阅读说明：本技术 一种th2细胞活化的3d打印支架及其制备方法与应用 (T-shaped supportH2-cell activated 3D printing support and preparation method and application thereof ) 是由 崔文国 李翠笛 马振江 颉天阳 齐进 王非 王金武 邓廉夫 于 2021-07-17 设计创作，主要内容包括：本发明提供了一种T-(H)2细胞活化的3D打印支架及其制备方法与应用。本发明通过动态共价键将聚乙烯亚胺/介孔二氧化硅微棒/卵清蛋白自组装疫苗稳定结合于3D打印钙磷基支架上；通过局部释放OVA激活体液免疫反应,实现支架中抗原特异性T-(H)2细胞的募集,促进材料植入早期血管新生。体外实验表明,本发明的支架能够有效募集和活化树突细胞表现抗原呈递功能,并能够促进骨髓间充质干细胞成骨分化。体内实验表明,与纯支架材料组和负载有疫苗载体的支架组相比,负载疫苗的打印支架在小鼠皮下包埋模型中,能够有效提高T-(H)2细胞在脾脏中的数量,在局部募集CD4～(+)T细胞,促进植入早期支架内部和周围大量新生血管形成。(The invention provides a T H 2 cell activated 3D printing support and preparation method and application thereof. The polyethyleneimine/mesoporous silica micro-rod/ovalbumin self-assembly vaccine is stably combined on the 3D printed calcium-phosphorus-based scaffold through a dynamic covalent bond; antigen specificity in scaffolds by local release of OVA to activate humoral immune responsesSex T H 2, the recruitment of cells promotes the angiogenesis in the early stage of material implantation. In vitro experiments show that the scaffold can effectively recruit and activate dendritic cells to express an antigen presenting function, and can promote osteogenic differentiation of mesenchymal stem cells. In vivo experiments show that compared with a pure stent material group and a stent group loaded with a vaccine carrier, the printing stent loaded with the vaccine can effectively improve T in a mouse subcutaneous embedding model H 2 number of cells in the spleen, local recruitment of CD4 + T cells, promote the formation of a large number of new blood vessels in and around the implanted early scaffold.)

1. T-shaped support_HThe preparation method of the 2-cell activated 3D printing scaffold is characterized by comprising the following steps:

(1) preparation of MSR/PEI vaccine vector: preparing a dispersion solution from the mesoporous silica micro-rods, mixing the dispersion solution with a polyethyleneimine solution, and co-incubating for 5-30min at 37 ℃ to prepare an MSR/PEI vaccine carrier;

(2) preparation of MSR/PEI/OVA vaccine: oscillating and incubating ovalbumin and MSR/PEI vaccine carrier for 1-3h at 37 ℃ to obtain MSR/PEI/OVA vaccine;

(3) taking an oxidized polymer and polyvinyl alcohol in an aqueous solution according to the mass ratio of 1: 5-20, mixing to form an aldehyde group-rich binder, uniformly mixing the calcium phosphate-based powder with the binder to prepare printable slurry, and printing to prepare a calcium phosphate-based support; wherein the calcium phosphate-based powder is one or more of self-curing calcium phosphate bone cement, beta-tricalcium phosphate or hydroxyapatite, and the oxidized polymer is one or more of oxidized hyaluronic acid, oxidized chondroitin sulfate or oxidized sodium alginate;

(4) the MSR/PEI/OVA vaccine was resuspended in PBS buffer and then dropped onto the scaffold to prepare a vaccine-loaded scaffold.

2. The method according to claim 1, wherein the mesoporous silica micro rod in step (1) is synthesized by: the preparation method is characterized in that a triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide is used as a structure directing agent, and tetraethoxysilane is used as a silicon source for synthesis.

3. The method according to claim 1, wherein the concentration of the mesoporous silica micro rod dispersion in the step (1) is 30 to 70 mg-mL^-1The concentration of the polyethyleneimine solution is 15-35 mu g/mL^-1。

4. The method according to claim 1, wherein the oxidized polymer in the step (3) is prepared by: dissolving hyaluronic acid, chondroitin sulfate or alginate in distilled water, dropwise adding 0.5M sodium periodate solution in an environment of 4-37 ℃, continuously stirring for 2-4h, stopping reaction, precipitating at 4-37 ℃ for 1-6h, collecting precipitate, dialyzing and freeze-drying to obtain the oxidized polymer.

5. The production method according to claim 1, wherein the weight average molecular weight of the polyvinyl alcohol in the step (3) is 98000.

6. T-shaped support_H2-cell activated 3D printing scaffold, characterized in that it is prepared by the method according to any one of claims 1 to 5.

7. The 3D-printed scaffold according to claim 6, wherein the 3D-printed scaffold has antigen-specific T cell recruitment function.

8. Use of the 3D printed scaffold obtained by the method of preparation according to any one of claims 1 to 5 or the 3D printed scaffold according to any one of claims 6 to 7, in the preparation of a bone repair promoting material.

9. Use according to claim 8, characterized in that it comprises the use in the preparation of a material promoting bone tissue regeneration.

10. Use according to claim 8, characterized in that it comprises the use in the preparation of materials promoting angiogenesis.

Technical Field

The invention belongs to the technical field of biomedical materials, relates to a 3D printing support, and particularly relates to a T_H2 cell activated 3D printing support and preparation method and application thereof.

Background

Clinically, large bone defects caused by trauma and disease are different in shape and difficult to self-heal, and often need to be implanted with bone repair implants meeting individual requirements, however, since the number of commonly used autologous/allogeneic bones is limited, clinical requirements are difficult to meet at present. In the manufacturing of the bone repair implant, due to the variety of selectable materials and designability of the structure, the 3D printed bone repair implant has a personalized shape and an internal structure, and is suitable for research and development for clinical application.

In tissue repair, Printed implants have been used clinically and have been shown to promote Bone action, with an internally connected tunnel structure suitable for tissue ingrowth, but have not shown a function to promote vascularization (R.Shahrokh, F.Hamid Reza, S.K.Kazem, H.Mehdi, A.Sadaf, Customized Titanium Mesh Based on the 3D Printed Model vs. Manual inductive Bending of Titanium Mesh for reconstruction of organic Bone Fracture: A random Clinical trial, Rev.Recent Clins. Trials 12, 154. 158, 2017.). When a large bone is defected, the autologous vascular network is damaged, and avascularization of the implant easily causes lack of nutrients, oxygen and the like required by cell and tissue regeneration inside the implant, so that central necrosis and repair failure are caused, which is also a big problem before wide clinical application. In addition, the local blood flow and the amount of nutrient substances are closely related to the bone regeneration rate, so that the rapid formation of vascularization in a printing implant is promoted, and sufficient nutrition is hopefully provided for the bone reconstruction process, thereby accelerating the tissue repair.

In the conventional method, growth factors having an effect of promoting angiogenesis, such as Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), and the like, are loaded into a bone repair material for sustained release to directly promote angiogenesis. At present, the problems of short half-life period of growth factors and local difficult preservation, which cause angiogenesis failure, can be overcome to a certain extent through material design. However, growth factors alone do not promote the formation of mature blood vessels, and the number and activity of local immune cells are a large determinant factor of whether the angiogenesis promoting effect of the bone repair body can be exerted. Therefore, it is a worthy direction to study to promote vascularization of 3D printed bone repair implants by modulating local immune microenvironment.

Recently, researchers have attempted to introduce monocyte/macrophage regulatory functions into 3D-printed scaffolds that have achieved some effect in promoting vascularization and bone repair. Macrophages play an important role in angiogenesis, and research shows that M1 type macrophages can promote angiogenesis, and M2 type macrophages can promote vascular maturation and remodeling. The 3D printing support is designed to sequentially release growth factors IFN-gamma and silicon ions, macrophages are polarized to M1 type and then to M2 type, and early neovascularization can be promoted in a mouse subcutaneous embedding model. However, macrophage activation is shown to be a continuous state by external stimuli, and M1 and M2 are at their extremes and not completely controllable in stability in complex microenvironments in vivo. Macrophages have a regulating effect on functions of osteogenesis, osteolysis, angiogenesis and the like, and related effect mechanisms are different, so that inappropriate manual regulation and control intervention can cause tissue damage. Therefore, the mechanism that the 3D printed scaffold needs to be combed to precisely regulate macrophage phenotypic changes to promote vascular bone tissue remodeling is relatively complex and remains to be further studied.

The 3D printing bracket with the T cell regulation function is constructed to promote vascularization and bone tissue regeneration to show great potential. T cell-associated acquired immune system plays an important role in bone defect repair and promotion of vascularization. Although T cells share numerous similar subtypes to macrophages, among them CD4⁺The conditioned medium of the T cells can obviously promote the osteogenic mineralization of the human mesenchymal stem cells. In the model of lower limb ischemia, CD4⁺T cells have been found to promote arterial formation. CD4⁺T2 Assist (T)_H2) The cells can not only secrete growth factors which are helpful for angiogenesis in the ischemia injury, but also activate eosinophils to secrete relevant growth factors. It can be seen that the printed stent has local recruitment and activation T_H2, the function of the cells can promote vascularization of the defect part and osteogenesis.

Mooney et al implanted the nano-aluminum/ovalbumin vaccine into a mouse lower limb ischemia model and observed that the released OVA (ovalbumin) antigen was capable of specifically recruiting T_H2 cells, triggering local angiogenesis and increased blood perfusion in the ischemic limb, reducing necrosis and promoting regeneration of muscle fibers in muscle (b.j.kwee et al, Treating ischemia vitamin recovery of anti-genetic T cells. sci.adv.5, eaav6313,2019.). The nanometer aluminum particles are used as a traditional vaccine carrier and an immunologic adjuvant and can promote OVA to T_H2 recruitment of cells. However, the OVA nanoparticle vaccine simply disperses in the bone defect part, is easy to rapidly disperse into the blood circulation, is difficult to play a role in local cell recruitment, and is not suitable to be used as a vaccine system for treating the bone defect alone.

Therefore, to target the site characteristics of bone defects, how to select a suitable OVA carrier and bind to a 3D-printed bone repair scaffold in order to recruit T by local release of specific antibodies_H2 cells to promote vascularization and bone tissue repair in the stent and at the implantation part, which becomes a technical problem to be solved urgently at the present stage.

Disclosure of Invention

The present invention is directed to solving the above problems and providing a T_H2 cell activated 3D printing support and preparation method and application thereof. The scaffold provided by the invention can effectively recruit and activate dendritic cells to express an antigen presenting function, can promote osteogenic differentiation of mesenchymal stem cells, and can promote formation of a large number of new blood vessels in and around the scaffold implanted at an early stage. The 3D printing support with the antigen-specific immune microenvironment regulation function can promote bone tissue regeneration, and provides a new idea for the design of implant materials.

One of the purposes of the invention is to provide a T_HThe preparation method of the 2-cell activated 3D printing scaffold comprises the following steps:

(1) preparation of MSR/PEI vaccine vector: preparing a dispersion solution from the mesoporous silica micro-rods, mixing the dispersion solution with a polyethyleneimine solution, and incubating for 5-30min at 20-37 ℃ to prepare an MSR/PEI vaccine vector;

(2) preparation of MSR/PEI/OVA vaccine: oscillating and incubating ovalbumin and MSR/PEI vaccine carrier for 1-3h at 37 ℃ to obtain MSR/PEI/OVA vaccine;

(3) taking an oxidized polymer and polyvinyl alcohol in an aqueous solution according to the mass ratio of 1: 5-20, mixing to form an aldehyde group-rich binder, uniformly mixing the calcium-phosphorus-based powder with the binder to prepare printable slurry, and printing to prepare a calcium-phosphorus-based support; wherein, the inorganic calcium-phosphorus-based component of the biological scaffold is one or more of self-curing calcium phosphate cement, beta-tricalcium phosphate and hydroxyapatite; the scaffold binding liquid is a mixture of polyvinyl alcohol and an oxidized polymer, and the oxidized polymer is one or more of oxidized hyaluronic acid, oxidized chondroitin sulfate or oxidized sodium alginate;

(4) the MSR/PEI/OVA vaccine was resuspended in PBS buffer and then dropped onto the scaffold to prepare a vaccine-loaded scaffold.

The invention inspires the regulation and control effect of T cells on vascularization, and constructs T by assembling mesoporous silica micro-rods, dendritic PEI and ovalbumin layer by layer under the action of simple charges_H2 cell-specific recruitmentA vaccine is further stably combined with the room temperature printed aldehyde-containing calcium phosphate-based bone repair scaffold based on Schiff base reaction, and the scaffold capable of locally recruiting T is designed and constructed_H2 cells, 3D printed bone repair scaffold that promotes vascularization. The mesoporous silica micro-rod as a carrier of OVA can activate specific T_H2, cell reaction, and the dendritic PEI coated mesoporous silica micro-rod can effectively promote the activation of dendritic cells and promote T cell reaction when being used as a vaccine carrier of OVA. Oxidized polymer is introduced into the calcium phosphate printing ink, so that the bracket is provided with abundant aldehyde groups, and the bracket is suitable for stable loading of MSR/PEI/OVA vaccine; OVA in the vaccine is gradually released, and T secreting the angiogenesis-related growth factor can be realized_H2 cells are recruited, promoting intravascular vascularization within the stent. In vitro cell experiment research shows that the vaccine on the scaffold releases OVA to quickly recruit and activate dendritic cells to have antigen presenting function, and gradually released silicon ions can promote osteogenic differentiation of bone marrow mesenchymal stem cells. In an in vivo subcutaneous embedding model, the vaccine-loaded micro-rod scaffold can rapidly activate and recruit T in the early stage of implantation_H2 cells, significantly promoting local and intrastent vessel formation. In addition, the in-vivo skull defect in-situ model finds that the 3D printing scaffold loaded with the micro-rod vaccine has an excellent effect of promoting vascularized bone regeneration.

Further, the mesoporous silica micro rod in the step (1) is synthesized by the following method: the preparation method is characterized in that a triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide is used as a structure directing agent, and tetraethoxysilane is used as a silicon source for synthesis.

Further, the concentration of the mesoporous silica micro-rod dispersion liquid in the step (1) is 30-70 mg/mL^-1The concentration of the polyethyleneimine solution is 15-35 mu g/mL^-1。

Further, the preparation method of the oxidized polymer in the step (3) is: dissolving hyaluronic acid, chondroitin sulfate or alginate in distilled water, dropwise adding 0.5M sodium periodate solution in an environment of 4-37 ℃, continuously stirring for 2-4h, stopping reaction, precipitating at 4-37 ℃ for 1-6h, collecting precipitate, dialyzing and freeze-drying to obtain the oxidized polymer.

Further, the polyvinyl alcohol in the step (3) has a weight average molecular weight of 98000.

It is another object of the present invention to provide a T_H2-cell activated 3D printing scaffold prepared as above. The 3D printed scaffold has antigen-specific T cell recruitment function.

The invention also aims to provide application of the 3D printing bracket, wherein the application comprises application in preparing materials for promoting bone repair, such as application in preparing materials for promoting bone tissue regeneration and application in preparing materials for promoting angiogenesis.

The invention has the following beneficial effects:

the invention stably combines a Polyethyleneimine (PEI)/mesoporous silica micro-rod (MSR)/Ovalbumin (OVA) self-assembly vaccine on a 3D printing calcium-phosphorus-based bracket through a dynamic covalent bond; activation of humoral immune response by locally released OVA to achieve antigen-specific T in scaffolds_H2, the recruitment of cells promotes the angiogenesis of the material in the early stage of implantation; the silicon ions in the mesoporous silica micro-rod are gradually released, so that good blood circulation can be formed at the defect part, and the regeneration of bone tissues is promoted. In vitro experiments, the scaffold can effectively recruit and activate dendritic cells to express antigen presenting function, and can promote osteogenic differentiation of mesenchymal stem cells. In an in vivo experiment, compared with a pure stent material group and a stent group loaded with a vaccine carrier, the printing stent loaded with the vaccine can effectively improve T in a mouse subcutaneous embedding model_H2 number of cells in the spleen, local recruitment of T_H2 cells, promoting the formation of a large number of new blood vessels in and around the implanted early stage stent.

Drawings

FIG. 1 is a preparation and characterization of the MSR vaccine system; (A) SEM images of MSR and MSR/PEI micro-rods; (B) EDS surface scanning of MSR/PEI micro-rod; (C) nitrogen adsorption and desorption curves, pore size distribution and TEM images of MSR and MSR/PEI micro-rods; (D) zeta potential maps of MSR, MSR/PEI and MSR/PEI/OVA micro-rods; (E) schematic representation of the adsorption of branched PEI and antigen one by one onto MSR;

FIG. 2 is the preparation and characterization of MSR vaccine system load scaffolds; (A) SEM images of CPC and MSR/PEI loaded CPC scaffolds; (B) FTIR spectra of hyaluronic acid and oxidized hyaluronic acid; (C) release profile of OVA on CPC-MSR/PEI/OVA scaffold; (D) releasing the concentration of Si ions in the solution;

fig. 3 is vaccine-loaded scaffold recruitment and activation of dendritic cells; (A) cell adhesion SEM images and MHC-II surface marker confocal images of dendritic cells after 24 hours of coculture with CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds; (B) surface CD86 and MHC-II expression flow cytometry analysis after dendritic cells were co-cultured with CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds for 24 hours; (C) cell recruitment 48 hours after co-culture of dendritic cells with CPC, CPC-MSR/PEI, and CPC-MSR/PEI/OVA scaffolds; (D) confocal observation of SIINFEKL-H2Kb expression by cells on the scaffolds after 48 hours of co-culture; (E) flow cytometry detection results of dendritic cells expressing SIINFEKL-H2Kb in the culture solution;

FIG. 4 is a vaccine loaded scaffold promoting mBMSCs cell adhesion and osteogenic differentiation; (A) SEM images of cells adhering to scaffolds of CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA; (B) ALP staining photographs after 7 days of co-culture of cells with CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds;

FIG. 5 is a vaccine loaded scaffold promoting antigen specific cell recruitment and angiogenesis; (A) experimental schematic diagram of subcutaneous implantation; (B) spleen CD4⁺IL4 in T cells⁺And IFN-gamma⁺Proportion of cells (. about.P)<0.001vs.Control，###P<CPC group, ● ● ● P, 0.001vs<cpc-MSR/PEI group 0.001 vs); (C) CD4 immunofluorescent staining pattern and associated fluorescence intensity statistical pattern (### P) 7 days after stent implantation<CPC group, ● ● ● P, 0.001vs<cpc-MSR/PEI group 0.001 vs); (D) sonograms and (E) digital photographs 14 days after stent implantation;

FIG. 6 is a vaccine loaded scaffold promoting vascularized bone regeneration in rat cranial defects; (A) a photograph of the stent implantation defect; (B) after 8 weeks of implantation, a skull three-dimensional reconstruction map, a section map and a reconstruction map of a region of interest containing a new bone scaffold (yellow: scaffold, white: new bone) are obtained based on Micro-CT results; (C) the percent volume of new bone growth (BV/TV) for each group, n 3. P <0.05, P <0.01, P <0.001vs. uninformed; CPC group, # P <0.05 vs; (D) HE, Masson staining and CD31 immunohistochemical staining of the scaffolds.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Examples

1. Experimental methods

Preparation of vaccine

Mesoporous silica nanorods (MSRs) were synthesized by sol-gel method using P123 copolymer (polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer) as structure directing agent and tetraethoxysilane as silicon source, according to the report of the prior art (c.li et al, RhBMP-2loaded 3D-printed mesoporous silica/calcium phosphate residue powders with enhanced vacuum and osteogenesis promoters. sci.rep.7,41331, 2017).

The concentration of 40. mu.L was 50 mg/mL^-1The MSR working solution has a concentration of 25 mug. multidot.mL and 100 muL^-1Mixing and co-incubating the PEI (polyethyleneimine) solution at 37 ℃ for 15min to prepare the MSR/PEI vaccine vector. 100 mu g of OVA (ovalbumin) and an MSR/PEI micro-rod are taken to be incubated for 1h under oscillation at 37 ℃ to obtain the MSR/PEI/OVA vaccine.

And performing physical and chemical detection after the micro-rod is freeze-dried. Observing the shapes of the MSR and MSR/PEI micro-rods through a scanning electron microscope; analyzing the mesoporous structures of the MSR and MSR/PEI micro-rods by using a specific surface and porosity analyzer and a transmission electron microscope; performing element surface scanning analysis on the surface of the MSR/PEI micro-rod by using an energy spectrometer; zeta potential of the vaccine system was analyzed with a nano Zeta potentiometer.

(II) Loading of vaccine onto Stent

Oxidized Hyaluronic Acid (OHA) was synthesized as follows: completely dissolving 1g Hyaluronic Acid (HA) in 200mL distilled water, dropwise adding 0.5M sodium periodate solution 5mL in ice bath, stirring for 3h, adding 40mL ethylene glycol to stop reaction, precipitating at 4 deg.C for 1h, collecting bottom OHA, dialyzing, and lyophilizing. The HA and OHA samples were subjected to infrared spectroscopic analysis by fourier transform infrared spectrometer.

Next, a 1 wt% aqueous OHA solution was mixed with a 10 wt% aqueous polyvinyl alcohol (Mw 98000, Sigma-Aldrich) solution to form an aldehyde-rich binder; CPC (calcium phosphate cement) powder and a binding agent are uniformly mixed to prepare printable slurry, the printable slurry is placed into a printer charging barrel to be printed at room temperature, a printed CPC support is hydrated overnight at 37 ℃ in an environment with 100% humidity, and then the printed CPC support is air-dried for standby.

Finally, 1mg and 2mg MSR/PEI/OVA microtubers were resuspended in PBS buffer and dropped onto printed CPC scaffolds to prepare vaccine-loaded scaffolds (CPC-MSR/PEI/OVA) for in vitro and in vivo experiments, respectively. MSR/PEI load scaffolds were prepared in the same manner as the vaccine load vector scaffold group (CPC-MSR/PEI).

(III) Release Profile of OVA

A bracket (10 multiplied by 2 mm)³) Co-incubation at 37 ℃ in 1mL PBS, and PBS collection and replacement at different time points. The amount of OVA in PBS was determined using the Pierce Micro BCA protein detection kit. The release amount was calculated and a release curve was plotted.

(IV) Release behavior of silicon ions

A bracket (10 multiplied by 2 mm)³) Co-incubation at 37 ℃ in 1mL PBS was performed, and PBS was collected and replaced every other day. The content of silicon ions in the PBS was measured with an inductively coupled plasma emission spectrometer.

(V) Dendritic Cell (DCs) differentiation, activation and antigen presentation assays

Dendritic cells were isolated from bone marrow cells of C57BL/6J mice. Bone marrow was collected from femur and tibia, and single cell suspensions were cultured in RPMI medium containing 10% fetal bovine serum, 1% penicillin streptomycin, and 20ng/mL mouse granulocyte-macrophage colony stimulating factor. Collecting non-adherent cells for experimental study on 7-9 days of differentiation culture.

To evaluate the effect of stent stimulation on the activity of DCs, 1X 10 was used⁶mL^-1Immature DCs were inoculated in 12-well platesCo-culture on the scaffolds in (1) for 24 hours. Subsequently, DCs were collected, stained with anti-CD11c, anti-CD86, and anti-MHC II on ice and analyzed using flow cytometry. Cell surface markers on the scaffold were stained with anti-MHC II, and nuclei were stained with DAPI, and then observed by confocal microscopy.

To assess cross presentation of DCs after stimulation, 0.5X 10⁶mL^-1DCs were seeded on scaffolds and co-cultured for 48 hours before collecting DCs in the supernatant, stained with anti-SIINFEKL/H-2Kb and analyzed using flow cytometry. Cell surface markers on the scaffolds were stained with anti-SIINFEKL/H-2Kb, and nuclei were stained with DAPI, followed by observation through a confocal microscope.

(VI) preparation of cell and scaffold extract for osteogenic differentiation test

Mouse bone marrow mesenchymal stem cells (mBMSCs) were isolated from the femur and tibia of C57BL/6J mice and cultured using DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin.

CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds (10X 2 mm)³) Each set of scaffolds was placed one by one in a 24-well plate and soaked in 1mL of medium, with the medium changed every other day. After 7 days of soaking, scaffolds were obtained for subsequent in vitro experiments.

CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffold leach liquor references (C.Li et al, RhBMP-2loaded 3D-printed mesoporous silica/calcium phosphate center porous scaffolds with enhanced vacuum and osteoprogenesis Properties. Sci. Rep.7,41331, 2017.). Soaking the treated scaffold in culture solution at 37 deg.C at a certain mass/volume ratio for 24 hr, collecting supernatant, filtering with 0.22 μm filter membrane, and storing at 4 deg.C.

(VII) cell adhesion

Scaffolds in 24-well plates were seeded 5X 10⁴Each mBMSCs was co-cultured for 24 hours. Subsequently, the scaffold is washed by PBS, fixed by 4% Paraformaldehyde (PFA), dehydrated by gradient ethanol and dried at constant temperature to prepare a sample, and the adhesion condition of cells on the scaffold is observed by a scanning electron microscope.

(VIII) detection of alkaline phosphatase Activity

Scaffolds in 24-well plates were seeded 5X 10⁴Individual bmscs were co-cultured. The culture medium was changed every 3 days. After 7 days of culture, cells were fixed with 4% paraformaldehyde, stained with alkaline phosphatase kit, and observed by naked eye and light microscope.

(nine) subcutaneous transplantation model

For in vivo immunomodulation and vascularization studies of materials, a mouse subcutaneous implant model was established (model establishment method C.Li, et al, patent-specific scans with a biological Gradient Environment for architectural implants ACS appl. Bio mater.3,4820-4831,2020.). 24 healthy male C57BL/6J mice were ordered (6 weeks old, 15-20 g). Bracket of CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVAImplanting a hypodermal embedded region (n ═ 6) prepared by blunt dissection. Non-operated mice served as a control group (n-6).

(Ten) in vivo T cell activation recruitment

Animals were sacrificed 7 days after implantation and the scaffolds and surrounding tissues were removed. The spleen is physically ground in RPMI culture medium containing 2% fetal calf serum to extract lymphocytes. CD8⁺Or CD4⁺T cells were obtained using a cell sorting kit. Sorted T cells were treated at 37 ℃ for 1 day, collected, stained with anti-mouse CD3e, CD8a, and CD4 on ice, fixed, and permeabilized. Then stained with anti-mouse IL-4 and IFN-. gamma.at 4 ℃ and analyzed by flow cytometry.

And (3) decalcifying, paraffin embedding and slicing the taken stent, dewaxing and rehydrating the slice, incubating the dewaxed slice and the anti-CD 4 primary antibody at 4 ℃ overnight, staining the secondary antibody and DAPI, observing the slice by using a confocal microscope, and analyzing the local immune regulation function of the stent. The mean fluorescence intensity was calculated using Image J software and the amount of cells recruited was quantified.

(eleven) angiogenesis in vivo

After the stent is implanted for 14 days, detecting local blood flow of the mouse by using a multi-mode ultrasonic/photoacoustic imaging system, and analyzing angiogenesis conditions in the stent; the animals were then sacrificed and the stents were removed to observe angiogenesis.

(twelve) in-situ defect repair model

In the in vivo osteogenesis study, a critical skull defect model is established (the model establishing method refers to D.Zou et al, repair critical-sized fibrous defects with a modified by a consistent active form of hypoxia-absorbent factor-1 alpha and a phosphate residue scaffold. biomaterials 32,9707-9718, 2011). 24 healthy male SD rats of 8 weeks of age were selected and randomly divided into 4 groups: (1) untreated group; (2) CPC; (3) CPC-MSR/PEI; (4) CPC-MSR/PEI/OVA. Each rat used trephine to make a defect of 4mm in diameter. Careful implantation of stentsThe incision is closed afterwards. After 8 weeks of material implantation, animals were sacrificed and the cranium was harvested for further analysis by fixation with 4% PFA. (n is 6)

(thirteen) Micro-CT tomographic analysis

All samples were scanned for bone formation analysis using a Micro-CT imaging system with a resolution of 20 μm. After scanning, the DataViewer software was used to reconstruct a three-dimensional image. Scaffolds and bones were reconstructed separately based on different gray scale ranges (scaffolds and newly formed bones rendered yellow and white, respectively). The new bone was quantitatively calculated using DataViewer software.

(fourteen) histological evaluation

After 4% PFA fixation, the samples were decalcified, paraffin embedded, sectioned, and the sections were stained with hematoxylin/eosin (HE), Masson trichrome (Masson), and CD31 immunohistochemistry, and the sections were observed with an optical microscope.

(fifteen) statistical analysis

Each set of experimental data was repeated at least 3 times, expressed as mean ± standard deviation. Data results were statistically analyzed using GraphPad Prism 5 software and ANOVA for analysis of variance.

2. Results of the experiment

Vaccine loading rack

To prepare vaccine loaded scaffolds, self-assembly was first prepared and analyzedA vaccine. The prismatic nanorods with a particle size of 600nm aggregated to form MSR nanorods, observed by scanning electron microscopy, with an average length of 23.2 μm and a diameter of 3.7 μm (FIG. 1A). Typical lamellar mesopores within the MSR can be seen by transmission electron microscopy (fig. 1C). The BET analysis results show (FIG. 1C), N of MSRs₂The adsorption and desorption isotherm is an IV-type isotherm containing an H1 hysteresis loop, and the mesoporous order characteristic is verified, the mesoporous aperture is 7.8nm, and the specific surface area is 849.9m²·g^-1Total pore volume of 1.25cm³·g^-1。

The MSRs were mixed with a branched PEI solution and incubated at 37 ℃ for 15 minutes to form PEI coated MSR/PEI mini-rods. The combination of PEI and MSR was observed by analyzing the distribution of the nitrogen (N) element of PEI, the oxygen (O) element of MSR and the silicon (Si) element of MSR in the energy spectrometer scanning. N, O and the same distribution of the Si element verified that the coating of PEI was adequate and uniform on the MSR (FIG. 1B). Subsequently, TEM and BET detections show that MSR/PEIs maintain the shape and the mesoporous structure of the micro-rods, and the specific surface area and the pore volume are slightly reduced in accordance with the reported phenomenon. Presumably, the reduction is caused by the coating of PEI on the mesopores on the surface of the micro-rods. The MSR/PEI micro-rod has high specific surface area and pore volume and is positively charged, so that the MSR/PEI micro-rod is suitable for serving as a carrier of antigen to realize sustained release.

Further taking OVA as an antigen model, and dispersing MSR/PEI in OVA antigen solution to directly adsorb to prepare the MSR/PEI/OVA vaccine micro-rod. The Zeta potentials of the MSR, MSR/PEI and MSR/PEI/OVA micro-rods were-22.33 + -1.80, 29.50 + -0.46 and 19.47 + -1.70, respectively, confirming successful self-assembly of the vaccine (FIG. 1D). After dispersion in PBS, the silicon hydroxyl groups on the surface of the MSR micro rods gave them a negative Zeta potential. In the case of good coatings of polycationic PEI, the Zeta potential of MSR/PEI changes from negative (MSR) to positive. The addition of the negatively charged polypeptide OVA makes the Zeta potential of MSR/PEI/OVAs lower than that of MSR/PEI. Vaccine-loaded scaffolds were then prepared and tested. From the FTIR results (FIG. 2C), in the spectrum of OHA, at 1715.3cm^-1A new absorption peak corresponding to the aldehyde group appears nearby, and the success of the synthesis is verified. The PVA/OHA solution acts as a binder allowing the CPC bio-ink to be smoothly extruded from the print nozzle. The scanning electron microscope shows that the printed CPC stent has controllabilityThe size of the outer shape of (2) is 500 μm, and the outer shape is a large hole with good mutual communication. In addition, it can be seen that MSR/PEI was well dispersed on the scaffold (FIG. 2B). To avoid rapid flushing with blood, the vaccine is designed to be covalently bound to the printed scaffold. Therefore, the behavior of the release of antigen and silicon ions from the scaffold was carefully observed. The results of the Micro-BCA kit assay showed (FIG. 2D) that OVA was gradually released from the scaffolds over 7 days. Although the release solution was changed every day, the Si ion concentration in the release solution was from 6 mg.L within 72 hours^-1Gradually decreases to 1 mg.L^-1And can be maintained at 1 mg.L^-1Left and right (FIG. 2E), this phenomenon indicates that the binding of MSR/PEIs to the CPC scaffold is relatively stable.

(II) vaccine-loaded scaffolds can recruit and activate dendritic cells

It has been reported that the activation of a large number of dendritic cells is closely related to the induction of a humoral immune response. Thus, the effect of MSR/PEI/OVA loaded scaffolds on dendritic cell recruitment and activation was further analyzed.

First, recruitment of dendritic cells on the vaccine loaded scaffold was observed. It was observed by scanning electron microscopy that cells were recruited to the vector and vaccine loaded scaffolds within 24 hours and attached predominantly to the micro-rods, whereas only a few cells were seen on the CPC scaffold (fig. 3A). Activation of dendritic cells was then assessed by expression levels of the co-stimulatory surface marker CD86 and Major Histocompatibility Complex (MHC) ii as previously reported. Confocal microscopy results show (FIG. 3A), MHC II⁺The recruited cells of (a) are distributed on the surface of MSR/PEI and MSR/PEI/OVAs and show a characteristic micro-rod morphology. Furthermore, the flow cytometric results of the cell composition in the culture broth (FIG. 3B) showed CD11c in the CPC group, MSR/PEI group and MSR/PEI/OVA group⁺Dendritic cells express CD86 separately⁺28.7%, 36.7% and 39.4%, respectively, express MHC II⁺Of 31.6%, 38.7% and 41%. Analysis of the effect of vaccine loaded scaffolds on dendritic cell antigen presentation after 48h of co-culture revealed that dendritic cell recruitment was observed microscopically between MSR/PEI and MSR/PEI/OVAs released from the macropores of the scaffold (FIG. 3C). In addition, confocal microscopy observed SIINFEKL-H2Kb on a CPC-MSR/PEI/OVAs scaffold⁺Antigen cross-presenting dendritic cells were more numerous than on the CPC-MSR/PEI and CPC scaffolds (fig. 3D). The cells in the culture broth were examined by flow cytometry and found that CPC, CPC-MSR/PEI and CPC-MSR/PEI/OVAs group CD11c⁺SIINFEKL-H2Kb in dendritic cells⁺The expression levels of (A) were 10.5%, 12.4% and 15.0%, respectively (FIG. 3E). It has been reported that MSR can enhance the expression of CD86 after being cultured with human dendritic cells as a potential biocompatible vaccine adjuvant. Our results show that MSR/PEI loaded on 3D printing scaffolds can not only promote dendritic cell activation, but also antigen presentation. In addition, dendritic cells of the CPC-MSR/PEI/OVAs group on scaffolds and in culture broth thereof SIINFEKL-H2Kb were compared to the other two groups⁺The largest number, confirming effective recruitment and activation of DCs. The increased number of activated cells in MSR/PEI/OVA-loaded scaffold culture compared to the other groups also indicates that the antigens released from the scaffold still retain their function.

(III) vaccine loaded scaffold for promoting mesenchymal stem cell attachment and osteogenic differentiation

As a biomaterial for bone repair, the scaffold plays an important role in the activity, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells. It is reported that the 3D printing scaffold releases silicon ions with proper concentration to promote osteogenic differentiation of bone marrow mesenchymal stem cells, however, the biocompatibility and osteogenic property of the novel vaccine system are not clear. Therefore, we examined the effect of vaccine loading on the function of bmscs. SEM observations showed that mBMSCs were tightly attached to all scaffold surfaces, whereas cells were more on both CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds (FIG. 4A). In addition, on CPC-MSR/PEI and MS-MSR/PEI/OVA scaffolds, mBMSCs showed a more extended morphology. Since the three sets of scaffolds have similar surface morphology, we hypothesized that the promotion of cell adhesion and spreading may be related to the positive charge of MSR/PEI and MSR/PEI/OVA. Subsequently, we observed the effect of vaccine loading on the osteogenic differentiation behaviour of cells. ALP activity detection of the bone marrow mesenchymal stem cells after 7 days of coculture with the scaffold shows that the ALP positive area on the CPC-MSR/PEI and MS-MSR/PEI/OVA scaffold is obviously larger than that of the CPC scaffold, and the vaccine system is verified to have osteogenesis promoting effectThe application is as follows. The CPC-MSR/PEI group had more positive area than the CPC-MSR/PEI/OVA group. This may be related to the release of MSR/PEI/OVA. Because loading OVA results in less NH on MSR/PEI/OVA available to bind to aldehyde groups on the scaffold₂MSR/PEI/OVA micro-rods are easier to release from the stent than MSR/PEI micro-rods. We also observed more ALP positive cells at the bottom of the CPC-MSR/PEI/OVA scaffold culture wells than CPC-MSR/PEI scaffolds. Although an ALP positive region was hardly seen on the CPC scaffold, a part of cells at the bottom of the culture well showed ALP activity. This is consistent with previous reports and cell adhesion results, although there are fewer cells adhered on the scaffold, the release component of the CPC scaffold slightly promotes osteogenic differentiation of the mesenchymal stem cells. In conclusion, the above results indicate that the scaffold loaded with the vaccine system can promote the adhesion and osteogenic differentiation of the mesenchymal stem cells. To assess the binding effect of vaccine and CPC scaffolds, we started the above experiment after soaking and rinsing the scaffolds loaded with MSR/PEI and MSR/PEI/OVAs in culture medium for several days, the results of which further demonstrate the stability of binding.

(IV) vaccine loaded scaffold induced T in subcutaneous embedding model_H2 reacting and promoting angiogenesis

We implanted MSR/PEI/OVA-loaded scaffolds into C57BL/6J mice to observe their generation and recruitment of antigen-specific CD4 subcutaneously⁺The capacity of T cells. 24 mice were randomly divided into 4 groups: (1) non-vaccinated mice, (2) mice vaccinated with a blank CPC scaffold, (3) mice vaccinated with a CPC-MSR/PEI scaffold, (4) mice vaccinated with a CPC-MSR/PEI/OVA scaffold. To characterize the T caused by the Stent_H1 and T_H2 response, spleen CD4 isolated on day 7 post-implantation was analyzed⁺Expression of Interferon (IFN) -gamma in T cells⁺(a key T)_H1 marker) and Interleukin (IL) -4⁺(a key T)_H2 markers) (fig. 5B). CPC-MSR/PEI group IFN-gamma⁺And IL4⁺The cell proportion is higher, which indicates that MSR/PEI can trigger T simultaneously_H1 and T_HAnd 2, reacting. However, CPC-MSR/PEI/OVA scaffolds allowed IFN-. gamma.⁺Decrease in cell mass, IL-4⁺Cell mass was significantly increased (P)<0.001). This indicates that the CPC-MSR/PEI/OVA scaffold is capable ofIs effective in eliciting specific immune stimulatory responses to the antigen OVA. Next, we continued to observe the recruitment of antigen-specific CD4 by the CPC-MSR/PEI/OVA scaffold⁺The capacity of T cells. CD4 in the macropore of CPC-MSR/PEI/OVA scaffold on day 7 post-inoculation⁺The aggregation amount of T cells is obviously higher than that of a CPC-MSR/PEI stent and a blank stent (P)<0.001) (fig. 5); CD4 in CPC-MSR/PEI scaffolds compared to blank scaffolds⁺There were more T cells, and this trend was consistent with the in vitro DCs activation results.

Previous studies found that OVA vaccines were based on CD4⁺The effect of T cell recruitment is effective in enhancing angiogenesis and blood perfusion in hind limb ischemia. Therefore, we examined the ability of implanting a CPC-MSR/PEI/OVA scaffold to promote angiogenesis in mice. On day 14 post-implantation, blood perfusion within the stent was detected using a live animal multimodal ultrasound imaging system (fig. 5D). From the results, blood flow signals were visible from top to bottom in the large pores of the CPC-MSR/PEI/OVA scaffolds. However, the CPC stent only sees the blood flow signal at the surface, and the CPC-MSR/PEI stent can only detect the signal from the surface to the middle of the stent. By visual inspection of the collected scaffolds (fig. 5E), more newly formed vessels were found around and inside the CPC-MSR/PEI/OVA scaffold than the other scaffolds. The above studies confirm that the vaccine microenvironment after stent implantation can promote rapid vascular growth. The mechanism of locally enhanced angiogenesis may be associated with an increase in OVA-specific IL-5 and IL-10. These are composed of antigen-specific T_H2 cells and eosinophils can promote angiogenesis in vivo by directly enhancing endothelial cell sprouting. The mechanism of this approach remains to be studied. Although release of silicon ions at appropriate concentrations can promote angiogenesis, the potentiating effect of OVA-specific immune responses has proven to be more effective.

(V) vaccine-loaded scaffold for promoting vascularization bone regeneration of rat skull defect repair model

We investigated the effect of vaccine loaded scaffolds on vascularized bone regeneration in a rat skull defect repair model (fig. 6A). Micro-CT results (FIG. 6B, C) showed that the neonatal bone volume ratio (BV/TV) in the defect was 5.02. + -. 0.35%, 8.78. + -. 0.61%, 11.13. + -. 2.45% in the untreated group, CPC-MSR/PEI group and CPC-MSR/PEI/OVA group, respectively, 8 weeks after implantation12.18 +/-0.39 percent. The CPC-MSR/PEI/OVA group showed the best repair with the highest BV/TV value (P)<0.01). From the 3D reconstructed image, more bone was newly formed around and inside the CPC-MSR/PEI/OVA scaffold than in the other groups. In addition, the CPC-MSR/PEI stent has a better osteogenesis promoting effect than the CPC stent. We also performed a more detailed analysis of tissue regeneration by histological analysis. HE and Masson staining results of the bone defect sites of the implanted scaffolds are shown in fig. 6D, and almost no osteogenesis was found in the untreated group and the CPC scaffold. The CPC-MSR/PEI/OVA group had more mature neogenetic bone tissue than the CPC-MSR/PEI group. In addition, abundant CD31 was seen in the CPC-MSR/PEI and CPC-MSR/PEI/OVA scaffolds⁺Cells were integrated into tubes in the macropores and new bone. The bone-promoting effect of the MSR/PEI scaffold is consistent with our expectation. The more effective osteogenesis promoting effect of the CPC-MSR/PEI/OVA scaffold may be related to the following two reasons. First, the blood vessels within the stent are abundant, further promoting bone regeneration. Second, recruited CD4⁺T may also directly promote osteogenesis through other pathways.

3. Conclusion

Through stable loading of MSR/PEI/OVA vaccines, the 3D printing scaffold with the antigen-specific T cell recruitment function is successfully constructed. The synthesized MSR has a layered ordered mesopore and a high specific surface area as a carrier, and is assembled with dendritic polycation high molecular PEI and antigen OVA layer by layer to prepare the MSR/PEI/OVA vaccine with the surface rich in amino. Based on Schiff base reaction, the vaccine and aldehyde group on the surface of the stent are covalently crosslinked and stably combined on the stent, thereby realizing the slow release of OVA and silicon ions. In vitro studies show that the loaded vaccine system not only can recruit and activate dendritic cells to treat antigens, but also can promote the adhesion, spreading and osteogenic differentiation of bone marrow mesenchymal stem cells on the stent. In vivo experimental results showed that after implantation of vaccine-loaded scaffolds in animals, the gradually released OVA rapidly recruited antigen-specific CD4⁺T cells, promote local angiogenesis and increase blood flow perfusion in the scaffold. The MSR/PEI/OVA vaccine loaded 3D printed scaffolds also promoted regeneration of bone tissue under the combined effect of increased blood supply and released silicon ions. In summary, we have developed a method for facilitatingAntigen-specific immune regulation and control 3D printing stent treatment platform for angiogenesis.