3D printing jawbone biological scaffold

Abstract

According to the 3D printing jawbone biological scaffold provided by the invention, a jawbone BMSCs and dental pulp dECM loaded biological scaffold is successfully constructed by utilizing a 3D printing technology, and the core requirements of jawbone defect repair on mechanical support strength, a biological activity microenvironment, osteogenesis bionic induction characteristics and immune regulation and control capability are synchronously met.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a 3D-printed jawbone bioscaffold. Background Technology

[0002] Jawbone defects refer to the partial loss of the jawbone and structural and functional abnormalities caused by trauma, disease, or surgery. They severely affect patients' physiological functions such as chewing, swallowing, and speech, and lead to a heavy psychosocial burden. Jawbone repair strategies mainly include autologous bone grafting, allogeneic bone grafting, artificial bone grafting, and bone tissue engineering, aiming to restore the continuity, support, shape, and related oral functions of the jawbone. While autologous bone grafting possesses osteocyte-inducing activity, it faces challenges such as insufficient donor sites and unpredictable bone resorption. Allogeneic bone grafting, although able to alleviate insufficient bone mass, suffers from drawbacks such as immune rejection and delayed osseointegration.

[0003] Artificial bone has been extensively studied in recent years, but due to the lack of inherent osteoblasts and osteoinductive properties, new bone formation is significantly limited after osteoconduction is achieved. While existing 3D-printed scaffolds possess a certain level of mechanical strength, their lack of cellular components presents a significant limitation in achieving biomimetic functions. Furthermore, the absence of functional matrix components in current 3D-printed scaffolds makes it difficult to effectively and precisely regulate stem cell fate (such as adhesion, proliferation, and differentiation) and coordinate local immune responses through matrix-cell interactions. These limitations restrict the application potential of 3D printing technology in jawbone regeneration, necessitating the development of bio-scaffolds loaded with living cells and functional matrices to synergistically address these challenges. Summary of the Invention

[0004] To address the above problems, this invention provides a 3D-printed jawbone bioscaffold and its preparation method.

[0005] To achieve the above objectives, the specific technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides a 3D-printed jawbone bioscaffold, the bioscaffold comprising: methacrylated gelatin, decellularized extracellular matrix of dental pulp tissue, and jawbone bone marrow mesenchymal stem cells.

[0007] In this invention, gelatin methacryloyl (GelMA) is a photosensitive crosslinkable biomaterial in which methacryloyl groups are introduced into the molecular chain of natural gelatin through chemical modification.

[0008] Furthermore, the concentration of the methacrylated gelatin is 10% (w / v).

[0009] In this invention, decellularized extracellular matrix (dECM) of dental pulp tissue is an extracellular matrix material retained after the cellular components in dental pulp tissue have been removed by physical, chemical, or biological methods. The dECM can be prepared by any decellularization method, including but not limited to physical, chemical, enzymatic, biological, or combinations thereof.

[0010] Furthermore, the concentration of decellularized extracellular matrix in the dental pulp tissue is 5 mg / mL.

[0011] In this invention, bone marrow mesenchymal stem cells (jawbone BMSCs) refer to adult stem cells with strong proliferative capacity and multi-directional differentiation potential that exist in the maxilla and mandible. They can be obtained by any separation method, including but not limited to tissue block adhesion, density gradient centrifugation, immunomagnetic bead sorting or flow cytometry, and the same technical effects as in the specific embodiments can be achieved.

[0012] Furthermore, the concentration of the jawbone bone marrow mesenchymal stem cells in the biological scaffold is 2 × 10⁻⁶. 6 cells / mL.

[0013] Furthermore, the degree of substitution of the methacrylamide group in the methacrylated gelatin is 60%.

[0014] Furthermore, the decellularized extracellular matrix of the dental pulp tissue is a lyophilized powder.

[0015] A second aspect of this invention provides a method for preparing a 3D-printed jawbone bioscaffold, the method comprising the following steps:

[0016] S1. Preparation of 3D printed jawbone bioscaffold pregel solution.

[0017] S2, 3D printing and photopolymer crosslinking molding.

[0018] In some embodiments, the pregel solution in step S1 is obtained by adding decellularized extracellular matrix of dental pulp tissue and mesenchymal stem cells of jawbone bone marrow to methacrylamide gelatin.

[0019] Furthermore, the concentration of the methacrylamide gelatin is 10% (w / v), wherein the degree of substitution of the methacrylamide group is 60%.

[0020] Furthermore, the decellularized extracellular matrix of the dental pulp tissue is a lyophilized powder with a concentration of 5 mg / mL in the pregel solution.

[0021] Furthermore, the jawbone bone marrow mesenchymal stem cells are suspended in the pre-gel solution at a concentration of 2 × 10⁻⁶. 6 cells / mL.

[0022] In some embodiments, the 3D printing in step S2 is performed using a Bio-Architect® WS bioprinter.

[0023] Furthermore, the nozzle temperature is 15℃.

[0024] Furthermore, the temperature of the receiving plate is 20°C.

[0025] Furthermore, the extrusion pressure is 0.15-0.2 MPa.

[0026] Furthermore, the printing speed is 8 mm / s.

[0027] Furthermore, the 3D printing is a layer-by-layer printing process.

[0028] In some embodiments, the photocuring crosslinking in step S2 is achieved by crosslinking formation through irradiation with 405 nm visible blue light.

[0029] Furthermore, the irradiation time is 60 seconds.

[0030] A third aspect of the present invention provides applications of the biological scaffold described in the first aspect of the present invention and / or the biological scaffold obtained by the preparation method described in the second aspect of the present invention, wherein the applications include any one of the following:

[0031] 1) Application in the preparation of implants for the repair of jawbone defects.

[0032] 2) Application in the preparation of auxiliary implant materials for the treatment of jaw fractures.

[0033] 3) Application in constructing jawbone tissue engineering models.

[0034] 4) Application in the preparation of biomaterials for tissue-engineered jawbone transplantation.

[0035] 5) Application in drug screening and evaluation.

[0036] In some embodiments, the drug screening and evaluation involves applying the test drug to a bio-scaffold-constructed jawbone tissue engineering model, observing the effects of the drug on cell growth, differentiation, and bone tissue formation on the scaffold, and assessing the efficacy and toxicity of the drug on the jawbone tissue.

[0037] In this invention, the implant for jawbone defect repair refers to the biocompatibility, suitable mechanical properties and ability to guide bone tissue growth of the biological scaffold, which is processed into an implant that conforms to the shape and size of the jawbone defect site. After being implanted into the jawbone defect area of ​​the human body, it provides structural support for the defective jawbone and promotes the growth of surrounding bone tissue into the scaffold, thereby realizing the structural and functional reconstruction of the jawbone.

[0038] In this invention, the auxiliary implant material for treating jaw fractures refers to a biological scaffold that, with its good mechanical stability and bioactivity, is placed at the fracture site of the jaw to fix the fracture ends, disperse stress, and provide a suitable microenvironment for bone cell growth, thereby accelerating the healing process of jaw fractures.

[0039] In this invention, the jawbone tissue engineering model refers to using this biological scaffold as a three-dimensional carrier for cell growth to simulate the physiological environment of jawbone tissue in vivo, providing a reliable in vitro experimental platform for studying the development and regeneration mechanisms of jawbone tissue and the pathogenesis of related diseases.

[0040] In this invention, the biomaterials used for tissue-engineered jawbone transplantation refer to the combination of biological scaffolds with cells (such as jawbone bone marrow mesenchymal stem cells, osteoblasts, etc.), growth factors, etc., to construct a bioactive tissue-engineered jawbone for clinical transplantation to replace damaged or missing jawbone tissue.

[0041] In this invention, drug screening and evaluation refers to using a model constructed with a biological scaffold to simulate the metabolism and action of drugs in the jawbone tissue in vivo. By observing the effects of drugs on cell growth, differentiation and bone formation on the scaffold, the efficacy and toxicity of drugs on the jawbone tissue are evaluated, providing experimental basis for new drug development.

[0042] Advantages and benefits of the present invention: The present invention provides a 3D printed jawbone bioscaffold that can accurately replicate the irregular shape of jawbone defects and realize personalized reconstruction for patients; it eliminates the need for autologous or allogeneic transplantation, thus solving the problem of limited sources and reducing the risk of immune rejection; it can meet the requirements of mechanical support, jawbone bionics and immune repair, and provides new ideas and directions for the research and development of materials for the treatment of jawbone defects. Attached Figure Description

[0043] Figure 1 The graphs show the mechanical performance test results of biological scaffolds constructed at different proportions. A is the statistical graph of compressive elastic modulus, B is the statistical graph of compressive elastic strength, C is the compressive stress-strain curve, D is the statistical graph of tensile elastic modulus, E is the statistical graph of tensile elastic strength, and F is the statistical graph of elongation at break.

[0044] Figure 2 The image shows the biocompatibility results of biological scaffolds constructed at different ratios detected by two-photon microscopy, where green fluorescence represents live cells and red fluorescence represents dead cells.

[0045] Figure 3The images show the in vitro osteogenic mineralization capacity of biological scaffolds constructed at different ratios. A is a schematic diagram of the culture process; B is a scaffold osteogenic phenotype image showing white mineralized nodule deposition; C is a MicroCT 3D reconstructed image showing the amount of mineral deposition; and D is a statistical graph showing the quantitative analysis results of bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular spacing (Tb.Sp).

[0046] Figure 4 The results show the immunomodulatory function of different groups of biological scaffolds. A is a schematic diagram of the culture process; B is a statistical graph of the expression levels of M1 and M2 macrophage-related genes detected by RT-PCR; and C is a representative image of immunofluorescence staining.

[0047] Figure 5 This diagram illustrates the osteogenic mineralization capacity and immune regulation results of the biological scaffold in vivo. A shows a schematic diagram of the in vivo osteogenic culture process; B shows the osteogenic phenotype of the scaffold in vivo; C shows a 3D reconstructed image of the scaffold using MicroCT, displaying the amount of osteogenic mineral deposition; D shows a statistical graph of the quantitative analysis results of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp); E shows a schematic diagram of the in vivo immune regulation process; F shows the scaffold stained with hematoxylin and eosin (HE); G shows representative immunofluorescence staining images of M1 and M2 macrophages on the scaffold; and H shows the normalized quantitative immunofluorescence.

[0048] Figure 6 Heatmap of differentially expressed genes in jawbone BMSCs from GelMA+BMSCs and GelMA+5dECM+BMSCs bioscaffolds.

[0049] Figure 7 This diagram shows the results of dECM regulation of osteogenic differentiation and immune regulation mechanisms. A shows the effect of si-ITGB7 on the expression levels of osteogenic-related genes detected by RT-PCR; B shows the ALP staining results; C shows the ARS staining results; D shows the effect of si-ITGB7 on the expression levels of M1 and M2-related genes in macrophages detected by RT-PCR; and E shows the effect of si-ITGB7 on the levels of cytokines in conditioned medium detected by ELISA. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example

[0052] I. Experimental Methods

[0053] 1. The main reagents and materials are shown in Table 1.

[0054] Table 1. Main Reagents and Materials

[0055]

[0056]

[0057] 2. Preparation of dental pulp dECM

[0058] (1) The cell culture plate surface was subjected to three chemical treatments at 37°C: first, it was coated with gelatin solution for 1 hour to provide a substrate adhesion layer, then cross-linked and fixed with glutaraldehyde solution (0.5 hours), and finally the active sites were blocked with ethanolamine solution (0.5 hours). Each step was thoroughly washed three times with PBS to finally construct a functionalized interface suitable for stable attachment of dECM.

[0059] (2) Dental pulp stem cells were administered at a rate of 3 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL in cell culture plates that had undergone surface pretreatment in step (1) and cultured in α-MEM medium containing 10% FBS until the cell confluence reached 95% or more.

[0060] (3) Replace the medium with a complete medium containing 100 μg / mL L-ascorbic acid to induce dental pulp stem cells to produce matrix. Induce continuously for 8 days, replacing the medium with fresh medium every 2 days to maintain the concentration of active ingredients. Terminate induction on day 8 to obtain extracellular matrix.

[0061] (4) Wash the cell layer three times with PBS pre-cooled at 4°C to remove residual culture medium, add PBS cell lysis buffer containing 0.5% Triton X-100 and 20 mmol / L NH4OH, and incubate at 37°C in the dark for 5-8 minutes until the cell nuclear structure completely disappears and no cell structure can be observed under a microscope, thus obtaining dECM.

[0062] (5) After decellularization of dECM, add an equal volume of PBS containing 1% penicillin antibody to terminate the reaction, and let stand at 4°C overnight to stabilize the dECM structure.

[0063] 3. Material Construction: The construction materials and processes for the 3D scaffold are shown in Table 2.

[0064] Table 2. Material Composition

[0065]

[0066] 4. 3D printing and photopolymerization crosslinking: Using the Bio-Architect® WS bioprinter, the nozzle temperature was controlled at 15℃ and the receiving plate at 20℃. Four sets of scaffolds were printed layer by layer at an extrusion pressure of 0.15-0.2 MPa and a speed of 8 mm / s. The scaffolds were then photopolymerized by irradiating them with 405nm visible blue light for 60 seconds.

[0067] 5. Key Performance

[0068] Mechanical properties: The compressive properties (compressive modulus of elasticity, compressive strength and compressive stress-strain curve) and tensile properties (tensile modulus of elasticity, tensile strength and tensile stress-strain curve) of four sets of supports were measured using an electronic universal testing machine at a crosshead speed of 1 mm / min.

[0069] Biocompatibility: At 1, 3 and 7 days after 3D printing of the bioscaffold, four groups of loaded jawbone BMSCs bioscaffolds were incubated for half an hour using Calcein AM / PI and a cytotoxicity assay kit, and live and dead images of jawbone BMSCs in the bioscaffolds were captured using two-photon microscopy.

[0070] 6. Functional Verification

[0071] Osteogenic function: On day 2 of 3D-printed bioscaffolds, GeMA, GelMA+BMSCs, and GelMA+5dECM+BMSCs bioscaffolds were placed in osteogenic induction medium to begin osteogenic induction, with the medium being changed every 2 days. On day 30, the in vitro automineralization level of the three scaffolds was detected using MicroCT.

[0072] Immune Function: A Transwell co-culture system was constructed. GelMA, GelMA+BMSCs, and GelMA+5dECM+BMSCs bioscaffolds were placed in the upper chamber of a Transwell culture medium; macrophages were seeded in the lower chamber, and co-cultured for 24 hours in osteogenic induction medium containing LPS. RT-PCR and immunofluorescence staining were used to assess the ability of the GelMA+5dECM+BMSCs bioscaffold to promote the transformation of macrophages from M1 macrophages (inflammatory macrophages) to M2 macrophages (repair macrophages). The effects of active factors secreted by BMSCs in the bioscaffold on macrophage polarization through osmosis were investigated.

[0073] Animal experiments: This study established a mouse dorsal subcutaneous ectopic transplantation model, implanting GelMA+BMSCs and GelMA+5dECM+BMSCs bioscaffolds, respectively. To evaluate their osteogenic mineralization potential in vivo, samples were collected on day 30 post-transplantation, and the degree of bone mineralization was detected using MicroCT. To dynamically assess the early host immune response after implantation, samples were collected on day 10 post-transplantation, and the phenotypic identification and semi-quantitative analysis of M1 (pro-inflammatory) and M2 (reparative) macrophages around the implant were performed using HE and immunofluorescence staining. Since the pure GelMA group lacked cellular components and had no active osteogenic potential in the mouse dorsal region, related animal experiments were not conducted.

[0074] Whole transcriptome sequencing: On day 14 of osteogenic induction using GelMA+BMSCs and GelMA+5dECM+BMSCs scaffolds, whole transcriptome sequencing was performed on the jawbone BMSCs within the scaffolds to investigate their osteogenic-immune regulatory mRNA expression mechanism. Whole transcriptome sequencing was not performed in the GelMA group because it lacked cellular components.

[0075] Mechanism verification:

[0076] ①si-NC sequence: Forward: UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 1); Reverse: ACGUGACACGUUCGGAGAATT (SEQ ID NO: 2).

[0077] ②si-ITGB7 sequence: Forward: GGCUCUCUGUGGAAAUCUATT (SEQ ID NO: 3); Reverse: UAGAUUUCCACAGAGAGCCTT (SEQ ID NO: 4).

[0078] Cells were cultured in groups of dECM+si-NC and dECM+si-ITGB7 to induce osteogenic growth. After 8 days of induction, the mRNA expression levels of ITGB7 and osteogenic-related genes (COL1A1, RUNX2, OPG) were detected by RT-PCR, and alkaline phosphatase activity was detected by ALP staining. After 14 days of induction, calcium nodule deposition was detected by ARS staining. Furthermore, conditioned medium from each group of cells was collected, macrophages were treated for 24 hours, and the expression of macrophage M1 / M2 polarization-related genes was detected by qRT-PCR. The secretion levels of related cytokines in the culture medium were detected by ELISA.

[0079] II. Experimental Results

[0080] 1. Mechanical properties

[0081] Mechanical properties are a key factor in the design and application of support systems. The results are as follows: Figure 1 As shown, at 80% strain, the compressive modulus and compressive strength of the GelMA+5dECM scaffold are both higher than those of the GelMA+10dECM group; the results of the compressive stress-strain curves are also consistent with this. Figure 1 In addition, the tensile modulus and tensile strength of the GelMA+5dECM group were higher than those of the GelMA+10dECM group. Figure 1 In the DE group); the elongation at break of the GelMA+5dECM group was significantly higher than that of the GelMA+10dECM group ( Figure 1 (F is given in the text). Therefore, the GelMA+5dECM group has both good compressive and tensile stress and maintains a moderate elongation at break, making it suitable for the design and application of biological scaffolds.

[0082] 2. Biocompatibility

[0083] Two-photon microscopy results on days 1, 3, and 7 after 3D-printed bioscaffolds are as follows: Figure 2 As shown, the jawbone BMSCs in all four bioscaffolds exhibited a relatively uniform arrangement, demonstrating good cell distribution. With time, all four bioscaffolds showed some degree of cell proliferation in their jawbone BMSCs, and the higher the dECM concentration, the stronger the viability of the jawbone BMSCs. Therefore, on days 1, 3, and 7, more green fluorescent live cells and fewer red fluorescent dead cells were observed in the GelMA+5dECM+BMSCs bioscaffold.

[0084] 3. Osteogenic capacity testing

[0085] Based on the results of mechanical properties and biocompatibility, GelMA+5dECM+BMSCs was selected as the best scaffold. For subsequent functional validation, GelMA (cell-free control group), GelMA+BMSCs, and GelMA+5dECM+BMSCs scaffolds were selected for the study.

[0086] On day 30 of osteogenic induction culture, the in vitro osteogenic mineralization capacity of the three scaffolds was systematically evaluated using MicroCT technology. The corresponding culture procedure is as follows: Figure 3 As shown in A in the figure. Gross observation showed that the surface of the scaffold in the GelMA+5dECM+BMSCs group had a large number of white mineralized nodules deposited, and its osteogenic phenotype was significantly better than that of the GelMA+BMSCs group and the GelMA group alone; the GelMA scaffold group, due to the lack of cellular components, showed almost no obvious mineralization. Figure 3 B). Micro CT 3D reconstructed image ( Figure 3(C) Further visually demonstrates that the mineral deposition in the GelMA+5dECM+BMSCs group was significantly higher than the other two groups, while the cell-free GelMA scaffold showed almost no mineralization signal. Quantitative analysis results consistently showed that the GelMA+5dECM+BMSCs group was significantly superior to the GelMA+BMSCs group and the GelMA group in key parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular spacing (Tb.Sp). Figure 3 (D in the text). The above results fully demonstrate that the GelMA+5dECM+BMSCs composite scaffold exhibits significant osteogenic mineralization potential in vitro.

[0087] 4. Immune function testing

[0088] Using carbodiimide (EDC / NHS) crosslinking agent, stable covalent bonds are established between the biological scaffold material molecules and the surface of the cell culture plate. The upper chamber of the Transwell plate is chemically crosslinked to pretreat the biological scaffold in the upper chamber, thereby achieving firm adhesion and preventing it from floating in the culture medium.

[0089] RT-PCR results as follows Figure 4 As shown in Figure B, compared with the LPS+GelMA control group and the LPS+(GelMA+BMSCs) scaffold group, the expression of M1 macrophage-related genes (iNOS and IL-1β) was significantly decreased in the LPS+(GelMA+5dECM+BMSCs) scaffold group, while the expression of M2 macrophage-related genes (IL-1ra and IL-10) was significantly increased. Among these, the LPS+GelMA group showed the most significant pro-inflammatory state and poor immune repair. Immunofluorescence staining further confirmed that in the LPS+(GelMA+5dECM+BMSCs) group, the red fluorescence signal of M1 markers (iNOS and CD86) was significantly weakened, while the green fluorescence signal of M2 markers (CD206 and CD163) was significantly enhanced, consistent with the aforementioned gene expression trends. Figure 4 In the C group, the LPS+GelMA group still exhibited the strongest M1 inflammatory phenotype and a weaker M2 repair phenotype. These results indicate that the GelMA+5dECM+BMSCs bioscaffold can significantly promote macrophage polarization from the M1 (pro-inflammatory phenotype) to the M2 (repair phenotype), demonstrating good immunomodulatory function and providing a new approach for the regulation and regenerative repair of the immune microenvironment in jawbone defects.

[0090] 5. In vivo experimental results

[0091] Figure 5Figure A shows a schematic diagram of the in vivo osteogenic culture process. Macroscopic observation of the biological scaffolds showed that the GelMA+5dECM+BMSCs group scaffold exhibited numerous osteogenic mineralization nodules, and its in vivo osteogenic capacity was significantly superior to that of the GelMA+BMSCs group. Figure 5 B). Micro CT 3D reconstructed image ( Figure 5 Results (C) showed that the osteogenic mineral deposition in the GelMA+5dECM+BMSCs group was significantly higher than that in the GelMA+BMSCs group. Quantitative analysis consistently showed that the GelMA+5dECM+BMSCs group was significantly superior to the GelMA+BMSCs group in key parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). Figure 5 (D in the middle).

[0092] Figure 5 The diagram in Figure E illustrates the in vivo immune regulation process. HE staining showed that, compared to the GelMA+5dECM+BMSCs group, the GelMA+5dECM+BMSCs group recruited significantly fewer inflammatory cells, indicating that it had superior immunomodulatory capabilities. Figure 5 The results of immunofluorescence staining showed that in the GelMA+5dECM+BMSCs group, the red fluorescence signal of M1 markers (iNOS and CD86) was significantly weakened, while the green fluorescence signal of M2 markers (CD206 and CD163) was significantly enhanced; indicating that the GelMA+5dECM+BMSCs bioscaffold can significantly recruit M2 repair macrophages, exhibiting excellent immunomodulatory function. Figure 5 (G in the middle). Figure 5 H represents the normalized quantification of immunofluorescence, showing a correlation with immunofluorescence. Figure 1 The resulting trend.

[0093] 6. Whole transcriptome analysis

[0094] Results of whole transcriptome analysis as follows Figure 6As shown, there are significant differences in gene expression in jawbone BMSCs between the two groups of bio-scaffolds. Compared with the GelMA+BMSCs bio-scaffold, the GelMA+5dECM+BMSCs bio-scaffold showed significant upregulation of 1422 genes and significant downregulation of 1314 genes (q < 0.05 and |log2 FC| > 1). Heatmap analysis of differentially expressed genes (DEGs) showed that ITGB7 was the most upregulated gene in the GelMA+5dECM+BMSCs bio-scaffold compared with the GelMA+BMSCs bio-scaffold. Therefore, we speculate that dECM may enhance the osteogenic differentiation capacity of jawbone BMSCs by promoting the expression of ITGB7, and indirectly induce macrophages to M2 repair polarization.

[0095] 7. Investigation into the mechanism of action

[0096] The expression of ITGB7 in jawbone bone mesenchymal stem cells (BMSCs) was knocked down using siRNA interference technology, followed by intervention with dECM. RT-PCR results showed that after ITGB7 knockdown, the expression of osteogenic-related genes COL1A1, RUNX2, and OPG was significantly downregulated; however, the downregulation of these genes could not be reversed after co-intervention with dECM and si-ITGB7. Figure 7 Similarly, ALP and ARS experimental results showed that ITGB7 knockdown led to a significant reduction in alkaline phosphatase activity-positive staining and calcium nodule deposition, and the combined treatment of dECM and si-ITGB7 failed to reverse this phenomenon. Figure 7 (BC in the jawbone). The above results indicate that dECM positively regulates osteogenic differentiation capacity by promoting the expression of ITGB7 in jawbone BMSCs.

[0097] In addition, conditioned medium was collected to intervene in macrophages. RT-PCR analysis showed that after ITGB7 knockdown, the expression of M1-related genes (iNOS and IL-1β) in macrophages increased significantly, while the expression of M2-related genes (IL-1ra and IL-10) decreased significantly; the above changes could not be reversed even after co-intervention with dECM and si-ITGB7. Figure 7 (D in the text). Quantitative detection of cytokines in conditioned medium by ELISA revealed that ITGB7 knockdown significantly increased the secretion of pro-inflammatory factors TNF-α and IL-6, while significantly decreasing the expression of anti-inflammatory factor IL-10. Furthermore, no significant reversal effect was observed in the dECM combined with si-ITGB7 group. Figure 7 These results indicate that dECM indirectly promotes macrophage polarization towards the M2 type by upregulating ITGB7 expression in jawbone BMSCs.

[0098] In summary, the results show that dECM in the bioscaffold enhances the osteogenic differentiation capacity of jawbone BMSCs by promoting ITGB7 expression and indirectly induces macrophages to M2 repair polarization, providing valuable insights for bone tissue engineering.

[0099] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A 3D printed jaw bone bioscaffold, characterized in that, The biological scaffold comprises: methacrylated gelatin, dental pulp tissue acellular extracellular matrix, and jaw bone marrow mesenchymal stem cells. Preferably, the concentration of the methacrylated gelatin is 10% (w / v). Preferably, the concentration of the dental pulp tissue acellular extracellular matrix is 5 mg / mL. Preferably, the concentration of the mandibular bone mesenchymal stem cells in the biological scaffold is 2 x 10 6 cells / mL.

2. The biological scaffold of claim 1, wherein, The degree of substitution of the methacryl group in the methacrylated gelatin is 60%.

3. The biological scaffold of claim 1, wherein, The dental pulp tissue acellular extracellular matrix is a freeze-dried powder.

4. A method for preparing a 3D-printed jaw bone bioscaffold, characterized by, The method comprises the following steps: S1, preparing a 3D printing jaw bone biological scaffold pre-gel solution; S2, 3D printing and photo-curing cross-linking shaping.

5. The preparation method according to claim 4, characterized in that, The pre-gel solution in step S1 is obtained by adding the dental pulp tissue acellular extracellular matrix and the jaw bone marrow mesenchymal stem cells to the methacrylated gelatin.

6. The production method according to claim 5, wherein The concentration of the methacrylated gelatin is 10% (w / v), and the degree of substitution of the methacryl group is 60%. Preferably, the dental pulp tissue acellular extracellular matrix is a freeze-dried powder, and the concentration in the pre-gel solution is 5 mg / mL. Preferably, the mandibular bone mesenchymal stem cells are suspended in the pre-gel solution at a concentration of 2 x 10 6 cells / mL.

7. The preparation method according to claim 4, characterized in that, The 3D printing in step S2 is performed by a Bio-Architect WS biological printer. Preferably, the nozzle temperature is 15℃. Preferably, the receiving plate temperature is 20℃. Preferably, the extrusion pressure is 0.15-0.2 Mpa. Preferably, the printing speed is 8 mm / s. Preferably, the 3D printing is layer-by-layer printing.

8. The preparation method according to claim 4, characterized in that, The photo-curing cross-linking in step S2 is achieved by irradiation with 405 nm visible blue light. Preferably, the irradiation time is 60 seconds.

9. Use of the biological scaffold according to any one of claims 1 to 3 and / or of the biological scaffold obtained according to the production process according to any one of claims 4 to 8, characterized in that, The application comprises any one of the following: 1) in the preparation of an implant for repairing a jaw bone defect; 2) in the preparation of an auxiliary implant material for treating a jaw bone fracture; 3) in the construction of a jaw bone tissue engineering model; 4) in the preparation of a biological material for tissue engineered jaw bone transplantation; 5) in drug screening and evaluation.

10. Use according to claim 9, characterized in that, The drug screening and evaluation is to apply a test drug to the jaw bone tissue engineering model constructed by the biological scaffold, observe the effect of the drug on cell growth, differentiation and bone tissue formation on the scaffold, and evaluate the efficacy and toxicity of the drug on the jaw bone tissue.

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