is to accurately induce exogenous macrophages into M2 macrophages in vitro through immune regulatory factors, and enter the body in the form of tissue engineering scaffold loading, thereby increasing the number of M2 macrophages, and then play its role well, which can quickly promote tissue repair and regeneration, such as defective tissues such as skin, mucosa, internal organs, cartilage or bone, and can be more effectively used in the treatment of periodontitis.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] Furthermore, mature macrophages were loaded onto the tissue engineering scaffold as follows: mature macrophages were loaded at a rate of 1×10 4 -1×10 8 The cells were seeded on tissue engineering scaffolds at a density of 100 / mL.

[0012] Furthermore, the tissue engineering scaffold is a TPP scaffold or an enzyme-treated TPP scaffold.

[0013] The beneficial effect of adopting the above further technical solution is that the tissue engineering scaffold can be a commonly used tissue engineering scaffold in biomedicine or a self-developed tissue engineering scaffold with good bioactivity and biocompatibility. In the present invention, the preferred tissue engineering scaffold is a TPP scaffold and an enzyme-treated TPP scaffold such as a lipase-treated TPP scaffold.

[0014] Further, the TPP scaffold is prepared by the following method:

[0015] PCL, PLGA and β-TCP are added into an organic solvent, dissolved and mixed evenly to obtain a slurry; the slurry is made into a 3D composite porous scaffold through a material extrusion technology, and then dried and sterilized to obtain the slurry.

[0016] Furthermore, the mass ratio of PCL, PLGA, and β-TCP was 35:35:30.

[0017] Furthermore, the immunomodulatory factor is IL-4, IL-13, TGF-β or glucocorticoid.

[0018] The beneficial effect of adopting the above further technical solution is: the immune regulatory factor is to regulate the polarization of mature macrophages to M2 type, mainly including a variety of cytokines and growth factors, that is to say, any substance that can regulate the polarization of mature macrophages to M2 type can be used. In the present invention, the preferred immune regulatory factor is IL-4, IL-13, TGF-β or glucocorticoid.

[0019] Furthermore, the concentration of the added immunomodulatory factor is 20-100 ng / mL.

[0020] Application of the macrophage hybrid scaffold prepared above in the preparation of tissue defect repair and regeneration implant materials.

[0021] Furthermore, the tissue is soft / hard tissue such as skin, mucosa, internal organs, bones, and periodontal tissue.

[0022] Furthermore, the macrophage hybrid scaffold prepared as above is used in the preparation of implant materials for treating periodontal bone defects.

[0023] The present invention has the following beneficial effects:

[0024] The macrophage hybrid scaffold constructed by the present invention plays an important role in promoting tissue repair and regeneration. The macrophage hybrid scaffold proposed, designed and constructed by the present invention enriches the theory and strategy of tissue regeneration and reconstruction based on immune regulation, especially enriches the theory and strategy of periodontal tissue regeneration and reconstruction based on immune regulation.

[0025] The present invention makes up for the shortcomings of the existing methods to a certain extent by constructing a macrophage hybrid scaffold for the treatment of periodontal bone defects in rats, wherein the monocytes / macrophages derived from peripheral blood mainly have the following advantages: ① They are terminally differentiated cells, do not proliferate, and do not have additional effects on the host after their life cycle ends; ② They are immune cells with immunomodulatory functions, secreting a variety of immunomodulatory factors, anti-angiogenic proteins or anti-inflammatory factor-related proteins; ③ They have a wide range of sources, are easy to obtain, have no application restrictions, and do not have ethical issues; ④ When the research is transformed into clinical practice, the patient's peripheral blood can be used to extract monocytes / macrophages. Therefore, the macrophage hybrid scaffold constructed by the present invention can deliver a large number of macrophages to the periodontal defect area, providing means and tools for maximizing the therapeutic effect of macrophages, which is of great significance for future cell delivery and cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are monocytes / macrophages isolated and cultured from rat peripheral blood.

[0027] Figure 2 The 3D composite porous scaffold (TPP scaffold) constructed for the present invention.

[0028] Figure 3 The results of constructing the macrophage hybrid TPP scaffold; A is the localization of macrophages on the TPP scaffold; B is a local magnified image of A.

[0029] Figure 4 Results of live cell tracking experiments of PKH26-labeled macrophages on TPP scaffolds.

[0030] Figure 5 The results of immunohistochemical staining of macrophage hybrid TPP scaffolds in rat subcutaneous sections; A is the result of immunohistochemical staining of Arg-1; B is the result of immunohistochemical staining of iNOS.

[0031] Figure 6 Micro-CT detection of new bone formation in the periodontal defect area of ​​rats with macrophage hybrid TPP scaffolds; A is the CT reconstructed image and cross-sectional image of the periodontal defect area of ​​rats; B is the quantitative analysis result of the new bone in the periodontal defect area of ​​rats.

[0032] Figure 7 These are the test results of bone repair of macrophage hybrid TPP scaffolds in periodontal bone defects in rats, including H&E staining and Masson staining results.

[0033] Figure 8 The results of constructing the macrophage hybrid TPP-L scaffold; A is the localization of macrophages on the scaffold; B is a local magnified image of A.

[0034] Fig. 9 Results of live cell tracking experiments of PKH26-labeled macrophages on TPP-L scaffolds.

[0035] Fig.10 The results of immunohistochemical staining analysis of macrophage hybrid TPP-L scaffold in rat subcutaneous sections; A is the result of Arg-1 immunohistochemical staining; B is the result of iNOS immunohistochemical staining.

[0036] Fig.11 Micro-CT detection of new bone formation in the periodontal defect area of ​​rats with macrophage hybrid TPP-L scaffolds; A is the CT reconstructed image and cross-sectional image of the periodontal defect area of ​​rats; B is the quantitative analysis result of the new bone in the periodontal defect area of ​​rats.

[0037] Fig.12 These are the test results of bone repair of macrophage hybrid TPP-L scaffold in periodontal bone defect of rats, including H&E staining and Masson staining results. DETAILED DESCRIPTION

[0038] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0039] Example 1: Isolation and culture of rat peripheral blood monocytes / macrophages

[0040] The isolation and culture of rat peripheral blood monocytes / macrophages specifically includes the following steps:

[0041] (1) Healthy adult male SD rats (weighing about 200 g, provided by the Animal Experiment Department of Kunming Medical University) were given an intraperitoneal injection of 1 mL / kg of 3% sodium pentobarbital for general anesthesia.

[0042] (2) After anesthesia takes effect, disinfect the chest with 75% alcohol, touch the part of the rat's heart that has the strongest heartbeat, and insert the blood collection needle between the 3rd and 4th ribs on the left side of the chest. Puncture the part of the rat's heartbeat that has the strongest heartbeat. When blood automatically flows into the blood collection tube accompanied by pulsation, connect the negative pressure tube to collect the rat's peripheral blood.

[0043] (3) Move the sample into a clean bench and dilute the blood sample 1:1 with PBS buffer containing 2% FBS and mix gently.

[0044] (4) Use a pipette to carefully add the gradient density centrifugation solution into the monocyte separation tube through the small hole in the center of the insert in the monocyte separation tube.

[0045] (5) Keep the separation tube vertical and use a pipette to slowly add the diluted blood sample along the wall of the tube.

[0046] (6) Centrifuge at 1200 g for 10 min at room temperature and carefully pour the upper layer containing the enriched PBMCs into a new centrifuge tube.

[0047] (7) Wash PBMCs with PBS buffer containing 2% FBS, centrifuge at 300 g for 8 min at room temperature, and discard the supernatant.

[0048] (8) Centrifuge at 120 g for 10 min at room temperature, turn off the centrifuge brake and repeat the wash to remove the platelets in the plasma layer from the enriched PBMCs.

[0049] (9) Discard the supernatant, resuspend the cells in a rat peripheral blood mononuclear cell culture medium, add the cell suspension to a T25 culture flask, add the culture medium to 5 mL, and place in a constant temperature incubator for static culture.

[0050] (10) After 2 to 4 hours, PBMCs (peripheral blood mononuclear cells) are basically attached to the wall. The old culture medium is discarded, and the cells are gently washed three times with PBS buffer. The culture medium is replaced with M-CSF (20 ng / mL). The medium is changed every 2 to 3 days. After about 5 to 7 days, the monocytes are induced to become macrophages.

[0051] The monocytes / macrophages isolated and cultured from the peripheral blood of rats are shown in Figure 1 .Depend on Figure 1 It can be seen that the peripheral blood mononuclear cells separated from rat peripheral blood by density gradient centrifugation successfully adhered to the wall under an inverted phase contrast microscope, and were round or oval in shape with large and round nuclei.

[0052] Example 2: Printing of 3D composite porous scaffold (TPP scaffold)

[0053] The printing of 3D composite porous scaffold (TPP scaffold) specifically includes the following steps:

[0054] (1) Preparation of mixed slurry: β-TCP particles were ground in a mortar and passed through a 300-mesh sieve. 2.1 g of the particles were weighed and placed in a 50 mL centrifuge tube. 7 mL of dichloromethane was added in a fume hood and stirred to uniformly disperse the β-TCP particles in the dichloromethane. 2.45 g of PCL particles and 2.45 g of PLGA particles were weighed and added to the above centrifuge tubes at a mixing ratio of β-TCP:PCL:PLGA = 30%:35%:35% by mass. The mixture was stirred for 1 h.

[0055] (2) 3D modeling: A 3D model of the porous scaffold was established in the CAD software. The shape of the porous scaffold was designed to be a disc. The printing parameters were set as follows: diameter 14 mm, layer height 0.21 mm, wire diameter 350-400 μm, wire pitch 600-650 μm, angle 90°, and number of layers 5.

[0056] (3) Printing: Import the CAD model into the 3D bioprinter, set the printer air pressure to 0.43 MPa, speed to 12 mm / s, and 0.26 mm conical needle for printing. After printing, remove the stent. The stent is named β-TCP / PCL / PLGA stent, referred to as TPP stent.

[0057] (4) Vacuum drying and sterilization: Place the TPP stent in a vacuum drying oven and remove it after vacuum drying for 72 hours. 60 Co irradiation sterilized, ready for use.

[0058] The 3D composite porous scaffold (TPP scaffold) constructed by the present invention is shown in Figure 2 .Depend on Figure 2It can be seen that the 3D composite porous scaffold (TPP scaffold) constructed by the present invention has a uniform wire diameter and a regular porous structure, which is conducive to tissue growth.

[0059] Example 3: Construction of macrophage hybrid TPP scaffold

[0060] The construction of macrophage hybrid TPP scaffold includes the following steps:

[0061] The macrophages (Mφ) prepared in Example 1 were digested with trypsin and centrifuged, and the cell concentration was measured and adjusted to 5×10 5 The density of cells / mL. The TPP scaffold was placed in the confocal dish in advance, with 500 μL of cell suspension in each well, and the culture medium was supplemented to 1 mL per well. 40 ng / mL of IL-4 was added, and the plate was cultured in a constant temperature incubator. F-actin staining was performed after 3 days of culture: the culture plate was removed, the culture medium was discarded, and the plate was washed 3 times with PBS buffer solution. Subsequently, 4% paraformaldehyde was added, the cells were fixed at room temperature for 20 minutes, and the plate was washed 3 times with PBS buffer solution. Use PBS buffer containing 0.1% Tritonx-100 to treat for 15 minutes to improve the permeability of the cells, and the cells were washed 3 times with PBS buffer solution. F-actin staining solution was added to each well to stain the cytoskeleton, incubated at room temperature in the dark for 1.5 hours, and then the F-actin staining solution was discarded and washed 3 times with PBS buffer solution. Finally, DAPI staining solution was added, incubated at room temperature in the dark for 5 minutes, and washed 3 times with PBS buffer solution. In a wet state, the sample was placed under a laser scanning confocal microscope for observation and image acquisition. The results are shown in Figure 3 .

[0062] Depend on Figure 3 It can be seen that the red fluorescence is macrophages, and the green fluorescence is TPP scaffolds. The staining results show that macrophages can attach to the surface of the TPP scaffold, confirming that the macrophage hybrid TPP scaffold was successfully constructed.

[0063] Example 4: Establishment of rat subcutaneous TPP / hybrid TPP scaffold model

[0064] The establishment of the rat subcutaneous stent implantation model specifically includes the following steps:

[0065] Eighteen healthy adult male SD rats (weighing about 200 g, provided by the Animal Experiment Department of Kunming Medical University) were selected to establish a dorsal subcutaneous stent implantation model. The rats were divided into a negative blank control group (Ctrl group), a TPP stent implantation group (TPP group), and a macrophage hybrid TPP stent implantation group (TPP+Mφ group). The stents were divided into three time points: 1, 4, and 7 days. Before implantation into the subcutaneous tissue of the rats, all stents were 60Co radiation sterilization. After the rat abdomen was disinfected with iodine cotton balls, 1 mL / kg of 3% sodium pentobarbital was injected intraperitoneally for general anesthesia. The back hair was removed with depilatory cream and shaver. After the surgical area was disinfected with iodine, three longitudinal incisions of about 1 cm in length were made in the middle of the back of each rat. The skin on both sides of the incision was gently lifted with forceps, and the skin and subcutaneous tissue were bluntly separated on both sides of the incision using blunt-tipped instruments to form a subcutaneous bag. Then, according to the grouping, the first bag was the Ctrl group as a blank control without stent implantation, the second bag was the TPP group implanted with TPP stent, and the third bag was the macrophage hybrid TPP stent implanted with TPP+Mφ stent, and then the incision was carefully sutured and closed. Routine feeding was performed after surgery. On the 1st, 4th, and 7th day after surgery, 6 SD rats were randomly selected and killed for samples. The samples taken included skin, stents, and subcutaneous tissue, and the samples were fixed with neutral formaldehyde fixative.

[0066] Example 5: Localization of macrophages after subcutaneous implantation of macrophage hybrid TPP scaffolds The localization of macrophages after subcutaneous implantation of macrophage hybrid TPP scaffolds is as follows:

[0067] PKH26 was used to label macrophages for live cell tracing experiments. PKH26-labeled Mφ were inoculated onto TPP scaffolds and implanted subcutaneously in rats. The rats were killed by cervical dislocation on days 1, 4, and 7 after surgery, and the specimens were collected. The specimens were fixed with neutral formaldehyde solution for 24 hours, run overnight, dehydrated with sucrose gradient, frozen, and sealed with DAPI-containing sealing agent. The specimens were observed and imaged under a laser scanning confocal microscope. The results are shown in Figure 4 .

[0068] Depend on Figure 4 It can be seen that the red fluorescence is exogenous macrophages, the blue fluorescence is the cell nucleus, and "M" is the TPP scaffold. It can be seen that the macrophage hybrid TPP scaffold can successfully carry macrophages into the body and gradually migrate from the scaffold to the defect area.

[0069] Example 6: Histological detection after implantation of macrophage hybrid TPP scaffold

[0070] For fixed skin specimens, first rinse them with running water overnight (at least 12 hours), then embed the tissues after systematic dehydration, and make routine paraffin sections, followed by Arg-1 and iNOS immunohistochemical staining:

[0071] (1) Dewaxing: Soak in xylene I for 15 min, then in xylene II for another 15 min. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 min. Finally, soak in distilled water for 2 min to remove the wax in the slices.

[0072] (2) Antigen repair: Place the sections on a copper rack, put them into a glass beaker containing pre-boiled citric acid antigen repair buffer (pH = 6.0), and place them in an oven at 100°C for 45 min for antigen repair. After cooling naturally to room temperature, place the sections in PBS buffer and shake and wash them three times, each time for 5 min;

[0073] (3) Circle drawing: After the slices are slightly dried, they are placed in a transparent box and the tissue area is circled with a histochemical pen to avoid antibody loss;

[0074] (4) Blocking: Add endogenous peroxidase blocker to the designated tissue area and block for 15 min at room temperature. Rinse once with distilled water, then slightly dry the sections and add goat serum. Block for 20 min at room temperature.

[0075] (5) Primary antibody incubation: Gently shake off the blocking solution, add the primary antibody solution Arg-1 (1:500) and iNOS (1:200) prepared in PBS containing 5% BSA on the slices, place the slices flat in a humidified box, and incubate in a refrigerator at 4°C overnight (about 12 h). Note that an appropriate amount of water should be added to the humidified box to prevent the evaporation of the antibody;

[0076] (6) Secondary antibody incubation: The next day, remove the wet box and place it at room temperature for 30 minutes. Wash the sections three times with PBS buffer, 5 minutes each time. After the sections are slightly dried, add the secondary antibody of the corresponding species to the designated area and incubate at room temperature in the dark for 15 minutes.

[0077] (7) DAB color development: Wash the sections three times with PBS buffer, 5 min each time. After the sections are slightly dried, add freshly prepared DAB color developer to the designated area and observe the color depth under a microscope.

[0078] (8) Counterstaining of cell nuclei: After complete color development, wash the sections with distilled water, counterstain the cell nuclei with hematoxylin, differentiate with hydrochloric acid and alcohol (about 2 seconds), wash the sections with tap water, and perform blue reversion with PBS buffer;

[0079] (9) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 min each to remove moisture;

[0080] (10) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results. Figure 5 .

[0081] Depend on Figure 5The results of Arg-1 staining showed that on the first day after the macrophage hybrid TPP scaffold was implanted, there was a linear distribution of Arg-1 positive cells at the edge of the TPP+Mφ group material; on the third day after implantation, the number of Arg-1 positive cells in the surrounding tissues of the TPP group and TPP+Mφ group increased; on the seventh day after implantation, the number of Arg-1 positive cells in the surrounding tissues of the TPP+Mφ group increased significantly.

[0082] Depend on Figure 5 The iNOS staining results showed that the iNOS expression in the TPP+Mφ group was lower than that in the TPP group at each time point.

[0083] Example 7: Establishment of a rat periodontal critical bone defect model

[0084] The establishment of the rat periodontal critical bone defect model specifically includes the following steps:

[0085] Thirty-six healthy adult male SD rats (weighing about 200 g, provided by the Animal Experiment Department of Kunming Medical University) were used to establish a periodontal critical bone defect model. The rats were managed by the Animal Department and kept in an SPF environment. They were randomly divided into three groups, A, B, and C, with 12 rats in each group, and divided into two time points of 2 weeks and 6 weeks. The groups were divided as follows: Group A was a negative blank control group; Group B was implanted with a TPP stent; Group C was implanted with a TPP+Mφ stent. After the rat abdomen was disinfected with iodine cotton balls, 1 mL / kg of 3% sodium pentobarbital was injected intraperitoneally for general anesthesia. After anesthesia took effect, the rats were fixed in a lateral position on the operating table. The scalpel was used to make a parallel incision about 2 mm above the lower edge of the right mandible of the rat until the mandibular bone surface. The blunt-tipped instrument was replaced to bluntly separate the soft tissue to fully expose the bone surface. The periosteum was carefully separated, and a cylindrical periodontal critical bone defect with a diameter of 4 mm and a depth of 1 mm was prepared on the buccal root of the first and second molars with a dental high-speed handpiece. The bone debris in the defect area was removed, and the wound was rinsed with a large amount of normal saline. Then, the stent was implanted or not implanted according to the group, and the wound was carefully sutured and closed. Routine care and feeding were performed after surgery.

[0086] Example 8: Micro-CT detection and three-dimensional reconstruction of periodontal bone defect sites in rats

[0087] Micro-CT detection and three-dimensional reconstruction of periodontal bone defect in rats, specifically including the following steps:

[0088] Rats were killed by cervical dislocation at 2 weeks and 6 weeks after surgery, and then dissected and sampled. After the specimens were obtained, they were immediately immersed in neutral formaldehyde fixative for 24 hours for fixation. Subsequently, the fixed specimens were scanned using Micro-CT scanning technology, and the specific scanning conditions were set to a tube voltage of 90kV and a tube current of 60μA. The acquired images were reconstructed and analyzed using Avatar 1.5.0 software, focusing on the quantitative statistical analysis of the four indicators of the defect area: bone volume fraction (BV / TV), bone surface area to tissue volume ratio (BS / TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp). The results are shown in Table 1. Figure 6 .

[0089] Depend on Figure 6 It can be seen that Micro-CT 3D reconstruction ( Figure 6 A) shows that new bone formation around the defect was observed in both the TPP group and the TPP+Mφ group at 2 weeks and 6 weeks, with more new bone formation in the TPP-L+Mφ group. Bone analysis results ( Figure 6 B) shows that at 2W and 6W, BV / TV, BS / TV, and Tb.N of the TPP group and TPP+Mφ group were higher than those of the control group, and Tb.Sp was lower than that of the control group. Among them, the BV / TV and BS / TV of the TPP+Mφ scaffold group at 2W were significantly higher than those of the control group, and at 6W, the number of trabeculae was significantly increased and the separation of trabeculae was reduced.

[0090] Example 9: Detection of bone repair at periodontal bone defect sites in rats

[0091] The detection of bone repair at the periodontal bone defect site of rats specifically includes the following steps:

[0092] (1) H&E staining

[0093] For the fixed mandibular specimens, first rinse them with running water overnight (at least 12 hours), then use 17% EDTA decalcification solution for decalcification (about 2 to 3 weeks), after systematic dehydration, embed the tissue, make conventional paraffin sections, and then perform H&E staining:

[0094] 1) Dewaxing: Soak in xylene I for 15 minutes, then in xylene II for another 15 minutes. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 minutes. Finally, soak in distilled water for 2 minutes to remove the wax in the slices.

[0095] 2) Staining: Stain in hematoxylin for 10 minutes, rinse with water for 1 minute. Use hydrochloric acid alcohol for differentiation, and the time is controlled within 2 to 5 seconds. Use warm water for blueing for 2 minutes. Soak in 70% ethanol for 2 minutes. Finally, stain with eosin solution, and the time is controlled within 20 to 30 seconds;

[0096] 3) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each to remove moisture;

[0097] 4) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results.

[0098] (2) Masson staining

[0099] For fixed specimens, first rinse them with running water overnight (at least 12 hours), then decalcify them with 17% EDTA decalcification solution (about 2 to 3 weeks). After systematic dehydration, embed the tissues, make conventional paraffin sections, and then perform Masson staining:

[0100] 1) Dewaxing: Soak in xylene I for 15 minutes, then in xylene II for another 15 minutes. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 minutes. Finally, soak in distilled water for 2 minutes to remove the wax in the slices.

[0101] 2) Staining: add 1 drop of Masson's complex staining solution to the slice, stain for 5 minutes, and rinse with distilled water; add 1 drop of phosphomolybdic acid, stain for 5 minutes, and spin dry; add 1 drop of aniline blue, stain for 5 minutes, and rinse with distilled water; add 1 drop of differentiation solution, and the differentiation time is controlled within 30-60 seconds;

[0102] 3) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each to remove moisture;

[0103] 4) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results.

[0104] The results of the bone repair test at the periodontal bone defect site in rats are shown in Figure 7 .Depend on Figure 7 It can be seen that the H&E and Masson staining results at 2W (2 weeks) showed that the TPP+Mφ group was significantly different from the other groups, with a large amount of new bone, new blood vessels, and collagen fibers formed at the bone defect. At 6W (6 weeks), the TPP+Mφ group had a larger amount of new bone formation and formed larger and richer blood vessels.

[0105] Example 10: Preparation of lipase-treated TPP scaffold (TPP-L scaffold)

[0106] The preparation of the lipase-treated TPP scaffold (TPP-L scaffold) specifically includes the following steps:

[0107] (1) Preparation of lipase solution

[0108] Weigh 0.3 g of lipase and dissolve it in 180 mL of PBS buffer, filter and sterilize it through a 0.22 μm filter, and divide it into smaller portions for later use.

[0109] (2) Lipase treatment of TPP scaffold

[0110] After the TPP scaffold was printed (see Example 2 for specific steps), it was ventilated and dried for 4 to 5 days. After UV sterilization, the scaffold was placed with a surface area to volume ratio of 3 cm 2 / mL was immersed in a lipase solution, and the lipase solution was changed every day. After immersion for 7 days, the stent was taken out and placed in PBS buffer for repeated washing 3 times to remove the moisture in the stent to obtain the TPP-L stent.

[0111] Example 11: Construction of macrophage hybrid TPP-L scaffold

[0112] The construction of macrophage hybrid TPP-L scaffold includes the following steps:

[0113] The macrophages (Mφ) prepared in Example 1 were digested with trypsin and centrifuged, and the cell concentration was measured and adjusted to 5×10 5 The density of cells / mL. The TPP-L scaffold was placed in the confocal dish in advance, with 500 μL of cell suspension in each well, and the culture medium was supplemented to 1 mL per well. 40 ng / mL of IL-4 was added, and the plate was cultured in a constant temperature incubator. F-actin staining was performed after 3 days of culture: the culture plate was removed, the culture medium was discarded, and the plate was washed 3 times with PBS buffer solution. Subsequently, 4% paraformaldehyde was added, the cells were fixed at room temperature for 20 minutes, and the plate was washed 3 times with PBS buffer solution. Use PBS buffer containing 0.1% Tritonx-100 to treat for 15 minutes to improve the permeability of the cells, and then washed 3 times with PBS buffer solution. F-actin staining solution was added to each well to stain the cytoskeleton, incubated at room temperature in the dark for 1.5 hours, and then the F-actin staining solution was discarded, and washed 3 times with PBS buffer solution. Finally, DAPI staining solution was added, incubated at room temperature in the dark for 5 minutes, and washed 3 times with PBS buffer solution. In a wet state, the sample was placed under a laser scanning confocal microscope for observation and image acquisition. The results are shown in Figure 8 .

[0114] Depend on Figure 8 It can be seen that the red fluorescence is macrophages, and the green fluorescence is TPP-L scaffold. The staining results show that macrophages can attach to the surface of the TPP-L scaffold, confirming that the macrophage hybrid TPP-L scaffold was successfully constructed.

[0115] Example 12: Establishment of rat subcutaneous TPP-L / hybrid TPP-L scaffold model

[0116] The establishment of the rat subcutaneous stent implantation model specifically includes the following steps:

[0117] Eighteen healthy adult male SD rats (weighing about 200 g, provided by the Animal Experiment Department of Kunming Medical University) were selected to establish a dorsal subcutaneous stent implantation model and divided into a negative blank control group (Ctrl group), a TPP-L stent implantation group (TPP-L group), and a macrophage hybrid TPP-L stent implantation group (TPP-L+Mφ group). The stents were divided into three time points: 1, 4, and 7 days. Before implantation into the subcutaneous tissue of the rats, all stents were 60 Co radiation sterilization. After the rat abdomen was disinfected with iodine cotton balls, 1 mL / kg of 3% sodium pentobarbital was injected intraperitoneally for general anesthesia. The back hair was removed with depilatory cream and shaver. After the surgical area was disinfected with iodine, three longitudinal incisions of about 1 cm in length were made in the middle of the back of each rat. The skin on both sides of the incision was gently lifted with forceps, and the skin and subcutaneous tissue were bluntly separated on both sides of the incision using blunt-tipped instruments to form a subcutaneous bag. Then, according to the grouping, the first bag was the Ctrl group as a blank control without stent implantation, the second bag was the TPP-L group implanted with TPP-L stent, and the third bag was the macrophage hybrid TPP-L stent implanted with TPP-L+Mφ stent, and then the incision was carefully sutured and closed. Routine feeding was performed after surgery. On the 1st, 4th, and 7th day after surgery, 6 SD rats were randomly selected and killed for samples. The samples taken included skin, stents, and subcutaneous tissue, and the samples were fixed with neutral formaldehyde fixative.

[0118] Example 13: Localization of macrophages after subcutaneous implantation of macrophage hybrid TPP-L scaffold

[0119] The localization of macrophages after the macrophage hybrid TPP-L scaffold was implanted subcutaneously. The specific process is as follows:

[0120] Macrophages were labeled with PKH26 for live cell tracing experiments. PKH26-labeled Mφ were inoculated onto TPP-L scaffolds and implanted subcutaneously in rats. The rats were killed by cervical dislocation on days 1, 4, and 7 after surgery, and the specimens were collected. The specimens were fixed with neutral formaldehyde solution for 24 hours, run overnight, dehydrated with sucrose gradient, frozen, and sealed with DAPI-containing sealing agent. The specimens were observed and imaged under a laser scanning confocal microscope. The results are shown in Fig. 9 .

[0121] Depend on Fig. 9 It can be seen that the red fluorescence is exogenous macrophages, the blue fluorescence is the cell nucleus, and "M" is the TPP-L scaffold. It can be seen that the macrophage hybrid TPP-L scaffold can successfully carry macrophages into the body and gradually migrate from the scaffold to the defect area.

[0122] Example 14: Histological detection after implantation of macrophage hybrid TPP-L scaffold

[0123] For fixed skin specimens, first rinse them with running water overnight (at least 12 hours), then embed the tissues after systematic dehydration, and make routine paraffin sections, followed by Arg-1 and iNOS immunohistochemical staining:

[0124] (1) Dewaxing: Soak in xylene I for 15 min, then in xylene II for another 15 min. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 min. Finally, soak in distilled water for 2 min to remove the wax in the slices.

[0125] (2) Antigen repair: Place the sections on a copper rack, put them into a glass beaker containing pre-boiled citric acid antigen repair buffer (pH = 6.0), and place them in an oven at 100°C for 45 min for antigen repair. After cooling naturally to room temperature, place the sections in PBS buffer and shake and wash them three times, each time for 5 min;

[0126] (3) Circle drawing: After the slices are slightly dried, they are placed in a transparent box and the tissue area is circled with a histochemical pen to avoid antibody loss;

[0127] (4) Blocking: Add endogenous peroxidase blocker to the designated tissue area and block for 15 min at room temperature. Rinse once with distilled water, then slightly dry the sections and add goat serum. Block for 20 min at room temperature.

[0128] (5) Primary antibody incubation: Gently shake off the blocking solution, add the primary antibody solution Arg-1 (1:500) and iNOS (1:200) prepared in PBS containing 5% BSA on the slices, place the slices flat in a humidified box, and incubate in a refrigerator at 4°C overnight (about 12 h). Note that an appropriate amount of water should be added to the humidified box to prevent the evaporation of the antibody;

[0129] (6) Secondary antibody incubation: The next day, remove the wet box and place it at room temperature for 30 minutes. Wash the sections three times with PBS buffer, 5 minutes each time. After the sections are slightly dried, add the secondary antibody of the corresponding species to the designated area and incubate at room temperature in the dark for 15 minutes.

[0130] (7) DAB color development: Wash the sections three times with PBS buffer, 5 min each time. After the sections are slightly dried, add freshly prepared DAB color developer to the designated area and observe the color depth under a microscope.

[0131] (8) Counterstaining of cell nuclei: After complete color development, wash the sections with distilled water, counterstain the cell nuclei with hematoxylin, differentiate with hydrochloric acid and alcohol (about 2 seconds), wash the sections with tap water, and perform blue reversion with PBS buffer;

[0132] (9) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 min each to remove moisture;

[0133] (10) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results. Fig.10 .

[0134] Depend on Fig.10 The results of Arg-1 staining showed that on the first day after the macrophage hybrid TPP-L scaffold was implanted, there was a linear distribution of Arg-1-positive cells at the edge of the TPP-L+Mφ group material; on the third day after implantation, the number of Arg-1-positive cells in the surrounding tissues of the TPP group and TPP-L+Mφ group increased; on the seventh day after implantation, the number of Arg-1-positive cells in the surrounding tissues of the TPP-L+Mφ group increased significantly.

[0135] Depend on Fig.10 The iNOS staining results showed that the iNOS expression in the TPP-L+Mφ group was lower than that in the TPP-L group at each time point.

[0136] Example 15: Establishment of a rat periodontal critical bone defect model

[0137] The establishment of the rat periodontal critical bone defect model specifically includes the following steps:

[0138] Thirty-six healthy adult male SD rats (weighing about 200 g, provided by the Animal Experiment Department of Kunming Medical University) were used to establish a periodontal critical bone defect model. The rats were managed by the Animal Department and kept in an SPF environment. They were randomly divided into three groups, A, B, and C, with 12 rats in each group, and divided into two time points of 2 weeks and 6 weeks. The groups were divided as follows: Group A was a negative blank control group; Group B was implanted with a TPP-L stent; Group C was implanted with a TPP-L+Mφ stent. After the rat abdomen was disinfected with iodine cotton balls, 1 mL / kg of 3% sodium pentobarbital was injected intraperitoneally for general anesthesia. After anesthesia took effect, the rats were fixed in a lateral position on the operating table. The scalpel was used to make a parallel incision about 2 mm above the lower edge of the right mandible of the rat until the mandibular bone surface. The blunt-tipped instrument was replaced to bluntly separate the soft tissue to fully expose the bone surface. The periosteum was carefully separated, and a cylindrical periodontal critical bone defect with a diameter of 4 mm and a depth of 1 mm was prepared on the buccal root of the first and second molars with a dental high-speed handpiece. The bone debris in the defect area was removed, and the wound was rinsed with a large amount of normal saline. Then, the stent was implanted or not implanted according to the group, and the wound was carefully sutured and closed. Routine care and feeding were performed after surgery.

[0139] Example 16: Micro-CT detection and three-dimensional reconstruction of periodontal bone defect sites in rats

[0140] Micro-CT detection and three-dimensional reconstruction of periodontal bone defect in rats, specifically including the following steps:

[0141] Rats were killed by cervical dislocation at 2 weeks and 6 weeks after surgery, and then dissected and sampled. After the specimens were obtained, they were immediately immersed in neutral formaldehyde fixative for 24 hours for fixation. Subsequently, the fixed specimens were scanned using Micro-CT scanning technology, and the specific scanning conditions were set to a tube voltage of 90kV and a tube current of 60μA. The acquired images were reconstructed and analyzed using Avatar 1.5.0 software, focusing on the quantitative statistical analysis of the four indicators of the defect area: bone volume fraction (BV / TV), bone surface area to tissue volume ratio (BS / TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp). The results are shown in Table 1. Fig.11 .

[0142] Depend on Fig.11 It can be seen that Micro-CT 3D reconstruction ( Fig.11 A) shows that new bone formation around the defect was observed in both the TPP-L group and the TPP-L+Mφ group at 2 weeks and 6 weeks, with the TPP-L+Mφ group producing more new bone. Bone analysis results ( Fig.11B) shows that BV / TV, BS / TV, and Tb.N of TPP-L group and TPP-L+Mφ group were higher than those of control group at 2W and 6W, and Tb.Sp was lower than that of control group. Among them, BV / TV and BS / TV of TPP-L+Mφ scaffold group were significantly higher than those of control group at 2W and 6W, and the number of trabeculae was significantly increased and the separation of trabeculae was reduced at 6W.

[0143] Example 17: Detection of bone repair at periodontal bone defect sites in rats

[0144] The detection of bone repair at the periodontal bone defect site of rats specifically includes the following steps:

[0145] (1) H&E staining

[0146] For the fixed mandibular specimens, first rinse them with running water overnight (at least 12 hours), then use 17% EDTA decalcification solution for decalcification (about 2 to 3 weeks), after systematic dehydration, embed the tissue, make conventional paraffin sections, and then perform H&E staining:

[0147] 1) Dewaxing: Soak in xylene I for 15 minutes, then in xylene II for another 15 minutes. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 minutes. Finally, soak in distilled water for 2 minutes to remove the wax in the slices.

[0148] 2) Staining: Stain in hematoxylin for 10 minutes, rinse with water for 1 minute. Use hydrochloric acid alcohol for differentiation, and the time is controlled within 2 to 5 seconds. Use warm water for blueing for 2 minutes. Soak in 70% ethanol for 2 minutes. Finally, stain with eosin solution, and the time is controlled within 20 to 30 seconds;

[0149] 3) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each to remove moisture;

[0150] 4) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results.

[0151] (2) Masson staining

[0152] For fixed specimens, first rinse them with running water overnight (at least 12 hours), then decalcify them with 17% EDTA decalcification solution (about 2 to 3 weeks). After systematic dehydration, embed the tissues, make conventional paraffin sections, and then perform Masson staining:

[0153] 1) Dewaxing: Soak in xylene I for 15 minutes, then in xylene II for another 15 minutes. Soak in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2 minutes. Finally, soak in distilled water for 2 minutes to remove the wax in the slices.

[0154] 2) Staining: add 1 drop of Masson's complex staining solution to the slice, stain for 5 minutes, and rinse with distilled water; add 1 drop of phosphomolybdic acid, stain for 5 minutes, and spin dry; add 1 drop of aniline blue, stain for 5 minutes, and rinse with distilled water; add 1 drop of differentiation solution, and the differentiation time is controlled within 30-60 seconds;

[0155] 3) Dehydration: Soak in 80% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each to remove moisture;

[0156] 4) Sealing: After the slices are naturally air-dried, seal them with neutral resin and cover with coverslips, taking care to remove bubbles. After the slices are completely dry, observe them under a microscope, use a slide scanner to capture images, and record the experimental results.

[0157] The results of the bone repair test at the periodontal bone defect site in rats are shown in Fig.12 .Depend on Fig.12 It can be seen that the H&E and Masson staining results at 2 weeks showed that the TPP-L+Mφ group was significantly different from the other groups, with a large amount of new bone, new blood vessels, and collagen fibers formed at the bone defect. At 6 weeks, the TPP-L+Mφ group had a larger amount of new bone formation and formed larger and richer blood vessels.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a macrophage hybrid scaffold, characterized in that: The following steps are involved: Peripheral blood monocytes were extracted and induced into mature macrophages, the mature macrophages were loaded on tissue engineering scaffolds, and immune regulatory factors were added to regulate the phenotype of mature macrophages to obtain M2 macrophage hybrid scaffolds.

2. The method for constructing a macrophage hybrid scaffold according to claim 1, characterized in that: The mature macrophages were loaded onto the tissue engineering scaffolds. Specifically, the mature macrophages were loaded at a rate of 1×10 4 -1×10 8 The cells were seeded on tissue engineering scaffolds at a density of 100 / mL.

3. The method for constructing a macrophage hybrid scaffold according to claim 1 or 2, characterized in that: The tissue engineering scaffold is a TPP scaffold or an enzyme-treated TPP scaffold.

4. The method for constructing a macrophage hybrid scaffold according to claim 3, characterized in that: The TPP scaffold was prepared by the following method: PCL, PLGA and β-TCP are added into an organic solvent, dissolved and mixed evenly to obtain a slurry; the slurry is made into a 3D composite porous scaffold through a material extrusion technology, and then dried and sterilized to obtain the product.

5. The method for constructing a macrophage hybrid scaffold according to claim 4, characterized in that: The mass ratio of PCL, PLGA and β-TCP is 35:35:

30.

6. The method for constructing a macrophage hybrid scaffold according to claim 1, characterized in that: Immunomodulatory factors include IL-4, IL-13, TGF-β or glucocorticoids.

7. The method for constructing a macrophage hybrid scaffold according to claim 6, characterized in that: The concentration of added immunomodulatory factors is 20-100 ng / mL.

8. A macrophage hybrid scaffold constructed by the construction method according to any one of claims 1 to 7.

9. Use of the macrophage hybrid scaffold according to claim 8 in preparing tissue defect repair and regeneration implant materials.

10. The use according to claim 9, characterized in that: Tissues include soft / hard tissues such as skin, mucosa, internal organs, bones, and periodontal tissues.

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