Preparation method and application of 4D printing stent for simplifying oral cavity bone increment surgery

Abstract

The invention is suitable for the technical field of oral implantology, and provides a preparation method and application of a 4D printing stent for simplifying oral bone increment surgery. According to the invention, a 4D printing technology is utilized, polydopamine and sodium alginate are mixed and printed into the hydrogel stent, and after strontium ion crosslinking, the stent shows excellent biodegradability and biocompatibility, can replace a titanium mesh bent stent, effectively maintains osteogenesis space, and reduces high technical sensitivity of an implant bone increment surgery. The stent can accurately adapt to different bone defect outlines, support is provided, healing is accelerated, and the risk of complications is reduced. The device is high in adaptability and easy and convenient to implant, unnecessary tissue stripping and injury can be reduced to the maximum extent, the operation duration is shortened, and postoperative recovery of a patient is facilitated. In a horizontal bone increment operation, the scaffold can provide a firm bone foundation for repair means such as subsequent tooth implantation and the like. A mechanical signal generated by deformation and bioactive components released by the stent have a synergistic effect, and bone tissue regeneration is accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral implantology, and in particular relates to a preparation method and application of a 4D printed bracket for simplifying oral bone augmentation surgery. Background Art

[0002] Tooth loss affects the physiological function and aesthetic effect of patients. Oral implants have become an important means to repair missing teeth, restore physiological function, maintain good appearance, ensure correct pronunciation and communication in clinical practice. However, insufficient bone volume caused by periodontal disease, trauma or natural resorption after tooth loss poses a major challenge to the stability and bone integration of implants. To date, guided bone regeneration (GBR) is a predictable and successful method for vertical and lateral bone augmentation of atrophic alveolar ridges before or during implant placement. Polytetrafluoroethylene (e-PTFE) and titanium mesh commonly used in clinical surgery have high mechanical strength and porous structure and do not induce immune response, but they still require secondary surgery to remove and there is a risk of soft tissue dehiscence and postoperative infection leading to bone regeneration failure. However, existing biodegradable barrier membranes are difficult to maintain defect space under exposure to oral fluids and / or blood due to their lack of rigidity. In addition, when biodegradable barrier membranes degrade, particles or fragments that may cause foreign body reactions are produced. Therefore, it is particularly important to develop new scaffold materials to more effectively guide alveolar bone regeneration, which may be inspired by bone biology, including immune response, mesenchymal stem cell recruitment, and osteogenic differentiation.

[0003] 4D bioprinting, combined with programmable biomaterials, living cells and bioactive factors, can achieve complex structural formation and functional maturation, providing greater potential for the construction of dynamic, personalized and precise bone tissue engineering scaffolds. Through detailed pre-programming, 4D printed structures can achieve expected time-dependent deformations in response to specific stimuli supported by scalable and flexible smart materials. The internal pore structure of the scaffold directly affects cell infiltration, nutrient diffusion and bone matrix deposition, and is an important factor in the tissue regeneration process. Sodium alginate (Alg) is a natural hydrophilic, biocompatible, injectable, biodegradable polysaccharide extracted from seaweed. Alg prepolymer solution shows shear thinning properties and is therefore an ideal precursor material for the preparation of 4D printed scaffolds. It can be cross-linked with divalent cations such as calcium, strontium and copper to maintain non-covalent bonds within the polysaccharide chain to form an eggshell structured gel. Compared with the gel formed by calcium ions, strontium has a higher molecular weight than calcium and a stronger affinity for alginate, so strontium alginate (Sr-Alg) hydrogel is more stable, and the strontium ions released during the degradation process promote new bone formation. However, due to the high hydrophilicity and excessive anionic charge of alginate, Alg scaffolds are difficult to provide cell adhesion sites, which is not conducive to the migration of osteoblasts during bone formation. Polydopamine (PDA) contains catechol, quinone, imine, amine functional groups and π conjugated structures, which make the material surface hydrophilic and is considered to be a promising antioxidant. It has strong photothermal conversion ability and the ability to scavenge reactive oxygen species (ROS), which helps to downregulate inflammatory mediators.

[0004] In summary, the existing technology faces two major limitations in the field of guided bone regeneration: first, the shape of the bone defect in the human body is irregular, which makes it difficult for traditional scaffolds to fit completely, and the gaps left may cause infection and affect healing; second, in order to achieve the ideal fit between the scaffold and the bone defect, traditional surgery often requires excessive trimming of the implant site, thereby increasing the wound surface. In response to the above problems, the present invention proposes a preparation method and application of a 4D printed scaffold for simplifying oral bone augmentation surgery. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a 4D printed bracket for simplifying oral bone augmentation surgery, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The method for preparing a 4D printed bracket for simplifying oral bone augmentation surgery comprises the following steps:

[0008] Step 1: at room temperature, dopamine hydrochloride is added to a Tris solution, and a chemical reaction is carried out under stirring to synthesize polydopamine nanoparticles;

[0009] Step 2: Collect the polydopamine nanoparticles by centrifugation and then wash them three times with water;

[0010] Step 3: dissolving sodium alginate in the polydopamine nanoparticle solution and stirring evenly to prepare 4D printing ink;

[0011] Step 4: Import the designed STL file model into the direct writing 3D printer for printing; the bracket is designed as a double-layer square structure;

[0012] Step 5: Under room temperature, the prepared scaffold is cross-linked with strontium ions, and then the scaffold is repeatedly washed with deionized water;

[0013] Step 6: Irradiate the stent with near-infrared light and use a photothermal camera to record the heating rate of the stent; heat the deformed stent from room temperature to 42°C, and the stent deforms;

[0014] Step 7: Place the deformed scaffold in a freeze dryer for drying.

[0015] Furthermore, the specific operations of step 1 are as follows:

[0016] At room temperature, dopamine hydrochloride was added to a 1.5 mmol Tris solution with a pH value of 8.5, and a chemical reaction was carried out under stirring conditions of 800 rpm for 20 hours to synthesize polydopamine nanoparticles.

[0017] Furthermore, in step 2, the centrifugal speed is 15000 rpm, the centrifugal temperature is 4° C., and the centrifugal time is 20 min.

[0018] Furthermore, the specific operations of step 3 are as follows:

[0019] Sodium alginate was dissolved in a 2 mg / ml polydopamine nanoparticle solution at a mass ratio of 7:3 and stirred evenly to prepare ink for 4D printing.

[0020] Furthermore, in step 4, the printing nozzle diameter is 0.41 mm, the printing speed is 3-4 mm / s, and the air pressure is 0.4-0.6 MPa.

[0021] Furthermore, in step 4, the specific printing parameters are set as follows: the size of the bracket is 10×10×1.5 mm, the layer thickness is 0.2 mm, 2 layers are printed in total, and the line spacing is 1 mm.

[0022] Furthermore, the specific operations of step 5 are as follows:

[0023] The prepared scaffolds were cross-linked with 600 mM strontium ions at room temperature for 5 minutes, and then the scaffolds were repeatedly washed with deionized water.

[0024] Furthermore, in step 7, the drying time is 48 hours.

[0025] A 4D printed bracket for simplifying oral bone augmentation surgery is prepared according to the preparation method described above.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention utilizes 4D printing technology to print a mixture of polydopamine and sodium alginate into a hydrogel scaffold through a direct-write 3D printer. After cross-linking with strontium ions, the scaffold exhibits excellent biodegradability and biocompatibility, and can be used as a degradable biomaterial to replace the titanium mesh curved scaffold, effectively maintain the bone formation space, and reduce the high technical sensitivity of implant bone augmentation surgery. The scaffold has the ability to deform into a saddle shape, can accurately adapt to different bone defect contours, provide ideal support for new bone tissue, accelerate the healing process, and reduce the risk of complications. The scaffold has strong adaptability and is easy to implant. It can minimize unnecessary tissue stripping and damage, shorten the operation time, and facilitate the patient's postoperative recovery. In horizontal bone augmentation surgery, the 4D printed scaffold allows personalized customization of the initial shape, and combined with the deformation function, ensures sufficient and appropriate bone augmentation, providing a solid bone foundation for subsequent dental implants and other repair methods. The mechanical signals generated by deformation work synergistically with the bioactive components released by the scaffold to effectively regulate the cell behavior in the implanted area, promote the polarization of macrophages to M2, release growth factors, and promote the osteogenic differentiation of mesenchymal stem cells, thereby accelerating bone tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The transmission electron microscope images and scanning electron microscope images of polydopamine nanoparticles; A is the transmission electron microscope image of polydopamine nanoparticles, and B is the scanning electron microscope image of polydopamine nanoparticles.

[0029] Figure 2 Scanning electron microscope image of the deformed scaffold.

[0030] Figure 3 Schematic diagram of the deformation of brackets of different shapes after near-infrared light irradiation.

[0031] Figure 4Figure 3 Biocompatibility and osteogenesis experiments were performed on the Control, AC, AS, PAS, and PASN groups; A is the CCK-8 test of the Control, AC, AS, PAS, and PASN scaffolds; B is the cell viability assay of the AC, AS, PAS, and PASN scaffolds; C is the quantitative analysis of BMSCs co-cultured with different hydrogel scaffolds for 7d and 14d; D is a schematic diagram of the co-culture of BMSCs with AC, AS, and PAS scaffolds; E is the immunofluorescence staining of ALP, COL-1, OCN, and RUNX2 proteins at 7d and the corresponding semi-quantitative analysis, with green representing ALP, COL-1, RUNX2, and OCN, red (phalloidin) representing F-actin, and blue (DAPI) representing cell nuclei; F is ALP staining of BMSCs cultured for 7d and 14d; G is the observation of calcium deposition in BMSCs at 28d by Alizarin Red S staining. *p<0.05, **p<0.01, ***p<0.001; “ns” indicates no statistically significant difference, n=3, error bars indicate mean ± standard deviation.

[0032] Figure 5 To evaluate the in vitro angiogenesis effect of different hydrogel scaffolds using HUVECs; A is a scratch test image; B is a transwell test image; C is a fluorescence microscope image of the tube formation experiment; D is a real-time PCR analysis of the mRNA expression of HIF-1α, PDGF, and VEGF; E is a quantitative analysis of the scratch test; F is a quantitative analysis of the transwell test; G is a quantitative analysis of the number of connection points; H is a quantitative analysis of the whole segment; I is an immunofluorescence staining of HUVECs cultured on different hydrogel films for 3 days, green is CD31 and VEGF, red (phalloidin) is F-actin, and blue (DAPI) is the cell nucleus; J is a quantitative analysis of CD31 and VEGF based on fluorescence intensity. *p<0.05, **p<0.01, ***p<0.001; "ns" indicates no statistically significant difference, n=3, error bars represent mean ± standard deviation.

[0033] Figure 6Figure 1 is the in vivo bone regeneration experiment of the scaffold; A is the bone regeneration ability of different hydrogel scaffolds in the rat skull defect model; B is the micro-CT reconstruction image at 4 and 8 weeks after surgery; C is the bone volume fraction (BV / TV), trabecular separation (Tb.Sp), trabecular number (Tb.N) and trabecular thickness (Tb.Th); D is the typical hematoxylin-eosin (HE) staining and Masson trichrome staining of the skull defect area at 4 and 8 weeks after surgery; E is the quantitative analysis of angiogenesis and osteogenesis-related proteins; F is the immunofluorescence staining of angiogenesis and osteogenesis-related proteins, green is CD31, OCN, blue (DAPI) is the cell nucleus. *p<0.05, **p<0.01, ***p<0.001; "ns" means no statistically significant difference, n=3, error bars represent mean ± standard deviation.

[0034] Figure 7 The flowchart of the scaffold preparation is shown in FIG. DETAILED DESCRIPTION

[0035] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0036] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0037] One embodiment of the present invention provides a method for preparing a 4D printed stent for simplifying oral bone augmentation surgery, as shown in the flow chart: Figure 7 As shown, the following steps are included:

[0038] Step 1: At room temperature, dopamine hydrochloride was added to 1.5 mmol Tris solution (pH=8.5), and a chemical reaction was carried out under stirring conditions of 800 rpm for 20 hours to synthesize polydopamine nanoparticles.

[0039] Step 2: Collect the polydopamine nanoparticles by centrifugation (centrifugation speed of 15000 rpm, temperature of 4°C, time of 20 min), and then wash with water three times.

[0040] Step 3: Sodium alginate was dissolved in a 2 mg / ml polydopamine nanoparticle (PDANPs) solution at a mass ratio of 7:3 and stirred evenly to prepare 4D printing ink.

[0041] Step 4: Import the designed STL file model into a direct-write 3D printer (Regenovo 3D Bio-Architect, China) for printing. The print nozzle diameter is 0.41 mm (22G), the print speed is set to 3-4 mm / s, and the air pressure is set to 0.4-0.6 MPa. The specific printing parameters are set as follows: the bracket size is 10×10×1.5 mm, the layer thickness is 0.2 mm, a total of 2 layers are printed, and the line spacing is 1 mm. The bracket is designed as a double-layer square structure.

[0042] Step 5: Under room temperature, the prepared scaffold was cross-linked with 600 mM strontium ions (strontium chloride solution) for 5 minutes, and then the scaffold was repeatedly washed with deionized water.

[0043] Step 6: Irradiate the stent with near-infrared light and use a photothermal camera to record the heating rate of the stent. The deformed stent is heated from room temperature to 42°C and deformed. The shape deformation behavior (such as the deformation angle) can be controlled by the laser power and irradiation time.

[0044] Step 7: The deformed scaffold was placed in a freeze dryer (FDU-2200, Japan) for drying for 48 h.

[0045] Figure 1 A is a transmission electron microscope image of polydopamine nanoparticles, and B is a scanning electron microscope image of polydopamine nanoparticles. It can be seen that the polydopamine nanoparticles are regular spherical with a diameter of about 100 nm, and the rough surface of the deformed scaffold is conducive to cell adhesion. Figure 2 is a scanning electron microscope image of the deformed scaffold. Figure 3 It is the deformation of stents of different shapes after near-infrared (NIR) light exposure.

[0046] Example 1: In vitro experiment;

[0047] 1) Biocompatibility and osteogenesis tests;

[0048] Excellent biocompatibility has always been a basic requirement for designing new bone tissue engineering scaffolds. 2+ (AS group), PDA / sodium alginate / Sr 2+ (PAS group), PDA / sodium alginate / Sr 2+The effect of near-infrared (PASN group) induction on cell deformation of the scaffold, and the cell counting kit (CCK-8) was used to detect the proliferation of rBMSCs (rat bone marrow mesenchymal stem cells) on the scaffold. The results of cell viability test showed that the PASN scaffold had good biocompatibility in the results of 1d and 3d. It decreased at 7d, which may be related to the released strontium ions promoting the differentiation of BMSCs ( Figure 4 (A). Live / dead staining was used to evaluate cell viability on the scaffolds. From the results on days 1, 3, and 7, the PASN scaffold was most suitable for cell proliferation because of the good biocompatibility of PDANPs and reduced cytotoxicity. No obvious cytotoxicity was observed after 7 days of culture ( Figure 4 (B), indicating that the PASN scaffold has good biocompatibility.

[0049] Bone marrow mesenchymal stem cells (BMSCs) were inoculated on the scaffolds for 7 and 14 days, and the expression of osteogenesis-related genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The results showed that all osteogenesis-related genes (type I collagen (COL-1), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP)) were significantly upregulated in the PAS group and AS group ( Figure 4 In order to further verify the biomineralization activation of PAS scaffolds at the protein level, we performed protein immunofluorescence staining on rBMSCs co-cultured with the scaffolds ( Figure 4 The results showed that the expression levels of osteogenic related proteins (ALP, COL-1, OCN and RUNX2) were higher in the PAS group and AS group ( Figure 4 Alkaline phosphatase (ALP) is one of the expression markers of early osteogenic differentiation of BMSCs. After rBMSCs were co-cultured with AC / AS / PAS scaffolds for 7 and 14 days, the strontium ions released by AS and PAS scaffolds increased the expression of ALP compared with the Control group and AC group ( Figure 4 F). Alizarin red (AR) staining reflects the formation of calcium nodules, indicating the later osteogenic activity. The results of 28d Alizarin red (AR) staining also showed that compared with the control group and AC group, the strontium ions released by PAS and AS scaffolds enhanced the biomineralization capacity by increasing the mineralization level of cell mineralized nodules ( Figure 4 Middle G).

[0050] 2) evaluate the in vitro angiogenesis effects of different hydrogel scaffolds;

[0051] For tissue engineering scaffolds, effective vascularization can enhance the bone regeneration ability of scaffold materials. Vascularization not only provides sufficient nutrition for migrating cells, but also promotes oxygen exchange and product metabolism in osteoblasts. In order to explore more effective vascularization strategies, we conducted a series of experiments.

[0052] The AC, AS, PAS and PASN groups were subjected to vascular endothelial cell scratch test, migration test and tubular structure formation test. In the PASN group, the PAS scaffold was first co-cultured with cells, and then exposed to near-infrared light for 5 minutes to increase the temperature of the scaffold and deform it. The scratch test results showed that the cells in the PASN group had the best migration ability, and the scratch was basically healed after 24 hours. The cell migration speed in the AS group was slightly faster than that in the AC group ( Figure 5 A and E). The transwell experiment also further verified that the PASN group had superior migration ability compared with other groups ( Figure 5 In addition, the results of the tube formation experiment showed that the PASN group significantly promoted the tube formation of human umbilical vein endothelial cells ( Figure 5 C, G, and H in the middle). The study also showed that near-infrared stimulation can promote angiogenesis by stabilizing the expression of hypoxia-inducible factor-1α (HIF-1α), thereby inducing the expression of downstream angiogenesis-related proteins. To further explore the ability of the PASN group to induce angiogenesis, the expression of angiogenesis-related genes in human umbilical vein endothelial cells (HUVECs) co-cultured with different groups was analyzed at the gene level. The results showed that the mRNA expression levels of HIF-1α, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) in the PASN group were significantly higher than those in the AC and AS groups ( Figure 5 At the same time, the results of protein fluorescence staining and fluorescence intensity quantitative analysis of CD31 and VEGF also showed the same trend ( Figure 5 This suggests that we can first implant the undeformed PAS scaffold into the bone defect area, and then gently apply near-infrared light to match the deformation degree of the scaffold with the angle of the bone defect area, while promoting osteogenesis-vascular coupling through the photothermal effect, which will bring convenience to clinical treatment.

[0053] Example 2: In vivo experiment;

[0054] To further investigate the in vivo bone regeneration capacity of the PASN group, we used a rat severe skull defect model for animal experiments. Briefly, a circular bone defect with a diameter of 5 mm was established on the bilateral parietal bones of each rat (two bone defects per rat). Then, different groups of scaffolds were implanted into the defect sites ( Figure 6The experiment was divided into 5 groups: Control, AC, AS, PAS and PASN. Micro-CT results showed that rats in the PASN group had better bone repair ability at 4 and 8 weeks, respectively. At 8 weeks, the skull defect in the PASN group was almost completely filled with new bone, and the new bone was better than that in the PAS and AS groups ( Figure 6 (B). Quantitative analysis of the defect site by micro-CT showed that the bone volume fraction (BV / TV) in the PASN group reached 47.16% and 71.24% at 4 and 8 weeks, respectively. In addition, the number of trabeculae (Tb.N) and trabecular thickness (Tb.Th) in the PASN group were also significantly higher than those in the Control group, and the degree of trabecular separation (Tb.Sp) was lower. In the PAS and PASN groups, the "crawling" growth pattern of new bone along the surface of the scaffold was due to the formation of intergroup hydrogen bonds on the surface of bone tissue and intergroup hydrogen bonds of PDANPs or π-π conjugation in the PAS / PASN scaffolds, connecting the scaffold to the biological tissue ( Figure 6 C). The new bone was further histologically analyzed and evaluated by HE and Masson staining. The results showed that hematoxylin-eosin (HE) staining showed no obvious inflammatory response or necrosis in the skull defect, which was consistent with in vivo biocompatibility. Compared with the Control group, the PASN group had significantly increased new bone formation at 4 and 8 weeks. In the Masson staining experiment, the PASN group and PAS group had more collagen formation than the Control group and AC group. In addition, the Masson staining results showed that the PASN group produced more blood vessels ( Figure 6 Subsequent immunofluorescence staining of osteogenesis- and angiogenesis-related proteins confirmed this finding. The protein expression of CD31 and OCN was significantly increased in the PASN group ( Figure 6 (E and F). In vivo experiments showed that the PASN scaffold is a good scaffold for osteogenesis and angiogenesis.

[0055] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a 4D printed stent for simplifying oral bone augmentation surgery, characterized in that: The following steps are involved: Step 1: at room temperature, dopamine hydrochloride is added to a Tris solution, and a chemical reaction is carried out under stirring to synthesize polydopamine nanoparticles; Step 2: Collect the polydopamine nanoparticles by centrifugation and then wash them three times with water; Step 3: dissolving sodium alginate in the polydopamine nanoparticle solution and stirring evenly to prepare 4D printing ink; Step 4: Import the designed STL file model into the direct writing 3D printer for printing; the bracket is designed as a double-layer square structure; Step 5: Under room temperature, the prepared scaffold is cross-linked with strontium ions, and then the scaffold is repeatedly washed with deionized water; Step 6: Irradiate the stent with near-infrared light and use a photothermal camera to record the heating rate of the stent; heat the deformed stent from room temperature to 42°C, and the stent deforms; Step 7: Place the deformed scaffold in a freeze dryer for drying.

2. The preparation method according to claim 1, characterized in that: The specific operations of step 1 are as follows: At room temperature, dopamine hydrochloride was added to a 1.5 mmol Tris solution with a pH value of 8.5, and a chemical reaction was carried out under stirring conditions of 800 rpm for 20 hours to synthesize polydopamine nanoparticles.

3. The preparation method according to claim 1, characterized in that: In step 2, the centrifugal speed is 15000 rpm, the centrifugal temperature is 4° C., and the centrifugal time is 20 min.

4. The preparation method according to claim 1, characterized in that: The specific operations of step 3 are as follows: Sodium alginate was dissolved in a 2 mg / ml polydopamine nanoparticle solution at a mass ratio of 7:3 and stirred evenly to prepare ink for 4D printing.

5. The preparation method according to claim 1, characterized in that: In step 4, the printing nozzle diameter is 0.41 mm, the printing speed is 3-4 mm / s, and the air pressure is 0.4-0.6 MPa.

6. The preparation method according to claim 1, characterized in that: In step 4, the specific printing parameters are set as follows: the size of the bracket is 10×10×1.5 mm, the layer thickness is 0.2 mm, a total of 2 layers are printed, and the line spacing is 1 mm.

7. The preparation method according to claim 1, characterized in that: The specific operation of step 5 is as follows: The prepared scaffolds were cross-linked with 600 mM strontium ions at room temperature for 5 minutes, and then the scaffolds were repeatedly washed with deionized water.

8. The preparation method according to claim 1, characterized in that: In step 7, the drying time is 48 hours.

9. A 4D printed stent for simplifying oral bone augmentation surgery, prepared according to the preparation method of any one of claims 1 to 8.

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