Piezoelectric composite biomaterial, manufacturing method therefor, and use thereof

Abstract

Disclosed in the present application are a piezoelectric composite biomaterial, a manufacturing method therefor, and use thereof. The manufacturing method comprises: forming a polydimethylphenylamide (PDA) coating on the surfaces of a plurality of nano barium titanate (BT) particles to obtain a plurality of BT / PDA particles; distributing the plurality of BT / PDA particles to the surfaces of a plurality of beta tricalcium phosphate (beta-TCP) particles to form bioactive composite BTP particles; and hot-melt mixing the composite BTP particles with a polymer matrix to form a BTP polymer composite biomaterial. The present application enables the composite material to possess bioactivity and piezoelectricity, overcoming the problem of barium titanate agglomeration.

Description

A piezoelectric composite biomaterial, its fabrication method and its application

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202411571300.5, filed on November 5, 2024, the entire contents of which are hereby incorporated by reference.

[0003] References to sequence lists

[0004] This application contains a sequence list that has been filed electronically in ST.26 (xml) format and is hereby incorporated by reference in its entirety. This ST.26 copy was created on November 4, 2025, and is named “H0660000001PCTCN.xml”, with a size of 19KB. Technical Field

[0005] This invention relates to the field of piezoelectric biomaterials technology, and in particular to a piezoelectric composite biomaterial, its fabrication method, and its application. Background Technology

[0006] Bone is a piezoelectric biomaterial. It responds to mechanical stimulation and generates electrical charges that signal cells and growth factors to aid in bone regeneration. Traumatic fractures caused by accidents and falls in older adults can result in large-area bone defects. These defects typically require a long recovery period, and nonunion, delayed healing, or failure to heal have been reported. In the past, limited bone defects could be treated with autologous cancellous bone grafts. However, when the length of the bone defect reaches 1.5 times the diameter of the bone axis, it exceeds the critical threshold for autologous repair, often leading to bone resorption and nonunion. Many synthetic grafts (including 3D-printed scaffolds used to repair and / or replace broken bones) do not possess the piezoelectric properties of natural bone. Among various piezoelectric materials, barium titanate (BaTiO3) (BT) has been extensively studied due to its excellent piezoelectric properties. Compared to other piezoelectric materials, barium titanate can generate a stronger electric field, thus stimulating cells and tissues more effectively. However, the inherent high density and agglomeration tendency of barium titanate powder, especially in the nanoscale range, result in poor dispersibility and prevent the formation of biomimetic piezoelectric biomaterials. Summary of the Invention

[0007] Therefore, one aspect of this application proposes a method for fabricating a piezoelectric composite biomaterial, comprising: forming a polydimethylbenzamide (PDA) coating on the surface of multiple barium titanate (BT) nanoparticles to obtain multiple BT / PDA particles; distributing the multiple BT / PDA particles onto the surface of multiple β-tricalcium phosphate (β-TCP) particles to form bioactive composite BTP particles; and thermally melting the composite BTP particles with a polymer matrix to form a BTP polymer composite biomaterial.

[0008] Another aspect of this application proposes a piezoelectric composite biomaterial comprising: multiple composite BTP particles, wherein each composite BTP particle includes multiple β-tricalcium phosphate β-TCP particles, and multiple BT / PDA particles are distributed on its surface, wherein the BT / PDA particles are formed by coating the surface of nano-barium titanate BT particles with a polydimethylbenzamide PDA coating.

[0009] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is used to manufacture bone tissue, including any one of oral and maxillofacial tissue, bone plate, and cartilage tissue.

[0010] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is used to manufacture wires and / or sutures.

[0011] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is fabricated into a sheet.

[0012] Other example embodiments will be described below.

[0013] This application offers numerous advantages. In some embodiments, the provided piezoelectric composite biomaterial exhibits excellent dispersion of barium titanate nanoscale piezoelectric particles on β-tricalcium phosphate β-TCP particles and is uniformly dispersed within PCL, and can be 3D printed to form layered microstructures. The piezoelectric effect activated by an ultrasonic actuator facilitates bone regeneration in a rat skull model. In some embodiments, the provided composite material possesses both bioactivity and piezoelectricity, overcoming the aggregation problem of barium titanate. Attached Figure Description

[0014] The performance and advantages of the invention can be further understood by referring to the remainder of this specification and the accompanying drawings, in which the same reference numerals are used for the same component. In some cases, a sub-label is placed after a label followed by a hyphen to indicate one of many similar components. When a label is mentioned without specifically naming an existing sub-label, it refers to all of these similar components.

[0015] Figure 1 is a schematic diagram of biocompatibility testing of L929 cells in a culture dish using ultrasound stimulation.

[0016] Figure 2 shows a schematic diagram of transmission electron microscopy (TEM) of nanoscale barium titanate BT coated with PDA.

[0017] Figure 3 is a schematic diagram of the BT / PDA / β-TCP particle distribution.

[0018] Figure 4 shows a schematic diagram of a 10% BTP / PCL 0-90 degree scaffold printed with 3 layers and an EDS scan.

[0019] Figure 5a shows the CLSM plot of L929 on PCL, 5% BTP / PCL, and 10% BTP / PCL.

[0020] Figure 5b shows fluorescence microscopy images of L929 biocompatibility tests. Representative fluorescently labeled images of EdU (yellow) and cell nuclei (blue) confirm that cell proliferation was significantly enhanced after the addition of BT and TCP.

[0021] Figure 5c shows the EDU ratios for PCL, 5% BTP / PCL, and 10% BTP / PCL.

[0022] Figure 5d shows the flow cytometry results on day 1, confirming the biocompatibility of the piezoelectric BTP / PCL scaffold.

[0023] Figure 6a is a schematic diagram of the immunofluorescence results, showing that the macrophage supernatant significantly enhances the expression of the MC3T3 cell surface marker BMP2.

[0024] Figure 6b is a schematic diagram of the immunofluorescence results, showing that the macrophage supernatant significantly enhances the expression of the RUNX2 marker on the surface of MC3T3 cells.

[0025] Figure 7 is a schematic diagram of the immunofluorescence results, showing that the macrophage supernatant significantly enhanced the expression of VEGF, a marker on the surface of HUVE cells.

[0026] Figure 8a shows the relationship between BMP2 concentration and BTP amount.

[0027] Figure 8b shows the relationship between VEGF concentration and BTP level.

[0028] Figure 9a shows the Micro-CT results of rat skull resection at 4 and 8 weeks post-surgery.

[0029] Figure 9b is a schematic diagram of BV / TV in week 4.

[0030] Figure 9c is a schematic diagram of BV / TV in week 8.

[0031] Figures 10A-10C show schematic transmission electron microscopy (TEM) images of nanoscale barium titanate BT coated with PDA; including HR-TEM images of BT / PDA / TCP particles.

[0032] Figures 10D-10E show the size distribution (C) and zeta potential (D) of BTO, BTO / PDA, and BTO / PDA / TCP NPS.

[0033] Figure 10F-10K shows the SEM images and EDS element mappings of BT / PDA / TCP particles.

[0034] Figure 10L shows the FTIR plots of BTO, BTO / PDA, and BTO / PDA / TCP NPs.

[0035] Figure 10M shows the XRD pattern of NPs.

[0036] Figure 10N shows the content of inorganic components in various scaffolds quantified by thermogravimetric analysis.

[0037] Figure 10O-10P shows the piezoelectric properties.

[0038] Figure 10Q is a schematic diagram of the water contact angle.

[0039] Figure 10R shows the SEM image and EDS element mapping diagram of the piezoelectric support.

[0040] Figure 11A shows live / dead cell stained CLSM images of L929 cells cultured on various scaffolds on day 1 and day 3, scale bar = 200 μm.

[0041] Figures 11B-11C show the proliferation of L929 fibroblasts cultured with a scaffold using the CCK-8 assay.

[0042] Figure 11D shows the apoptosis of L929 cells cultured on different scaffolds detected by flow cytometry.

[0043] Figure 11E shows CLSM images of L929 EdU staining on different scaffolds. Scale bar = 200 μm.

[0044] Figure 11F shows CLSM images of L929 cells stained with cytoskeletal ghost cyclic peptide-DAPI on different scaffolds. Scale bar = 50 μm.

[0045] Figure 11G shows a representative photograph of the hemolytic activity assay from the prepared sample.

[0046] Figure 11H shows the quantitative analysis of hemolysis rate in the prepared samples. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 indicate significant differences compared to the PCL group.

[0047] Figure 12A shows a representative immunofluorescence image of the RAW 264.7 cytoskeleton on the scaffold. F-actin (red) is a fibronectin representing the cytoskeleton; F4 / 80 (green) is a cell surface glycoprotein and a marker of mature mouse macrophages; and DAPI (blue) represents the cell nucleus. Scale bar = 50 μm.

[0048] Figure 12B shows representative immunofluorescence images of iNOS (green) and CD206 (red) in RAW 264.7 cells on a scaffold. Scale bar = 50 μm.

[0049] Figure 12C shows a representative flow cytometry plot of cell surface markers in RAW 264.7 cells, including CD86 and CD206.

[0050] Figures 12D-12F show the relative mRNA expression levels of anti-inflammatory and pro-inflammatory genes in macrophages on piezoelectric scaffolds.

[0051] Figure 12G-12H shows the secretion of healing cytokines by macrophages stimulated by piezoelectric scaffolds. Data are expressed as mean ± standard deviation (n=3). *P<0.05 and **P<0.01 indicate statistical significance compared with the CON group.

[0052] Figure 13A is a schematic diagram of cells that promote angiogenesis.

[0053] Figures 13B-13C show representative microscopic images of HUVECs after co-culturing with different macrophage cultures for 12 hours. Scale bar = 250 μm.

[0054] Figure 13D shows a representative Transwell migration image of macrophages cultured in the lower chamber alone for 24 hours. Scale bar = 200 μm.

[0055] Figure 13E shows representative immunofluorescence images of VEGF (green) and cell nuclei (blue) in HUVECs after 7 days of co-culturing with different macrophage cultures. Scale bar = 50 μm.

[0056] Figure 13F shows the relative mRNA expression levels of angiogenesis-related genes (including VEGF and Ang-1) of HUVECs after 7 days of co-culturing with different macrophage cultures.

[0057] Figure 13G shows a representative image of angiogenesis in the CAM experiment with stent in presence.

[0058] Figure 13H shows representative fluorescence images and quantitative analysis of HUVEC tubes after co-culturing cells with different macrophage culture media. Scale bar = 200 μm. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 indicate significant differences compared to the CON group.

[0059] Figure 14A is a schematic diagram of cells that promote osteogenic differentiation.

[0060] Figure 14B shows representative MC3T3-E1 cells incubated with different scaffold extracts for 7 days for ALP staining. Scale bar = 250 μm.

[0061] Figure 14C shows ARS staining of representative MC3T3-E1 cells after incubation with different scaffold extracts for 21 days. Scale bar = 250 μm.

[0062] Figure 14D shows the expression of osteogenic genes BMP2 and OPN. The expression of these osteogenic genes significantly increased under hydrogel piezoelectric stimulation.

[0063] Figures 14E and 14F are immunofluorescence images of BMP2 (green) and Runx2 (red), where the expression of these osteogenic-associated proteins significantly increased upon hydrogel piezoelectric stimulation. Scale bar = 50 μm.

[0064] Figure 14G is a schematic diagram of the mechanism by which the piezoelectric signal generated by the ultrasound-activated piezoelectric scaffold acts on VGCC and the intracellular signal transduction.

[0065] Figure 14H shows calcium ion imaging of MC3T3 cells after different treatments.

[0066] Figure 14I is a schematic diagram of the mechanism by which GsMTX4 acts on Piezo1 and Trpc1.

[0067] Figure 14J shows calcium ion imaging of MC3T3 cells using GsMTX4.

[0068] Figure 14K shows the BMP2 immunofluorescence imaging of MC3T3 cells using GsMTX4.

[0069] Figure 14L is a schematic diagram of the mechanism by which Hv1a acts on VGCC.

[0070] Figure 14M shows calcium ion imaging of MC3T3 cells using Hv1a.

[0071] Figure 14N shows Hv1a immunofluorescence imaging of MC3T3 cells with BMP2. *P<0.05 and **P<0.01 indicate statistical significance compared with the CON group.

[0072] Figure 15A is a schematic diagram of the establishment of a 5mm skull defect model in an SD rat.

[0073] Figure 15B shows the micro-CT coronal image of regenerated bone tissue, along with BV / TV and BMD results.

[0074] Figure 15C shows HE staining of the bone defect area 4–8 weeks post-surgery. Scale bar = 500 μm.

[0075] Figure 15D shows Masson's trichrome staining of the bone defect area at 4 and 8 weeks post-surgery. Scale bar = 500 μm.

[0076] Figure 15E shows representative Runx2 immunofluorescence staining images of the defect area at 4 and 8 weeks post-surgery. Scale bar = 100 μm.

[0077] Figure 15F shows representative BMP2 immunofluorescence staining images of the defect area at 4 and 8 weeks post-surgery. Scale bar = 100 μm. *P<0.05 and **P<0.01 indicate statistical significance compared to the CON group.

[0078] Figure 16A shows representative images of iNOS and CD206 immunofluorescence staining in the bone defect area 4 weeks after scaffold implantation. Scale bar = 100 μm.

[0079] Figure 16B shows representative images of CD31 immunofluorescence staining in the bone defect area 4 and 8 weeks after stent implantation. Scale bar = 100 μm.

[0080] Figure 16C shows representative VEGF immunofluorescence staining images of the bone defect area 4 and 8 weeks after stent implantation. Scale bar = 100 μm. *P<0.05 and **P<0.01 indicate statistical significance compared with the CON group.

[0081] Figure 17A shows the DEG quantitative analysis of macrophages in the CON group and the 10% PBT group.

[0082] Figure 17B is a DEG heatmap.

[0083] Figure 17C is a diagram of KEGG pathway analysis in macrophages of the CON group and the 10% PBT group.

[0084] Figure 17D is a GO enrichment analysis plot of the 20 most differentially upregulated and downregulated biological processes.

[0085] Figure 17E shows immunofluorescence images of P-p65 (green) and F-actin (red), scale bar = 50 μm.

[0086] Figure 17F shows representative immunofluorescence staining images of p-p65 in the defect area at 4 and 8 weeks post-implantation. Scale bar = 100 μm.

[0087] Figure 17G is a schematic diagram of the possible molecular mechanism by which piezoelectric scaffolds promote bone repair by modulating macrophage M2 polarization and inhibiting the NF-κB axis.

[0088] Figure S1A shows the compressive stress-strain curves for various stents. Conditions: 1 mm / min, n = 5 samples / group. Data: mean ± standard deviation; *p < 0.01 vs. PCL (ANOVA).

[0089] Figure S1B shows the external degradation curve of the piezoelectric support.

[0090] Figures S2A-S2B are SEM images of each scaffold.

[0091] Figure S3 shows the piezoelectric constants of different supports.

[0092] Figure S4A shows the electromechanical response of the PBT piezoelectric support before and after ultrasonic simulation.

[0093] Figure S4B shows the polarization-electric field (PE) hysteresis loop of the piezoelectric support.

[0094] Figure S5A shows a real-time temperature monitoring image during ultrasound stimulation.

[0095] Figure S5B shows calcium ion imaging of osteoblasts treated with different ultrasound frequencies.

[0096] Figure S5C is a CLSM image showing the live / dead staining of osteoblasts on days 1 and 3 after treatment with different ultrasound intensities. Scale bar = 200 μm.

[0097] Figure S5D shows the osteoblast proliferation after treatment with different ultrasound intensities using the CCK-8 assay. (n=3). *p<0.05, **p<0.01, ***p<0.001.

[0098] Figure S6A is a representative image of primary osteoblasts in mice.

[0099] Figure S6B shows confocal laser scanning microscopy (CLSM) images of the staining of live / dead cells in osteoblasts cultured on different scaffolds on days 1, 3, and 5. Scale bar: Scale bar = 200 μm.

[0100] Figure S6C shows the proliferation of osteoblasts cultured on a scaffold, detected using the CCK-8 assay.

[0101] Figure S7A shows a representative photograph of the coagulation test of the prepared samples. Figures S7B-S7C show the quantitative analysis of the blood coagulation index (BCI) of the prepared samples.

[0102] Figure S8A shows the secretion of BMP-2 and VEGF by macrophages after stimulation with a piezoelectric scaffold. Treatment with bevacizumab (0.2 μg mL⁻¹), a humanized monoclonal antibody targeting VEGF-A, downregulated VEGF secretion levels in the macrophage supernatant. The specific BMP inhibitor LDN193189 (1 μM) significantly inhibited BMP2 secretion levels in the macrophage supernatant.

[0103] Figure S8B is an immunofluorescence image of VEGF. Scale bar = 50 μm.

[0104] Figure S8C is an immunofluorescence image of BMP2. Scale bar = 50 μm.

[0105] Figure S8D is an immunofluorescence image of RUNX2. Scale bar = 50 μm.

[0106] Figure S9 shows the angiogenesis of different stents detected using the CAM experiment. The figure illustrates representative images of angiogenesis in the CAM experiment with stents in place. Quantitative indicators include percentage of vessel area, total vessel length, branching index, and number of connection points. (n=3)*P<0.05 and **P<0.01 indicate significant differences compared to the CON group.

[0107] Figure S10A shows a representative image of primary mouse osteoblasts. Figure S10B shows representative ALP staining results of primary osteoblasts, with cells co-cultured with different macrophage cultures for 7 days. Scale bar = 250 μm. Figures S10C-S10D are immunofluorescence images of BMP2 (green) and Runx2 (red). Scale bar = 50 μm.

[0108] Figure S11 shows the surgical procedure and representative digital images of a critical-sized bone and periosteal defect model, as well as the implantation of a piezoelectric stent.

[0109] Figure S12 is a sagittal view of the micro-CT analysis.

[0110] Figure S13 shows representative immunohistochemical staining images of COL-1 in the defect area at 4 and 8 weeks post-implantation.

[0111] Figure S14 is an immunofluorescence image of P-IKKα / β (red). Scale bar = 50 μm.

[0112] Numbered Examples

[0113] Example 1: A method for fabricating a piezoelectric composite biomaterial, comprising:

[0114] Multiple BT / PDA particles are obtained by forming a poly(xylene)-coating on the surface of multiple barium titanate (BT) nanoparticles.

[0115] The plurality of BT / PDA particles are distributed onto the surface of a plurality of β-tricalcium phosphate β-TCP particles to form bioactive composite BTP particles;

[0116] The composite BTP particles are thermally melt-mixed with a polymer matrix to form a BTP polymer composite biomaterial.

[0117] Example 2: The manufacturing method according to Example 1 is characterized in that the mass ratio of BT, PDA and β-TCP is (1-30):(0.05-2):(0.1-2).

[0118] Example 3: The manufacturing method according to Example 1 is characterized in that the mass ratio of BT, PDA and β-TCP is 6:0.1:0.64.

[0119] Example 4: The manufacturing method according to Example 1, characterized in that: the polymer is polycaprolactone (PCL) or a bioabsorbable polymer.

[0120] Example 5: The manufacturing method according to Example 3, characterized in that: the step of forming a poly(xyleneamide) PDA coating on the surface of BT particles includes:

[0121] PDA was added to deionized water to obtain a 2.0 g / L PDA solution;

[0122] Adjust the pH of the PDA solution to 8.5;

[0123] BT particles were added to the PDA solution and stirred for 24 hours to obtain BT / PDA particles;

[0124] The BT / PDA particles were washed with distilled water and dried in a vacuum oven at 45°C.

[0125] Example 6: The manufacturing method according to Example 1 is characterized in that the step of distributing the plurality of BT / PDA particles onto the surface of β-TCP particles includes: adding β-TCP and BT / PDA particles to deionized water at 50°C, mechanically stirring to obtain composite BTP particles; washing the composite BTP particles with distilled water and drying them in a vacuum oven at 60°C.

[0126] Example 7: The manufacturing method according to Example 1 is characterized in that the thickness of the PDA coating on the surface of the BT particles is 5-40 nm.

[0127] Example 8: The manufacturing method according to Example 1 is characterized in that the average particle size of the BT particles is 60-100 nm.

[0128] Example 9: The manufacturing method according to Example 8 is characterized in that the mass of the composite BTP particles is 5-20% relative to the polymer matrix.

[0129] Example 10: The manufacturing method according to Example 4 is characterized in that the step of hot-melt mixing the composite BTP particles with polycaprolactone (PCL) includes:

[0130] PCL is placed in an open-type heated twin-roll mill with the heated rolls at 100°C. When the PCL melts and covers the twin-roll mill, BTP particles are mixed into the hot melt, and the composite melt is turned over for at least 30 minutes. After cooling to room temperature, a solvent-free PCL / BTP polymer composite biomaterial is obtained for melt extrusion 3D printing.

[0131] Example 11: A piezoelectric composite biomaterial, comprising:

[0132] Multiple composite BTP particles, wherein each composite BTP particle comprises:

[0133] Multiple β-tricalcium phosphate β-TCP particles have multiple BT / PDA particles distributed on their surface. The BT / PDA particles are formed by coating the surface of nano-barium titanate BT particles with a polydimethylbenzamide PDA coating.

[0134] Example 12: The piezoelectric composite biomaterial according to Example 11 further includes a polymer matrix, wherein multiple composite BTP particles are thermally melt-mixed with the polymer matrix to form a BTP polymer composite biomaterial.

[0135] Example 13: An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods described in Examples 1-10 or any of the piezoelectric composite biomaterials described in Examples 10-12 is used to manufacture bone tissue, including any one of oral and maxillofacial tissue, bone plate, and cartilage tissue.

[0136] Example 14: An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods described in Examples 1-10 or any of the piezoelectric composite biomaterials described in Examples 10-12 is used to manufacture wires and / or sutures.

[0137] Example 15: An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods described in Examples 1-10 or any of the piezoelectric composite biomaterials described in Examples 10-12 is made into a sheet. Detailed Implementation

[0138] As used herein and in the claims, the word "comprising" means including the following elements, but does not exclude other elements. "Comprising," "containing," or "having" means including the following elements, but does not exclude other elements. It should be understood that for each embodiment using the terms "comprising" or "including," this disclosure / application also includes alternative embodiments in which the terms "comprising," "including," "containing," or "having" are replaced with "consisting of" or "substantially consisting of." These alternative embodiments using "consisting of" or "substantially consisting of" are to be understood as narrower embodiments of the "comprising," "including," "containing," or "having" embodiments.

[0139] For example, alternative embodiments of "a composition comprising A, B, and C" are "a composition consisting of A, B, and C" and "a composition consisting essentially of A, B, and C". This disclosure / application includes these embodiments even if the latter two are not explicitly stated. Furthermore, it should be understood that the scope of the three embodiments listed above is different.

[0140] As used herein and in the claims, unless the context clearly indicates otherwise, the singular forms “an / a”, “an / a”, and “the” also include the corresponding plural indicators. When a numerical range is mentioned in the specification, the numerical range is understood to include every discrete point within that range. For example, 1-7 represents 1, 2, 3, 4, 5, 6, and 7.

[0141] As used herein, the terms “about,” “substantially,” and “approximately” are understood to mean within the normal tolerance range of the field and not exceeding ±10% of the specified value. By way of example only, approximately 50 refers to all values ​​from 45 to 55, inclusive. As used herein, the phrase “about” also includes specific values; for example, approximately 50 includes 50.

[0142] For clarity, "characterized in" does not limit or change the nature of whether the following list of terms is open or closed. For example, in a claim for "an apparatus comprising A, B, C and characterized in that D, E, and F", elements D, E, and F remain open-ended terms, and the claim is intended to include other elements due to the use of the word "comprising" preceding the claim.

[0143] In this application:

[0144] BT and BTO are abbreviations for barium titanate (BaTiO3).

[0145] PDA is C8H 11 NO2 is an abbreviation for polydopamine.

[0146] TCP and β-TCP are abbreviations for β-tricalcium phosphate.

[0147] PCL is an abbreviation for polycaprolactone;

[0148] BTP is an abbreviation for BT / PDA / β-TCP.

[0149] The challenge in producing bioactive and 3D-printable piezoelectric PCL polymer matrices lies in the dispersibility of barium titanate (BT) and the formulation of the correct composition. This is extremely difficult and challenging. This problem is primarily attributed to BT, which has an inherent tendency to aggregate in the high-density and nanoscale range. This can lead to uneven distribution within the polymer matrix, hindering its ability to be processed by conventional polymer processing methods such as injection molding and 3D printing. This results in unpredictable and poor chemical, physical, and electrical properties. Recently, Osteopore has produced and commercialized 3D-printed PCL scaffolds. However, these 3D-printed scaffolds do not possess piezoelectric properties.

[0150] Therefore, this application proposes the following technical solution:

[0151] One aspect of this application discloses a method for fabricating a piezoelectric composite biomaterial, comprising: forming a poly(dimethylbenzamide) PDA coating on the surface of multiple nano-barium titanate (BT) particles to obtain multiple BT / PDA particles; distributing the multiple BT / PDA particles onto the surface of multiple β-tricalcium phosphate (β-TCP) particles to form bioactive composite BTP particles; and thermally melting the composite BTP particles with a polymer matrix to form a BTP polymer composite biomaterial. The use of PDA-coated BT (BT\PDA) dispersed on the β-TCP surface endows the composite material with bioactivity and piezoelectricity, overcoming the agglomeration problem of barium titanate and improving the dispersibility of nano-sized BT. The high-shear thermal melting method enables the composite BTP particles to be uniformly dispersed in the polymer matrix, making it suitable for use in 3D printing to form biomimetic tissues with interconnected pores and layered microstructures. In at least one embodiment, the mass ratio of BT, PDA, and β-TCP is (1-30):(0.05-2):(0.1-2), and the remaining component is deionized water that will be evaporated and removed.

[0152] In at least one embodiment, the quality ratio of BT, PDA, and β-TCP is 6:0.1:0.64. In at least one embodiment, the quality ratio of BT, PDA, and β-TCP is 10:0.2:0.8. In at least one embodiment, the quality ratio of BT, PDA, and β-TCP is 15:0.5:1. In at least one embodiment, the quality ratio of BT, PDA, and β-TCP is 20:0.6:1.2. In at least one embodiment, the quality ratio of BT, PDA, and β-TCP is 25:1:1.5.

[0153] In at least one embodiment, the polymer is polycaprolactone (PCL) or a bioabsorbable polymer. PCL has been approved by multiple regulatory agencies, including the U.S. FDA, for use in medical devices.

[0154] In at least one embodiment, the step of forming a poly(xylene)-xyleneamide (PDA) coating on the surface of BT particles includes: adding PDA to deionized water to obtain a PDA solution of 2.0 g / L; adjusting the pH of the PDA solution to 8.5; adding BT particles to the PDA solution and stirring for 24 hours to obtain BT / PDA particles; washing the BT / PDA particles with distilled water and drying them in a vacuum oven at 45°C.

[0155] In at least one embodiment, the step of distributing multiple BT / PDA particles onto the surface of β-TCP particles includes: adding β-TCP and BT / PDA particles to deionized water at 50°C, mechanically stirring to obtain composite BTP particles; washing the composite BTP particles with distilled water and drying them in a vacuum oven at 60°C.

[0156] In at least one embodiment, the thickness of the PDA coating on the BT particle surface is 5-40 nm. In at least one embodiment, the thickness of the PDA coating on the BT particle surface is 25 nm. In at least one embodiment, the thickness of the PDA coating on the BT particle surface is 15 nm. In at least one embodiment, the thickness of the PDA coating on the BT particle surface is 10 nm. In at least one embodiment, the thickness of the PDA coating on the BT particle surface is 30 nm.

[0157] In at least one embodiment, the average particle size of BT particles is 60-100 nm.

[0158] In at least one embodiment, the mass of the composite BTP particles is 5-20% relative to the polymer matrix. In at least one embodiment, the mass of the composite BTP particles is 5% relative to the polymer matrix. In at least one embodiment, the mass of the composite BTP particles is 10% relative to the polymer matrix. In at least one embodiment, the mass of the composite BTP particles is 15% relative to the polymer matrix. In at least one embodiment, the mass of the composite BTP particles is 20% relative to the polymer matrix.

[0159] In at least one embodiment, the step of hot-melt mixing composite BTP particles with polycaprolactone (PCL) includes: placing PCL in an open-type heated twin-roll mill with the temperature of the heated rolls at 100°C; when the PCL melts and covers the twin-roll mill, mixing BTP particles into the hot melt; turning the composite melt over for at least 30 minutes; and cooling to room temperature to obtain a solvent-free PCL / BTP polymer composite biomaterial for melt extrusion 3D printing.

[0160] Another aspect of this application proposes a piezoelectric composite biomaterial comprising: multiple composite BTP particles, wherein each composite BTP particle includes multiple β-tricalcium phosphate β-TCP particles, and multiple BT / PDA particles are distributed on its surface. The BT / PDA particles are formed by coating the surface of nano-barium titanate BT particles with a poly(xylene)phthalamide (PDA) coating. In at least one embodiment, the mass ratio of BT, PDA, and β-TCP is (1-30):(0.05-2):(0.1-2), and the remaining component is deionized water that will be evaporated and removed.

[0161] In at least one embodiment, a polymer matrix is ​​further included, wherein a plurality of composite BTP particles are thermally melt-mixed with the polymer matrix to form a BTP polymer composite biomaterial.

[0162] In at least one embodiment, it is obtained by any of the above-described manufacturing methods.

[0163] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is used to manufacture bone tissue, including any one of oral and maxillofacial tissue, bone plate, and cartilage tissue.

[0164] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is used to manufacture wires and / or sutures.

[0165] Another aspect of this application proposes an application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the above-described manufacturing methods or any of the above-described piezoelectric composite biomaterials is fabricated into a sheet.

[0166] Other benefits and advantages offered by the various embodiments of the present invention will be readily apparent from the following description.

[0167] A piezoelectric composite biomaterial is fabricated using a method described below.

[0168] First, a poly(xylene-2-methyl-2-ethyl)-2-methyl phthalate (PDA) coating was formed on the surface of multiple barium titanate (BT) nanoparticles to obtain multiple BT / PDA particles, thus achieving PDA surface modification of the BT particles. Specifically, the process involved adding 0.1 g of polydopamine (PDA) to deionized water to obtain a 2.0 g / L solution, thereby overcoming agglomeration problems and improving the dispersibility of nano-sized BT (average approximately 60-100 nm). Then, 0.06 g of Tris was added until the pH of the solution reached 8.5. Next, 6 g of BT particles were added to the aforementioned polydopamine solution and mechanically stirred for 24 hours. The PDA-modified BT / PDA particles were then washed with distilled water and dried in a vacuum oven at 45°C.

[0169] Next, BT / PDA / β-TCP (BTP) particles were prepared by distributing multiple BT / PDA particles onto the surface of multiple β-tricalcium phosphate (β-TCP) particles to form bioactive composite BTP particles. The specific process was as follows: 0.64 g of β-TCP and 1 g of BT / PDA particles were added to 800 mL of deionized water and mechanically stirred at 50 °C for approximately 8 hours to obtain BT / PDA / β-TCP (BTP) particles. These particles, referred to as BTP, were washed with distilled water and then dried in a vacuum oven at 60 °C.

[0170] Next, the composite BTP particles are hot-melt blended with a polymer: PCL is selected as the polymer, and the PCL and composite BTP particles are mixed in an open-type heated twin-roll mill to obtain a solvent-free PCL / BTP blend. In one embodiment, 5% BTP / PCL is prepared using 5% by weight of composite BTP particles. In another embodiment, 10% BTP / PCL is prepared using 10% by weight of composite BTP particles. The temperature of the heated rollers is set to 100°C, and the roller spacing is set to 2-4 mm. While the PCL melts and covers the twin-roll mill, a small amount of composite BTP particles is mixed into the hot melt, and the composite melt is frequently turned over for about 30 minutes. Frequent turning of the hot melt ensures good mixing. The composite melt is cut into small pieces (10x10 mm square), cooled to room temperature, and stored in a drying oven until they are used for melt extrusion 3D printing.

[0171] The following examples demonstrate the fabrication of 0-90 degree PCL / BTP scaffolds using 3D printing and the verification of the biocompatibility of the fabricated scaffolds.

[0172] Example 1

[0173] PCL / BTP granules were fed into the melt feeder using a 3D near-field micro-extruder (MPB02, Foshan, China). The printing temperature was set to 140℃ and the voltage to 3.6KV. 3D printing was performed by extruding the blend into filaments, building the original scaffold layer by layer. A 0-90 degree pattern was used to create the scaffold. The extrusion pressure was set to 30 bar, and the printing speed was maintained at 40 mm / s. The gap between adjacent layers in the z-direction was approximately 0.1 mm.

[0174] The following are the methods and equipment used for biocompatibility testing.

[0175] This study used L929 fibroblasts (purchased from Prenoxel) for biocompatibility evaluation according to ISO 10993 and GB / T 16886.5 recommendations. The biocompatibility of the scaffold was evaluated using L929 cells in culture dishes, as shown in Figure 1. Ultrasonic stimulation was applied to the bottom of the culture dishes three times daily for 5 minutes each time. L929 cells were cultured in DMEM containing 10% FBS and 1% P / S solution in an incubator at 37°C and 5% CO2. L929 cells were then cultured at a rate of 3 × 10⁻⁶ cells / mL. 3 Cells / scaffolds were seeded at different densities on different scaffolds, with 10 μl of CCK-8 (Abbkine, China) solution added to each well. The absorbance of the solution at 450 nm was recorded using a Thermo Field microplate reader. On days 1 and 3, cells on the culture plates were stained with a live and dead cell staining kit (Abbkine, China). Fluorescence images were captured using a confocal laser scanning microscope (CLSM, Opera Phenix Plus, USA) to assess their live / dead status (green fluorescence: live; red fluorescence: dead). To assess apoptosis in the piezoelectric scaffolds, L929 cells were harvested and stained with Annexin V and propyl iodine (PI) (Abbkine, China). Finally, the percentage of live cells in different groups was measured by flow cytometry (FACSCelesta, Becton, Dickinson, USA). Cells were counted at 3 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of L929 cells and cultured for 3 days, followed by staining of the cell nuclei and F-actin. The method was as follows: cells on the scaffold were first fixed with 4% paraformaldehyde solution (Solarbio, China) for 30 min, washed with PBS, infiltrated with 0.1% Triton X-100 for 15 min, blocked with 1% BSA solution for 1 h, stained with FITC-labeled Sigma-Aldrich F-actin at room temperature for 1 h, and then stained with DAPI to stain the cell nuclei. The cytoskeleton was observed using CLSM.

[0176] Scanning electron microscope (SEM)

[0177] The morphology of the piezoelectric scaffold was characterized using emission scanning electron microscopy (FE-SEM; Zeiss, Sigma, Germany). The distribution of Ca, Ba, and Ti elements in the scaffold was scanned using an energy dispersive spectrometer (UltimMax 40, Oxford, UK).

[0178] Transmission electron microscopy (TEM)

[0179] The morphology of BT, BT / PDA, and BT / PDA / TCP was characterized by transmission electron microscopy (TEM, Themis Z(3.2)). For TEM samples, BT, BT / PDA, and BT / PDA / TCP nanopowders were dissolved in anhydrous ethanol (5 mg / mL) for 10 min and then added dropwise to a copper grid (accelerating voltage: 300 kV). The distribution of Ba, Ti, Ca, and C was analyzed by X-ray diffraction (EDS).

[0180] MicroCT Experiment

[0181] All microCT experiments were conducted on a microCT system (NEMO, NMC-200, China). This microCT system utilizes a high-resolution large-matrix flat panel detector and a microfocus X-ray machine, featuring three-dimensional reconstruction technology. It can achieve in vivo structural imaging of small rodents (mice or rats) and non-destructive three-dimensional detection of ex vivo animal tissues and biological materials in a short time. The following settings were used: voltage, 80 kV; current, 0.06 mA; scanning accuracy, 35 μm.

[0182] Ultrasonic stimulation

[0183] Ultrasonic stimulation was performed using a handheld digital ultrasound therapy machine (WED-101China). In vitro experiments (cell culture) were conducted at 1 MHz, three times daily, 2.5 W / cm², for 5 minutes each time. In vivo experiments (rat) were performed with the ultrasound device set to 1 MHz, 2.5 W / cm², stimulating the animal at the defect site for 5 minutes every other day until sampling.

[0184] Osteogenic differentiation research

[0185] In the osteogenic differentiation study, mouse MC3T3-E1 cells were provided by Procell. MC3T3-E1 cells were cultured in α-MEM containing 10% FBS and 1% P / S solution at an incubator temperature of 37°C and a CO2 concentration of 5%. The culture medium was changed daily. MC3T3-E1 cells (1.5 × 10⁻⁶) were cultured in α-MEM containing 10% FBS and 1% P / S solution at a CO₂ concentration of 5%. 4Cells / well were co-cultured with conditioned macrophage medium and complete medium (1:1, consisting of 10 mmol / L β-glycerophosphate disodium, 50 μg / ml ascorbic acid, and 10 nmol / L dexamethasone). After 7 days of co-culture, the cells were fixed and incubated with 5% goat serum. Samples were then incubated with primary antibodies against RUNt-associated transcription factor 2 (Runx2, 1:100; 20700-1AP, Proteintech) and bone morphogenetic protein 2 (BMP2, 1:200; 66383-1-lg, Proteintech), respectively, followed by incubation with fluorescently labeled secondary antibodies. Cells were then re-stained with DAPI and observed using CLSM (Opera Phenix Plus, USA).

[0186] In vitro angiogenesis evaluation

[0187] This study used mouse-derived human umbilical vein endothelial cells (HUVECs) provided by Procell. HUVEC cells were cultured in DMEM containing 10% FBS and 1% P / S solution at 37°C with a CO2 concentration of 5%. The culture medium was changed daily. HUVEC cells (1.5 × 10⁻⁶) 4 Cells / well were co-cultured in conditioned macrophage medium and complete medium (1:1) in 10% DMEM. After 7 days of co-culture, cells were fixed and incubated with 5% goat serum. Samples were then correlated with vascular endothelial growth factor (VEGF 1:100; 66828-1-lg, Proteintech). After incubation with fluorescently labeled secondary antibody, cells were re-stained with DAPI and observed using CLSM (Opera Phenix Plus, USA). Angiogenesis assays were performed using HUVECs induced to angiogenesis in different macrophage cultures as an example. Briefly, HUVEC cells were seeded in Matrigel (Matrigel, 356262, Corning, USA) and incubated with different macrophage supernatants. Tubular structures were then observed and quantitatively analyzed.

[0188] Assessment of macrophage function

[0189] The mouse-derived macrophage line RAW 264.7 in this study was provided by Procell. RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% P / S solution in an incubator at 37°C and 5% CO2. The medium was changed daily. Cells were seeded into each sample of a 6-well tissue culture plate at a density of 3 × 10⁶ cells / well. 5Cells / well. The secretion levels of bone morphogenesis factor BMP-2 and VEGF in macrophages were observed using enzyme-linked immunosorbent assay (ELISA). After co-culturing for 2 days, cells were subjected to daily ultrasound stimulation (WED-101, China) at a frequency of 1 MHz and 2.5 W / cm². 2 Each study lasted 5 minutes, and the supernatant from each group was collected for further research. The concentrations of BMP-2 and VEGF secreted by cells in the collected supernatant were measured using a commercially available ELISA kit (vascular endothelial growth factor VEGF, EM0205; bone morphogenetic protein 2 (BMP2), EM0022; Wuhan Fine Testing).

[0190] animal experiments

[0191] Animal experiments were approved by the Animal Protection and Use Committee of Hunan University (No.: HNU-IACUC-2024-114). An animal model of skull bone defect injury was established. Rats were first anesthetized using a Rayward gas anesthesia machine with isoflurane (Rayward R510-22) inhaled at a rate of 2 L / min. After disinfection, the skin was dissected, and two critical-sized full-thickness bone defects (5 mm in diameter) were created on both sides of the sagittal suture of the parietal bone in each rat using a small animal skull trephine cooled with physiological saline. A scaffold was implanted to fill the defects, and the wound was closed with 3-0 sutures. Whole skull fragments were harvested for evaluation at 4 and 8 weeks post-implantation. Twenty-four male rats (200–220 g) were randomly divided into a control group, a PCL group, a 5% PBT group, and a 10% PBT group. For three days post-surgery, ultrasound stimulation (WED-101 type) was administered every other day at a frequency of 1 MHz, 2.5 W / cm², for 5 minutes each time until sampling. At 4 and 8 weeks post-surgery, rats were euthanized with CO2, and the entire skull was scanned using a microCT system (NEMO, NMC-200, China) with a voltage of 80 kV, a current of 0.06 mA, and a scanning resolution of 35 μm. Bone volume / total tissue volume (BV / TV) and bone mineral density (BMD) were analyzed based on the reconstructed microCT images.

[0192] result

[0193] Regarding PDA surface modification of BT particles: This application uses PDA to modify the surface of barium titanate (BT) to facilitate the polydispersity of nano-sized BT. Figure 2 shows a transmission electron microscope (TEM) image of nano-sized barium titanate (BT) coated with polydiamine (PDA). The PDA coating is approximately 25 nm in size and improves the dispersibility of BT.

[0194] Regarding the BT / PDA / β-TCP (BTP) particles: TEM and EDS in Figure 3 show that BT is well distributed on the larger β-TCP particles. The TEM image in Figure 3 shows BT / PDA coated on tricalcium particles, forming a BT / PDA / TCP (BTP) piezoelectric composite material.

[0195] Regarding 3D printing of BTP / PCL scaffolds: The BTP / PCL composite material exhibits good rheological properties at 140°C, making it suitable for 3D printing. The printing speed is also reasonable. Figure 4 shows an example of a 10% BTP / PCL 0-90 degree scaffold successfully 3D printed in 3 layers. EDS scanning also shows good distribution of BTP within the PCL. The BTP / PCL filaments are uniform, and the layers bond well. Figure 4 shows an example of a 10% BTP / PCL 0-90 degree scaffold successfully 3D printed in 3 layers. EDS scanning also shows good distribution of BT within the PCL.

[0196] Regarding the biocompatibility assessment results: EdU (5-ethynyl-2-deoxyuridine) is based on an easy-to-perform chemical reaction that does not require DNA denaturation, is rapid and reproducible, and has an excellent signal-to-noise ratio. This technique provides extensive opportunities for analyzing cell proliferation, population homeostasis, and cell labeling procedures. Figure 5a shows CLSM images illustrating phalloidin-DAPI staining on the cytoskeleton of L929 cells cultured on different scaffolds on day 3. The scaffolds were microstructured in the 0–90 degree range. The adhesion of L929 cells to the piezoelectric scaffolds was investigated by phalloidin immunofluorescence staining. Ultrasonic stimulation was performed three times daily for 5 minutes each time. L929 cells in the pure PCL group were spindle-shaped. In contrast, cells in the 5% PBT and 10% PBT groups were polygonal, with filamentous pseudopodia, a more developed cytoskeleton and cell clusters, and stronger adhesion. Piezoelectric BT converts mechanical force into electrical signals through ultrasound, providing electrical stimulation to surrounding cells. The surface charge can be built into the in-situ generated potential, regulating cell behavior through signaling pathway-mediated biological processes. Figure 5b shows representative fluorescently labeled images of EdU (yellow) and cell nuclei (blue). EdU staining results of L929 cells co-cultured on different substrates for 24 hours confirmed that cell proliferation was significantly enhanced with the addition of BTP. Cells cultured on the piezoelectric scaffold exhibited a higher proliferation rate compared to the pure PCL group. Figure 5c shows EDU expression graphs for PCL, 5% BTP, and 10% BTP. EDU expression increased with increasing BTP content. 10% BTP showed the highest increase in EDU expression. Figure 5d shows apoptosis of L929 cells cultured on different samples detected by flow cytometry on day 1. Apoptosis in L929 cells cultured on the piezoelectric scaffold remained unchanged. These results confirm the biocompatibility of the piezoelectric BTP scaffold.

[0197] Results of the osteogenic differentiation study: In the osteogenic differentiation study, mouse MC3T3-E1 cells were used, with the culture medium changed daily. MC3T3-E1 cells were co-cultured with conditioned macrophage culture medium and complete culture medium. After 7 days of co-culture, the cells were fixed and incubated with 5% goat serum. The samples were then associated with RUNt-associated transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2). After incubation with fluorescently labeled secondary antibodies, the cells were re-stained with DAPI and observed using CLSM (Figures 6a and 6b). Figure 6a shows that the immunofluorescence results significantly enhanced the expression of the MC3T3 cell surface marker BMP2 in the macrophage supernatant; Figure 6b shows that the immunofluorescence results significantly enhanced the expression of the MC3T3 cell surface marker RUNX2 in the macrophage supernatant.

[0198] Regarding the results of in vitro angiogenesis assessment: Angiogenesis was induced in different macrophage cultures using HUVECs, and angiogenesis assays were performed to observe and quantitatively analyze tubular structures. Furthermore, immunofluorescence staining was performed after 7 days of culture, as shown in Figure 7. Figure 7 shows that the piezoelectric scaffold significantly enhanced the expression of the VEGF marker on the surface of HUVE cells.

[0199] Results of macrophage function assessment: This study used the mouse macrophage cell line RAW 264.7 and employed enzyme-linked immunosorbent assay (ELISA) to observe the secretion levels of bone morphogenesis factors (BMP-2) and VEGF in macrophages. After co-culturing for 2 days, macrophages were subjected to ultrasound stimulation at 1 MHz and 2.5 W / cm² three times daily for 5 minutes each time. Supernatants from each group were collected for further analysis. The concentrations of BMP-2 and VEGF secreted by cells in the collected supernatants were measured using a commercially available ELISA kit. (See Figures 8a and 8b) Figure 8a shows the relationship between BMP-2 concentration and BTP levels, indicating that BMP-2 concentration significantly increased under ultrasound stimulation; Figure 8b shows the relationship between VEGF concentration and BTP levels, indicating that VEGF concentration increased during ultrasound simulation.

[0200] Animal experiment on rat skull defects: Figure 9a shows the Micro-CT results at 4 and 8 weeks post-surgery. Figure 9b shows the relationship between BMP2 concentration and BTP amount. It can be seen that the concentration of BMP2 increases significantly under ultrasound stimulation; Micro-CT results show that the healing effect of the 10% PBT group is best at 4 and 8 weeks post-surgery. At 8 weeks, the 10% BTP / PCL showed that the defect was almost completely filled with bone. Compared with the control group, its bone volume was more than 3 times (see Figure 9b) and twice that of the PCL scaffold. These results demonstrate the effect of piezoelectricity on bone regeneration under ultrasound stimulation. Figure 9a shows the Micro-CT results of the rat skull experiment, showing a significant increase in bone formation on the BTP-containing scaffold, with the 10% BTP PCL scaffold almost completely filling the defect. Figure 9b shows the BV / TV at week 4, indicating that BV / TV increases with increasing BTP; Figure 9c shows the BV / TV at week 8, indicating that the BV / TV of 10% BTP is almost 3 times that of the control group.

[0201] in conclusion

[0202] A novel synthetic piezoelectric composite material (BTP) has been prepared and fabricated. This composite material is made of barium titanate nanoparticles and coated with polydipamine to aid dispersion and prevent agglomeration. BTP was successfully mixed with β-TCP to form the bioactive piezoelectric composite material PCLBTP / PCL. The microstructure of BTP was confirmed using TEM. BTP can be thermally melt-mixed into PCL without solvents, demonstrating that BTP / polymer can be used for 3D printing. Under ultrasound stimulation, macrophages secrete BMP2 and VEGF, and compared with pure PCL, skull experiments in 24 rats showed significant bone growth after 8 weeks.

[0203] Example 2

[0204] This application proposes a scaffold made of BTP polymer composite biomaterials, which effectively combines bone induction with local immunomodulation to achieve efficient bone regeneration. The BTO-containing composite scaffold is manufactured using melt extrusion 3D printing technology. Polycaprolactone (PCL), a biodegradable polymer commonly used in 3D printing, is used as the printing material. The solvent-free 3D printing process ensures uniform dispersion of β-tricalcium phosphate (β-TCP) within the PCL / BTO scaffold, eliminating hot spots and providing uniform bioactivity. Ultrasonic stimulation (1 MHz, 2.5 W / cm²) was applied. 2 Under these conditions, BTO nanoparticles generate a local piezoelectric field (0.7–2.3 mV), which accelerates the dissociation of β-TCP electrochemically through interfacial water splitting: anodic reaction (2H₂O → 4H₂O) + +O2+4e - Protons are produced, and these protons attack the β-TCP lattice (Ca3(PO4)2+2H). +→2CaHPO4+Ca 2+ The cathode reaction (2H₂O + 2e⁻) - →H2+2OH - This promotes hydroxyl-mediated mineralization. Ultrasonic cavitation further enhances this process, where microbubble rupture generates microfluidic shear forces that erode the β-TCP surface. The generated Ca... 2+ and PO4 3- Electromigration of ions in PO4 3- Attracted by the positively charged BTO region, while Ca 2+ The piezoelectric PCL / PBT composite scaffolds are repelled into surrounding tissues, creating a continuous ion flux and providing a microenvironment for rapid biomineralization. The resulting piezoelectric PCL / PBT composite scaffolds were compared with similarly printed PCL / 5% PBT and PCL / 10% PBT scaffolds. The PCL / PBT scaffolds exhibited significant piezoelectric properties. These scaffolds significantly alleviated the inflammatory response and promoted angiogenesis in the local tissue environment, thereby significantly accelerating skull healing in SD rats. Therefore, 3D-printed PCL / PBT scaffolds show great potential as implant materials for the reconstruction of large-area bone defects.

[0205] Materials and methods

[0206] 2.1. Materials

[0207] PCL was purchased from Osteopro, Singapore. BaTiO3 nanoparticles were purchased from Sigma-Aldrich (9012-76-4, 9000-70-8, H8502; St. Louis, Missouri, USA). Fetal bovine serum, α-modified Eagle medium (α-MEM), Duhenne-modified Eagle medium (DMEM), phosphate-buffered saline (PBS), trypsin-EDTA, and penicillin / streptomycin (P / S) used for cell culture experiments were purchased from Gibco Life Technologies, USA. TritionX-100 and bovine serum albumin were purchased from Saiton Biotechnology Co., Ltd. (Beijing). CCK-8 cell proliferation and cytotoxicity assay kit (CA1021), cell migration and invasion staining kit (G4740), and DAPI solution (C0065) were purchased from Beijing Solarbio Biotechnology Co., Ltd. Calcein AM (C2012), Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (A0428), and Western blotting antibody diluent were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Fixative was purchased from Shanghai Yesen Biotechnology Co., Ltd. VEGF mouse monoclonal antibody (66828) was purchased from Plintek Biotechnology Co., Ltd. (USA). Rhodamine phalloidin (T10446) was purchased from Beijing Paiseton Biotechnology Co., Ltd. Edu cell proliferation imaging assay kit (KTA2030) and Annexin V-AbFluor™ 488 / PI dual-color apoptosis detection kit (KTA0002) were purchased from Wuhan Abbott Biotechnology Co., Ltd.; anti-mannose receptor (CD206) antibody (AB300621), anti-iNOS antibody (AB49999), and anti-F4 / 80 antibody (AB300421) were purchased from Abcam (USA). VEGFA antibody (AF5131), CD31 antibody (AF6191), and type I collagen antibody (AF7001) were purchased from Affinity Biosciences (USA). RUNX2 (20700-1-AP) antibody and BMP2 (66383-1-lg) antibody were purchased from Proteintech (Wuhan, China). All water used in the experiments was purified using a Milli-Q circulating purification system (Millipore, USA).

[0208] 2.2. Synthesis and Characterization of BT@PDA@β-TCP NPs

[0209] PDA-modified BT particles: First, 0.1 g of dopamine was dissolved in deionized water to prepare a solution with a concentration of 2.0 g / L. Next, 0.06 g of Tris was added to the above solution until the pH reached 8.5. Then, 6 g of BT particles were added to the dopamine solution and mechanically stirred for 24 hours. Finally, the PDA-modified BT particles were washed with distilled water and then dried in a vacuum oven at 45°C. This study selected a dopamine pH of 8.5.

[0210] Preparation of BT@PDA@β-TCP particles: First, 0.64 g of β-TCP and 1 g of BT@PDA particles were mixed in 800 mL of deionized water at 50 °C for about 8 h to obtain BT@PDA@β-TCP particles. The grafted BT@PDA@β-TCP particles were then washed with distilled water and dried in a vacuum oven at 60 °C. The morphology of the NPs was observed using a scanning electron microscope (TESCAN, Czech Republic, MIRA4 LMH). The composition and crystal structure were observed by SEM using energy-dispersive spectroscopy (EDS) (UltimMax 40, Oxford, UK) combined with Fourier transform infrared spectroscopy (FTIR, Thermo Fisher, Nicolet 6700, USA).

[0211] 2.3.3 Fabrication and characterization of 3D printed scaffolds.

[0212] PCL and NPs were thoroughly mixed in a near-field microextruder to obtain a PCL / NPs blend. The near-field microextruder temperature was set to 100°C and the roller spacing was 4 mm. Using fused deposition modeling (FDM) technology, the PCL / NPs blend was placed in an FDM printer to obtain a 3D structured porous scaffold. The original scaffold was then constructed layer by layer by extruding the blend into fibers. The scaffold was printed at 90° intervals between two consecutive layers. The extrusion pressure was set to 30 bar, and the printing speed was maintained at 40 mm / s. The gap between adjacent layers in the z-direction was approximately 0.1 mm. The printing temperature was set to 140°C. PCL, PCL / 5% PBT, and PCL / 10% PBT scaffolds were fabricated based on the added NPs. For simplicity, the PCL, PCL / 5% PBT, and PCL / 10% PBT scaffolds are referred to as PCL, 5% PBT, and 10% PBT scaffolds, respectively.

[0213] Characterization of piezoelectric scaffolds: The morphology of the piezoelectric scaffolds was characterized using a scanning electron microscope (TESCAN (Czech Republic) MIRA4 LMH), and the distribution of Ca, Ba, and Ti elements in the piezoelectric scaffolds was scanned using an energy dispersive spectrometer (UltimMax 40, Oxford, UK). Thermogravimetric analysis (TGA, Q-50, USA) was used to assess the actual content of inorganic components in the composite scaffolds by heating the samples in air at a rate of 10 °C / min from room temperature to 650 °C. The water contact angle was measured using an automatic contact angle meter (SL200 A / B / D, Soren Technology, China). The droplet volume was 1–2 μL. After image capture, the baseline and apex of the droplet bottom were located, and the contact angle value was automatically calculated using the software provided with the instrument. Compression tests of these scaffolds were conducted on a universal testing machine (Instron 5500R, UK) equipped with a 500 N load sensor. The beam speed was set to 1 mm / min, and the total strain was set to 3% of the scaffold height.

[0214] 2.4. Biocompatibility Studies

[0215] Cell viability: Square scaffolds with sides of 5 mm and a height of 0.6 mm were immersed in 75% ethanol for 6 hours, sterilized by UV irradiation, and washed with PBS before use. According to ISO 10993 and GB / T 16886.5, L929 fibroblasts (purchased from Prexel) are a recommended cell line for evaluating the biocompatibility of implants in vivo. L929 cells were seeded at a density of 3 × 10³ cells / scaffold on different scaffolds, and CCK-8 assays were performed after 1, 3, and 5 days of culture. 10 μl of CCK-8 solution was added to each well, and the cells were cultured at 37°C and 5% CO2 for 1 hour. The absorbance of the solution was recorded at 450 nm using a Thermo Field microplate reader. On days 1 and 3, the cells on the culture plates were stained using a live and dead cell staining kit (Abbkine, China). Fluorescence images were captured using a confocal laser scanning microscope (CLSM, Opera Phenix Plus, USA) to assess the live / dead status (green fluorescence: live; red fluorescence: dead). To assess apoptosis in the piezoelectric scaffold, L929 cells were harvested and stained with Annexin V and propane iodide (PI). Finally, the percentage of live cells in different groups was measured by flow cytometry (FACSCelesta, Becton, Dickinson, USA). To further assess cell proliferation in the piezoelectric scaffold, EdU staining was performed using the Abbkine KTA2030 kit.

[0216] Cell adhesion: L929 cells were seeded at a density of 1×10⁴ cells / scaffold on different scaffolds and cultured for 3 days. Nuclear and F-actin staining was then performed. The method was as follows: Cells on the scaffolds were first fixed with 4% paraformaldehyde solution (Solarbio, China) for 30 minutes, washed with PBS, infiltrated with 0.1% Triton X-100 for 15 minutes, blocked with 1% BSA solution for 1 hour, stained with FITC-labeled Sigma-Aldrich F-actin at room temperature for 1 hour, and then stained with DAPI for nuclear staining. The cytoskeleton was observed using CLSM.

[0217] In vitro hemolysis rate: Blood compatibility was studied by an acute hemolysis test performed on extracts from different stent samples. Briefly, 1 ml of fresh anticoagulated whole blood (from SD rats) was suspended in 10 ml of PBS to obtain a homogeneous suspension. The prepared stent extract was incubated with the diluted blood suspension at 37°C for 1 hour, then the sample was removed and centrifuged at 3000 rpm for 10 minutes. The absorbance of the supernatant at 540 nm was measured, and the hemolysis rate was calculated.

[0218] The formula is: In vitro hemolysis rate (%) = (H1-H0) / (H2-H0)×100%, where H0, H1, and H2 represent the absorbance values ​​of the negative control (PBS), the sample, and the positive control (deionized water), respectively.

[0219] 2.5. Angiogenesis was measured using a chicken embryo chorioallantoic membrane model.

[0220] Fertilized chicken embryos were pre-incubated for 3 days at 37°C and 60% humidity. Then, the air cell of the egg was carefully opened to expose the allantoic membrane. Various supports were placed on the allantoic membrane to assess blood vessel formation. The egg was sealed with a sealing film and incubated for another 3 days. Observation was performed using a stereomicroscope (VISION, TY10 Mantis Elite, South Korea). AngioQuant software (version 1.33, Mathwork Inc, Houston, Texas, USA) was used to quantitatively analyze the tubular length, size, and total number of connections of the formed blood vessels.

[0221] 2.6. In vitro immune regulation by macrophages.

[0222] In this application, the mouse macrophage cell line RAW 264.7 was provided by Punosei. RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% P / S solution, and incubated at 37°C in a 5% CO2 incubator. The medium was changed daily.

[0223] First, morphological changes in macrophages co-cultured on different scaffolds were observed using immunofluorescence. Briefly, after 2 days of co-culture, samples were stained with F-actin and F4 / 80 and photographed using a confocal microscope. Next, for phenotypic polarization, surface markers of M1 macrophages (CD86) and M2 macrophages (CD206) were detected using flow cytometry. RAW 264.7 cells were first treated with 50 ng / ml lipopolysaccharide (LPS) for 24 hours as a simple in vitro stimulus to the inflammatory microenvironment during bone healing. The harvested cells were then seeded at a density of 3 × 10⁵ cells per well into each sample in a 6-well tissue culture plate. After 2 days of culture, cells were digested with trypsin and collected, then blocked with 1% bovine serum albumin (BSA) for 1 hour. After washing twice with PBS, cells were stained for 30 minutes with FITC-conjugated CD86 (105005, BioLegend, China) and PE-conjugated CD206 (141705, BioLegend, China). Cells were analyzed by flow cytometry (BD, USA) using FlowJo software (Tree Star Inc., USA). Macrophage polarization was analyzed by immunofluorescence staining and qPCR. For immunofluorescence staining, after 2 days of incubation, cells were fixed with 4% paraformaldehyde, infiltrated with 0.5% Triton X-100, and sealed with 1% BSA. Subsequently, anti-iNOS antibody (Abcam, ab210823) was incubated with mouse monoclonal antibody, followed by incubation with Cy3-labeled goat anti-mouse IgG secondary antibody (Servicebio, GB21303). The nuclei were then re-stained with DAPI and observed by confocal microscopy. For the M2 marker, the CD206 (Invitrogen, PA5-101657) antibody was stained using the same method. Finally, the stained cells were observed using a confocal microscope (CLSM). Furthermore, the gene expression levels of the M1 surface marker (iNOS) and the M2 surface marker (CD206) were detected by qPCR.

[0224] The levels of bone morphogenesis factors BMP-2 and VEGF secreted by macrophages were observed using enzyme-linked immunosorbent assay (ELISA). After co-culturing for 2 days, the culture supernatant from each group was collected for subsequent studies using previously reported methods. The concentrations of BMP-2 (Fine Test EM0874) and VEGF (Fine Test EM0205) secreted by cells in the collected supernatant were determined using a commercially available ELISA kit.

[0225] 2.7. In vitro angiogenesis assessment

[0226] Provided by ProCell, cultured in DMEM medium containing 10% FBS and 1% P / S solution, and placed in an incubator at 37°C and 5% CO2, with the medium changed once daily.

[0227] The Transwell assay and in vitro scratch assay were used to detect the ability of piezoelectric scaffolds to promote the migration of human umbilical vein endothelial cells (HUVECs). In the Transwell assay, HUVECs were seeded in the upper chamber, and macrophage culture medium was added to the lower chamber. Cells migrating on the lower surface were observed by staining with 0.1% crystal violet solution. In the scratch assay, a straight scratch was formed when HUVECs reached 90% confluence. Macrophage culture medium was added to stimulate HUVEC migration. After fixing the HUVECs, the migration was observed under an inverted microscope.

[0228] Angiogenesis was assessed using HUVECs from different macrophage cultures to induce angiogenesis. Briefly, HUVECs were seeded in Matrigel (Matrigel, 356262, Corning, USA) and incubated with different macrophage supernatants. Tubular structures were then observed and quantitatively analyzed. Furthermore, after 7 days of culture, immunofluorescence staining was performed to detect new angiogenesis, and the expression of VEGF and angiopoietin-1 (Ang-1) was detected by RT-qPCR. All primer sequences used in RT-qPCR are listed in Tables S1-S2.

[0229] 2.8. Study on osteogenic differentiation

[0230] Provided by ProCell. MC3T3-E1 cells were cultured in α-MEM medium containing 10% FBS and 1% P / S solution in an incubator at 37°C and 5% CO2. The medium was changed once daily.

[0231] The experimental procedure was the same as above. MC3T3-E1 cells (5 × 10⁴ cells / well) were co-cultured with conditioned macrophage medium and complete medium (1:1, consisting of 10 mmol / L β-glycerophosphate disodium, 50 μg / ml ascorbic acid, and 10 nmol / L dexamethasone). ALP staining was performed on day 7 using the BCIP / NBT staining kit according to the manufacturer's instructions. On day 14, fixed cell samples were immersed in ARS solution for ARS staining. After 7 days of co-culture, cells were fixed and incubated with 5% goat serum. The samples were then incubated with primary antibodies against Runx2 (Proteintech) and bone morphogenetic protein 2 (BMP2; Proteintech). After incubation with fluorescently labeled secondary antibodies, cells were re-stained with DAPI and observed using CLSM (Opera Phenix Plus, USA). The expression levels of Runx2 and Bmp2 in MC3T3-E1 cells were detected by qRT-PCR. The forward and reverse primers for the selected genes are also listed in S1-S2.

[0232] Table S1. Primers designed for selected osteogenic and angiogenesis differentiation-related genes.

[0233] Table S2. Primers designed for macrophage polarization-related genes in RAW264.7 cells

[0234] 2.9. Calcium ion imaging examination of osteoblasts

[0235] The calcium ion fluorescent probe (2 mM, Beyotime) was diluted to 2 μM in serum-free medium and incubated with cells at 37°C for 30 minutes. The cells were then washed with PBS (P1020, Solarbio) to remove excess calcium ion fluorescent probe.

[0236] 2.10. The body's ability to repair bone defects.

[0237] This animal experiment was approved by the Animal Protection and Use Committee of Hunan University (Approval No.: HNU-IACUC-2024-114). An animal model of skull bone defect injury was established. First, rats were anesthetized using a Rayward gas anesthesia machine with isoflurane (R510-22) inhaled at a rate of 2 L / min. After disinfection, the skin was dissected, and two critical-sized full-thickness bone defects (5 mm in diameter) were created on both sides of the sagittal suture of the parietal bone of each rat using a small animal skull trephine cooled with physiological saline. A scaffold was implanted to fill the defects, and the wound was closed with 3-0 sutures. Whole skull fragments were harvested for evaluation at 4 and 8 weeks post-implantation. Twenty-four male rats (200–220 g) were randomly divided into a control group, a PCL group, a 5% PBT group, and a 10% PBT group. For three days post-surgery, ultrasonic stimulation (WED-101 type, domestic) was administered every other day at a frequency of 1 MHz, 2.5 W / cm², for 5 minutes each time, until sampling. At 4 and 8 weeks post-surgery, rats were sacrificed by CO2, and whole-cranial scans were performed using a microCT system (NEMO, NMC-200, domestically produced). Scanning parameters were set as follows: voltage 80 kV, current 0.06 mA, and scan resolution 35 μm. Based on the reconstructed microCT images, bone volume / total tissue volume (BV / TV) and bone mineral density (BMD) were analyzed. After CT scanning, specimens were fixed in 4% paraformaldehyde at 4°C for 48 hours, then decalcified by immersion in 10% EDTA at 37°C for approximately 4 weeks. After paraffin embedding, 5 μm thick paraffin sections were prepared. To assess the local inflammatory response after implantation, iNOS and CD206 were selected as specific markers for double-labeled immunofluorescence staining to observe the phenotype of infiltrating macrophages. Further immunofluorescence staining was performed on pro-angiogenic factors VEGF / CD31 and osteogenic differentiation promoting factors bone morphogenetic protein 2 (BMP-2), Runx2, and COL1 to evaluate the effects of the piezoelectric scaffold on bone vascularization and osteogenic differentiation during bone healing. Finally, histological analysis was performed. Tissue samples were sectioned and stained with hematoxylin and eosin (HE) to assess bone repair capacity. Masson's trichrome staining was then performed to observe the maturity of regenerated bone tissue in the defect area. In Masson's trichrome staining, red areas represent mineralized collagen (mature bone matrix), and blue areas represent unmineralized collagen (immature bone matrix). All stained samples were semi-quantitatively imaged using ImageJ software.

[0238] 2.11. Transcriptome Analysis

[0239] RNA sequencing was used to investigate the gene expression profiles of macrophages cultured on different scaffolds. The specific method was as follows: RAW 264.7 macrophages (5 × 10⁶ cells / mL) were co-cultured with different scaffold samples in 6-well plates for 2 days. Total RNA was then collected from each group of macrophages using TRIzol reagent according to the manufacturer's instructions. RNA samples were stored at -80°C and then sequenced. Total RNA extraction, purification, library construction, and sequencing were all performed by Beijing Tsinghua Biotechnology Co., Ltd. The expression of several selected genes was examined using heatmaps, and the expression was evaluated using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

[0240] 2.12. Statistical Analysis

[0241] All experiments in this study were performed at least three times. All data are expressed as mean ± standard deviation (SD) and graphs were generated using Origin 2018 software (Origin Lab Corporation, USA). Analysis employed Student's t-test (unpaired and two-tailed), one-way or two-way ANOVA, and Tukey post-hoc analysis. An AP value <0.05 was considered statistically significant.

[0242] result

[0243] Preparation and Characterization of PBT: In this application, PDA was used to modify the surface of barium titanate (BT) to facilitate the polydispersity of nano-sized BT. A thin amorphous coating of approximately 3 nm was observed on the surface of BTO nanoparticles (Fig. 10A). The thickness of the PDA-coated BTO nanoparticles was significantly greater than that of the original BTO nanoparticles, demonstrating the successful synthesis of BTO@PDA nanoparticles. Due to the negative charge of the PDA layer, the Zeta potential of the BTO nanoparticles decreased with PDA coating, while TCP became positively charged. The presence of TCP nanoparticles led to an increase in the Zeta potential of the BTO@PDA@TCP nanoparticles. These potential changes directly reflected the step modification process of the BTO nanoparticles (Figs. 10D-10E). Fig. 2C shows a TEM image of the BTO@PDA@TCP nanoparticles. A large number of dot-like particles were distributed on the TCP surface, indicating that BTO could be deposited on the TCP nanoparticle surface through the PDA layer. EDS elemental spectra also confirmed the successful modification of BTO@PDA@TCP (Figs. 10F-10K). Further analysis of the functional groups on the composite nanoparticle sample using Fourier transform infrared (FT-IR) spectroscopy (Figure 10L) revealed a sharp band at 1258 cm⁻¹, attributed to the CO bond in PDA, indicating successful PDA coating on the BTO surface. A broad and strong band appeared near 961 cm⁻¹, attributed to the PO₄³⁻ stretching vibration line originating from β-TCP. X-ray diffraction (XRD) showed characteristic diffraction peaks at 27.7° and 34.3°, corresponding to the diffraction peaks of TCP crystal planes, compared to BTO (Figure 10M). These results indicate the successful synthesis of the BTO / PDA / TCP nanocomposite. 3D printing has been widely used in the fabrication of scaffolds with customizable shapes and internal structures in bone tissue engineering. Poly(ε-caprolactone) PCL possesses advantages such as biodegradability, non-toxicity, low melting point (facilitating molding during manufacturing), and suitability for bone tissue regeneration, leading to its widespread application in tissue engineering. This application utilizes fused deposition modeling (FDM) 3D printing technology to construct porous PCL / BTO@PDA@TCP (PBT) as a bone repair scaffold material, with the liquefaction temperature set at 140°C. In this application, PBT scaffolds were prepared using 5% and 10% by mass of BTO@PDA@TCP, with the 10% PBT scaffold exhibiting the best mechanical properties (Figure S1). As shown in the corresponding SEM and EDS elemental mapping images, the stacked printed lines form a porous structure, displaying uniform Ca, Ba, and Ti signals similar to the line morphology (Figure 10R). This application evaluates the piezoelectric properties of the polarized scaffold by exposing it to cyclic loading and measuring the piezoelectric constant d33. Figure 2G shows the piezoelectric response of different scaffolds. The d33 piezoelectric coefficient of the PBT composite increases with increasing BaTiO3 content.Notably, compared to the unmodified PCL sample, the piezoelectric response of BaTiO3 inclusions up to 5 wt.% gradually increased to 2.4 pC / N. However, when BaTiO3 exceeded 10 wt.%, the piezoelectric response significantly increased to 2.8 pC / N. Similar results have been observed in previous studies, such as with BaTiO3. Some HA-BaTiO3 composites exhibit very high d33 values ​​(>50 pC / N) due to the dense packing density of piezoelectric BaTiO3 particles in the sintered samples. The PBT composite scaffold in this application would never exhibit such a high piezoelectric response because the BaTiO3 particles are not densely packed within the polymer matrix. Furthermore, bone regeneration does not require such a high scaffold-mediated piezoelectric response, as bone itself exhibits a piezoelectric response. The d33 value of the 3D-printed PBT composite in this application is close to the piezoelectric response of bone. Therefore, the piezoelectric response of the PBT composite observed in this application highlights its great potential in bone remodeling and osteogenesis processes, as well as in accelerating the treatment of orthopedic defects. Cross-sectional scanning electron microscopy (SEM) images revealed the microstructure of PBT. The incorporation of different NPs was verified by TGA. The results showed that the residual mass fractions of PCL, 5% PBT, and 10% PBT scaffolds after thermal decomposition were 1.01 wt%, 5.3 wt%, and 10.1 wt%, respectively (Fig. 10N). Furthermore, water contact angle tests were performed on all printed scaffolds (Fig. 10Q), with the water contact angle of the scaffold made of hydrophobic PCL >90°, decreasing with the addition of inorganic NPs. It is generally believed that a hydrophilic matrix surface is more conducive to cell attachment and growth than a matrix on a hydrophobic smooth surface. Figs. 10A-10C are HR-TEM images of BT@PDA@TCP particles. Figs. 10D-10E show the size distribution and zeta potential of BTO, BTO@PDA, and BTO@PDA@TCP NPS. Figs. 10F-10K are SEM images and EDS elemental mappings of BT@PDA@TCP particles. Fig. 10L is the FTIR plot. Figure 10M shows the XRD patterns of the NPs. Figure 10N shows the content of inorganic components in various scaffolds quantified by thermogravimetric analysis. Figures 10O-10P show the piezoelectric properties. Figure 10Q shows the water contact angle. Figure 10R shows the SEM image and EDS elemental mapping of the piezoelectric scaffold.

[0244] Results of the biocompatibility study of piezoelectric scaffolds:

[0245] This application investigated the cell compatibility of piezoelectric scaffolds by measuring the survival, proliferation, and adhesion of L929 cells. Before cell culture experiments, all scaffolds were immersed in alcohol and sterilized with ultraviolet light for 6 hours. Cell compatibility of the piezoelectric scaffolds was assessed using a live / dead cell staining method. The results showed that L929 cells grew well on all scaffolds (emitting green fluorescence) and adhered tightly to the scaffold surface (Figure 11A), indicating good biocompatibility of all scaffolds. Cell compatibility between the piezoelectric scaffolds and L929 cells was assessed using a CCK8 assay kit. The results showed that the number of L929 cells increased significantly with prolonged cell culture time (Figures 11B-11C), indicating that the introduction of BTO and TCP did not produce cytotoxicity. Normal cells and early apoptotic cells have intact cell membranes, and PI, as a nucleic acid dye, cannot penetrate these intact cell membranes. In apoptotic and necrotic cells, PI can cross the cell membrane and bind to the cell nucleus. Labeling PI with Annexin V-FITC can distinguish between apoptotic, necrotic, and normal cells. In the bivariate flow cytometry scatter plot, the first quadrant represents necrotic or late-stage apoptotic cells, the third quadrant represents normal cells, and the fourth quadrant represents early-stage apoptotic cells. The sum of the cell counts in the first and fourth quadrants is correlated with the apoptosis rate. Quantitative apoptosis assessment by flow cytometry showed that all L929 groups had a high proportion of viable cells (>95%) (Figure 11D), indicating that the piezoelectric scaffold has good biocompatibility and low cytotoxicity to L929 cells. The addition of BTO and TCP had no adverse effect on the biocompatibility of the piezoelectric scaffold; on the contrary, it promoted cell proliferation and survival. To observe the adhesion and extension behavior of L929 cells on the piezoelectric scaffold, immunofluorescence staining was used to study the adhesion of L929 cells on the piezoelectric scaffold. L929 cells in the pure PCL group were spindle-shaped (Figure 11F). In contrast, cells in the 5% PBT and 10% PBT groups were polygonal, had filamentous pseudopodia, and had a more developed cytoskeleton and cell clusters, exhibiting strong adhesion. EdU staining results (Figure 11E) of L929 cells co-cultured on various scaffolds for 24 hours verified the significant increase in cell proliferation capacity resulting from the addition of BTO and TCP. Compared with the pure PCL group, the cell proliferation rate was higher in the piezoelectric groups, especially the 10% PBT group. Piezoelectricity can help cells quickly enter the differentiation stage and increase the cell proliferation rate. In summary, the prepared piezoelectric scaffold can promote the adhesion and proliferation of L929 cells. This may be due to the presence of PDA, which promotes cell adhesion, in the piezoelectric scaffold, coupled with its enhanced surface hydrophilicity and charged substrate surface, which facilitates the adsorption of proteins and various bioactive molecules from the culture medium through electrostatic interactions, thereby further promoting cell adhesion, proliferation, and differentiation. Since clinically used artificial bone regeneration scaffold materials inevitably come into contact with blood, the blood compatibility of the piezoelectric scaffold is studied by detecting its hemolytic activity.Optical imaging and quantitative analysis of hemolysis rate both showed results similar to the negative control group (PBS) (Figures 11G-11H), indicating that the hemolysis rate was safe (less than 5%). This good blood compatibility is mainly attributed to the nanoparticle PDA coating. Studies have shown that the PDA coating has a positive effect on reducing the hemolytic activity of biomaterials. The results indicate that the prepared piezoelectric scaffold material is non-hemolytic and has good compatibility with blood cells. Figure 11A shows CLSM images of live / dead staining of L929 cells cultured on various scaffolds on days 1 and 3. Scale bar = 200 μm. Figures 11B-11C show the proliferation of L929 fibroblasts cultured with the scaffolds using the CCK-8 assay. Figure 11D shows the detection of apoptosis of L929 cells cultured on different scaffolds by flow cytometry. Figure 11E shows CLSM images of EdU staining of L929 cells on different scaffolds. Scale bar = 200 μm. Figure 11F shows CLSM images of L929 cells stained with cytoskeletal ghost cyclic peptide-DAPI on different scaffolds. Scale bar = 50 μm. Figure 11G shows representative photographs of hemolytic activity assays from the prepared samples, and Figure 11H shows the quantitative analysis of hemolysis rate in the prepared samples. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 indicate significant differences compared to the PCL group.

[0246] Results of in vitro regulation of macrophage polarization by piezoelectric scaffolds

[0247] In bone repair, inflammation, oxidative stress, angiogenesis, and osteogenic processes are closely related, and the first-stage immune response after scaffold implantation is crucial for bone tissue repair. A sustained immune response during bone repair can delay bone regeneration, ultimately leading to nonunion. Generally, the disruption of this inflammatory cascade directly hinders stem cell proliferation and differentiation, thus impeding subsequent repair processes. Following bone injury, the early response of innate immune effector cells (especially macrophages) begins to play a role. The timely transition from pro-inflammatory M1-like macrophages to pro-regenerative M2-like macrophages represents a critical stage in normal bone regeneration. Furthermore, the phenotypic transformation of macrophages during inflammation influences a series of subsequent events that determine the fate of the scaffold after implantation. This study investigates the immunomodulatory properties of piezoelectric scaffolds. Figure 12A shows the immunofluorescence staining of RAW 264.7 mouse macrophage lines F-actin and F4 / 80 on the piezoelectric scaffold. F-actin and F4 / 80 double-labeled immunofluorescence images showed that macrophages adhered to the surface of the piezoelectric scaffold. Control group cells were shaped like fried eggs with numerous filopodia, while macrophages in the 5% PBT and 10% PBT groups were spindle-shaped. Macrophages in the PCL and control groups exhibited an M1-like morphology with a larger dispersal area, while macrophages in the 5% and 10% PBT groups showed a fused appearance, considered a typical M2 phenotype. Through immunomodulation, macrophages in the piezoelectric scaffold group were more likely to polarize into M2 macrophages than those in the non-piezoelectric scaffold group. Flow cytometry was used to detect macrophage polarization (Figure 12C). Flow cytometry results showed that the piezoelectric scaffold significantly reduced the expression of the surface marker CD86 on M1 macrophages and enhanced the expression of the surface marker CD206 on M2 macrophages. The expression of CD86 in macrophages in the control and PCL groups was higher than that in the 5% PBT and 10% PBT groups. Meanwhile, the number of CD206+M2 macrophages in the 5% PBT and 10% PBT groups was increased compared to the control and PCL groups. Immunofluorescence and flow cytometry results were similar (Fig. 12B), showing an increase in the number of CD206+M2 macrophages in the piezoelectric group. qRT-PCR was used to detect the mRNA expression levels of polarization-related genes such as CD206, TNF-α, and INOS in macrophages. Similar to the staining results, the gene expression levels of M2 macrophages in the 5% PBT and 10% PBT groups were higher than those in the CON and PCL groups (Fig. 12D-12F). Furthermore, ELISA was used to detect the levels of BMP-2 and VEGF in the macrophage supernatant. The secretion of BMP-2 and VEGF indicated that the enhanced electromicroenvironment regulated the process, accelerating the secretion of pro-regenerative cytokines by macrophages (Fig. 12G-12H). These results suggest that the electrical signals and microenvironment provided by the piezoelectric scaffold can modulate immunity, promote M2 macrophage polarization and macrophage secretion of healing-promoting cytokines, and promote bone tissue and angiogenesis regeneration through paracrine mechanisms. Figure 12A shows a representative immunofluorescence image of the RAW 264.7 cytoskeleton on the scaffold.F-actin (red) is a fibronectin representing the cytoskeleton; F4 / 80 (green) is a cell surface glycoprotein and a marker of mature mouse macrophages; DAPI (blue) represents the cell nucleus. Scale bar = 50 μm. Figure 12B shows representative immunofluorescence images of iNOS (green) and CD206 (red) in RAW 264.7 cells on the scaffold. Scale bar = 50 μm. Figure 12C shows representative flow cytometry images of cell surface markers in RAW 264.7 cells, including CD86 and CD206. Figures 12D-12F show the relative mRNA expression levels of anti-inflammatory and pro-inflammatory genes in macrophages on the piezoelectric scaffold. Figures 12G-12H show the secretion of healing cytokines by macrophages stimulated by the piezoelectric scaffold. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05 and **P < 0.01 indicate statistical significance compared to the CON group.

[0248] Research findings on piezoelectric stents promoting in vitro angiogenesis

[0249] To investigate the chemotactic response of macrophage supernatant to HUVECs (Fig. 13A), Transwell migration and cell scratch assays were performed (Figs. 13B-13C, 13D). The supernatant promoted HUVEC migration and infiltration, with the piezoelectric group showing higher migration ability than the control group (P<0.05). This is because the macrophage supernatant has a positive effect on cell migration, possibly because the piezoelectric scaffold can promote the secretion of VEGF by M2 macrophages (Figs. 12G-12H). The 10% PBT piezoelectric scaffold showed the strongest promoting effect on HUVEC migration and infiltration. Angiogenesis plays a crucial role in the reconstruction of bone defect tissue after bone injury, as it provides blood, nutrients, and oxygen to damaged tissue and alleviates uncontrolled inflammatory responses at the injury site. Therefore, angiogenesis experiments were performed to evaluate the ability of piezoelectric scaffolds to promote new blood vessel formation (Fig. 13G). This application used a chicken embryo allantoic membrane (CAM) model to assess angiogenesis. Three types of scaffolds were placed directly on the CAM, and images were taken to examine angiogenesis in the scaffold-infiltrated area. As shown in Figure 13E, the 10% PBT scaffold exhibited the richest angiogenesis, followed by the 5% PBT scaffold, while limited angiogenesis was observed in all PCL scaffolds. Based on these images, ImageJ was used for quantitative analysis of vessel length, size, and number of connections. More vessels were observed in the piezoelectric scaffold group of HUVECs, while some incomplete tubular structures were visible in the PCL group. The percentage of vessel area and total number of connections were significantly increased in both the 5% PBT and 10% PBT groups, suggesting that the piezoelectric scaffold combined with macrophages can promote in vitro angiogenesis. VEGF is a gene associated with angiogenesis, and its expression profile is closely related to angiogenesis. After one week of co-culture, VEGF immunofluorescence staining was further performed to assess the angiogenic capacity of the piezoelectric scaffold. When HUVECs were co-cultured with macrophage supernatant, VEGF expression in the 5% PBT and 10% PBT groups was significantly upregulated compared to the control and PCL groups (Figure 13E), indicating that the piezoelectric scaffold has good in vitro angiogenic bioactivity. The results of RT-qPCR analysis were consistent with the immunofluorescence staining results. As shown in Figure 13H, the expression levels of VEGF and Ang-1 in HUVECs from the 10% PBT group were significantly higher than those from the control and PCL groups. Therefore, the piezoelectric scaffold can effectively promote angiogenesis by modulating the immune microenvironment. These data indicate that the piezoelectric scaffold can provide a favorable electro-microenvironment for bone vascularization, demonstrating its great potential in bone regeneration and bone angiogenesis. Figure 13A is a schematic diagram of cells promoting angiogenesis. Figures 13B-13C are representative microscopic images of HUVECs after co-culturing with different macrophage cultures for 12 hours. Scale bar = 250 μm. Figure 13D is a representative Transwell migration image of macrophage culture medium added to the lower chamber alone after culturing for 24 hours. Scale bar = 200 μm.Figure 13E shows representative immunofluorescence images of VEGF (green) and cell nuclei (blue) in HUVECs after 7 days of co-culturing with different macrophage cultures. Scale bar = 50 μm. Figure 13F shows the relative mRNA expression levels of angiogenesis-related genes (including VEGF and Ang-1) in HUVECs after 7 days of co-culturing with different macrophage cultures. Figure 13G shows representative images of angiogenesis in the CAM experiment with scaffold presence. Figure 13H shows representative fluorescence images and quantitative analysis of HUVEC tube formation after co-culturing cells with different macrophage cultures. Scale bar = 200 μm. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 indicate significant differences compared to the CON group.

[0250] Research findings on the in vitro osteogenic effect of piezoelectric scaffolds

[0251] To verify the hypothesis that piezoelectric scaffolds induce macrophage polarization and secrete osteogenic factors to promote osteoogenesis, and to confirm the macrophage-mediated osteogenic enhancement, the key differentiation stages of MC3T3-E1 pre-osteoblasts cultured in M2 macrophage conditioned medium were analyzed: ALP activity (day 7) and mineralization (day 21) (Figure 14A). ALP, as an important osteoblast marker for cell maturation and calcification, is widely accepted as an indicator of early osteoblast differentiation. The 5% PBT and 10% PBT groups showed the highest ALP expression intensity, followed by the PCL and con groups (Figure 14B). Multiple studies have reported that piezoelectric BaTiO3 can improve biocompatibility in polymer matrices and plays a key role in promoting ALP activity and matrix mineralization. Furthermore, calcium deposition and collagen secretion are also necessary conditions for bone formation, which can be detected by Alizarin Red (ARS) staining. Corresponding to alkaline phosphatase (ALP) staining, significant Alizarin Red (ARS) positive staining was detected in cells cultured in both the 5% PBT and 10% PBT groups (Fig. 14C), indicating that MC3T3 cells formed significant calcium nodules during osteogenic differentiation. The enhanced osteogenic activity of MC3T3 cells was mainly attributed to the secretion of osteogenic-related cytokines by M2 macrophages. To further investigate the effect of macrophage supernatant on the osteogenic capacity of MC3T3 cells, the mRNA expression levels of osteogenic-related genes Runx2 and BMP-2 were detected using qRT-PCR. Similar to the staining results, the expression levels of osteogenic genes in the 5% PBT and 10% PBT groups were higher than those in the CON and PCL groups (Fig. 14D). Subsequently, immunofluorescence staining was used to detect the expression levels of Runx2 and BMP-2 in MC3T3 cells (Fig. 14E, 14F). The expression of Runx2 and BMP-2 in MC3T3 cells of the 5% PBT and 10% PBT groups was significantly higher than that in the CON and PCL groups. In summary, this study suggests that piezoelectric scaffolds can significantly promote early osteogenic differentiation and mineralization of MC3T3 cells by regulating the immune microenvironment, and can serve as a therapeutic platform to accelerate bone regeneration.

[0252] Calcium ions (Ca 2+ The related signaling pathways play a crucial role in the osteogenic differentiation of MC3T3-E1 cells (MC3T3s) induced by ultrasound-activated piezoelectric scaffolds (Figure 14G). Therefore, this application measures intracellular Ca2+. 2+ The concentration of Ca2+ in cells of the ultrasound-activated piezoelectric scaffold group is shown in Figure 14H. 2+ The significantly increased concentration indicates that the piezoelectric potential generated by ultrasonic activation of the piezoelectric scaffold promoted the influx of calcium ions. This application demonstrates that the piezoelectric signal generated by ultrasonic activation triggers the Ca... 2+Influx occurs, followed by transduction via calcium-associated membrane receptors. Among the potential calcium-associated receptors on the membrane, voltage-gated calcium channels (VGCCs), piezoelectric mechanosensitive ion channel component 1 (Piezo1), and transient receptor potential typical 1 (TRPC1) are well-known for receiving physical signals and inducing calcium influx. To explore which channel plays a key role in piezoelectric stimulation, these three channels were blocked individually using inhibitors. Blocking VGCCs with ω-Hexatoxin-HV1A (HV1A) significantly inhibited calcium influx and suppressed osteogenic differentiation of MC3T3 in the PBT+US group (Figs. 14M and 14N). Conversely, incubation with Piezo1 and TRPC1 (GSMTX4) inhibitors did not affect calcium influx or osteogenic differentiation of MC3T3 (Figs. 14J and 14K). These results indicate that VGCCs are key channels for converting piezoelectric signals into intracellular calcium signals. Fig. 14A is a schematic diagram of cells promoting osteogenic differentiation. Figure 14B shows representative MC3T3-E1 cells incubated with different scaffold extracts for 7 days for ALP staining. Scale bar = 250 μm. Figure 14C shows representative MC3T3-E1 cells incubated with different scaffold extracts for 21 days for ARS staining. Scale bar = 250 μm. Figure 14D shows the expression of osteogenic genes BMP2 and OPN. The expression of these osteogenic genes significantly increased under hydrogel piezoelectric stimulation. Figures 14E and 14F are immunofluorescence images of BMP2 (green) and Runx2 (red), where the expression of these osteogenic-related proteins significantly increased under hydrogel piezoelectric stimulation. Scale bar = 50 μm. Figure 14G is a schematic diagram of the mechanism by which the piezoelectric signal generated by ultrasound-activated piezoelectric scaffold acts on VGCC and intracellular signal transduction. Figure 14H is calcium ion imaging of MC3T3 cells after different treatments. Figure 14I is a schematic diagram of the mechanism by which GsMTX4 acts on Piezo1 and Trpc1. Figure 14J shows calcium ion imaging of MC3T3 cells using GsMTX4. Figure 14K shows BMP2 immunofluorescence imaging of MC3T3 cells using GsMTX4. Figure 14L is a schematic diagram of the mechanism of Hv1a acting on VGCC. Figure 14M shows calcium ion imaging of MC3T3 cells using Hv1a. Figure 14N shows BMP2 immunofluorescence imaging of MC3T3 cells using Hv1a. *P<0.05 and **P<0.01 indicate statistically significant differences compared to the CON group.

[0253] In vivo bone regeneration capacity assessment results

[0254] In terms of biological characteristics, piezoelectric scaffolds significantly promote osteoogenesis through various biochemical and biophysical properties and effectively induce the transformation of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype. To further evaluate the role of piezoelectric scaffolds in bone repair, 24 male Sprague-Dawley rats (300±20g) were randomly divided into four groups (n=6) to create critical-sized cranial defects (5mm in diameter): (1) blank control group, (2) PCL group, (3) 5% PBT group, and (4) 10% PBT group (Fig. 15A, Fig. S5A-5D). Three different concentrations of scaffolds (PCL, 5% PBT, and 10% PBT) were implanted at the defect site. Rats without any scaffold implantation served as the blank control group. Micro-CT scans showed that at 4 and 8 weeks post-operation, the 10% PBT group had the best bone healing effect, which was superior to the control group and the PCL group (Fig. 15B). The data showed that after implantation of piezoelectric scaffolds, more new bone was formed compared with the control group and the PCL group. Sagittal microCT analysis further confirmed the high bone regeneration capacity of the piezoelectric scaffold. Eight weeks after implantation, the bone volume to television ratio (BV / TV) in the 10% PBT group was significantly higher than that in the PCL and CON groups. The 10% PBT scaffold implantation site also showed more mature bone tissue and higher bone mineral density. Bone mineral density (BMD) data showed the same trend as the BV / TV results. Treatment of critical-sized skull defects in rats with ultrasound-activated 10% PBT scaffolds significantly accelerated bone regeneration. This enhancement was directly related to ultrasound-triggered ion release. The released Ca... 2+ and PO4 3-Ions supersaturate the local fluid, accelerating hydroxyapatite (HA) nucleation. Dissolved ions adsorb onto the scaffold, forming a CaP-rich bone layer that guides new bone growth. In summary, the 10% PBT piezoelectric scaffold exhibited better osteogenic behavior in cell experiments, and its enhanced ability to promote bone regeneration was also validated in a rat model. To further investigate the effect of the piezoelectric scaffold on skull defect repair, histological evaluation was performed using H&E staining and Masson trichrome staining. H&E staining image analysis showed that the 10% PBT group had more newly formed bone plates and bone islands (Fig. 15C). In Masson trichrome staining, red staining represents mineralized collagen and mature bone matrix, while blue staining represents unmineralized collagen and immature bone matrix. In this application, the 10% PBT group showed abundant red staining and more collagen deposition, indicating that the regenerated skull in the piezoelectric group was calcifying and mostly mature (Fig. 15D). In addition, similar results were obtained by COL1A1 immunofluorescence staining of bone tissue. To assess osteogenic activity in vivo, Runx2 and BMP2 immunofluorescence staining was performed on bone tissue. The results showed that the proportion of positive cells in the 10% PBT group was significantly higher than that in the control group (Figures 15E and 15F), indicating that the piezoelectric scaffold can accelerate the expression of osteogenic-related genes. The expression of these proteins can continuously activate osteogenic activity and ECM deposition. These results suggest that the piezoelectric scaffold can promote bone regeneration and collagen deposition in vivo. Figure 15A is a schematic diagram of the establishment of a 5mm skull defect model in SD rats. Figure 15B shows micro-CT coronal images, BV / TV, and BMD results of regenerated bone tissue. Figure 15C shows HE staining of the bone defect area at 4–8 weeks post-surgery. Scale bar = 500 μm. Figure 15D shows Masson trichrome staining of the bone defect area at 4 and 8 weeks post-surgery. Scale bar = 500 μm. Figure 15E shows representative Runx2 immunofluorescence staining images of the defect area at 4 and 8 weeks post-surgery. Scale bar = 100 μm. Figure 15F shows representative BMP2 immunofluorescence staining images of the defect area at 4 and 8 weeks post-surgery. Scale bar = 100 μm. *P<0.05 and **P<0.01 indicate statistical significance compared to the CON group.

[0255] Results of a study on the role of M2 macrophages in promoting angiogenesis in vivo

[0256] In further experiments, this application investigated macrophage polarization and angiogenesis at bone defects. Macrophage infiltration was detected by tissue immunofluorescence. The results showed that CD206 expression in the skull tissue of the 10% PBT group was higher than that in the control group, while iNOS expression was lower than that in the control group (Figure 16A). This result is consistent with the in vitro experimental results, indicating that M2 macrophage infiltration increases near bone defects after implantation of piezoelectric scaffolds. As mentioned earlier, VEGFA is not only an indicator of vascular endothelial cell function but also a mediator of angiogenesis-osteogenic coupling. Immunohistochemical fluorescence staining showed that VEGFA expression was highest in the 10% PBT group (Figure 16C). Subsequently, tissue immunofluorescence staining was performed to measure CD31 expression, which is expressed at tight junctions between endothelial cells. CD31, as a marker of H-type vessels, is a major vessel involved in osteoogenesis, and most osteoprogenitor cells selectively localize near H-type vessels. The results showed that CD31 expression was highest in the 10% PBT group (Figure 16B). In summary, these findings indicate that piezoelectric scaffolds can modulate immune invasion and angiogenesis, thereby promoting bone defect repair in vivo. A favorable immune microenvironment not only enhances osteogenic differentiation but also promotes angiogenesis. Angiogenesis provides the necessary nutrients for maintaining osteogenic differentiation and osteoblast proliferation. Figures 16A-16C show M2 macrophages and the promotion of angiogenesis in vivo. Figure 16A is a representative image of iNOS and CD206 immunofluorescence staining in the bone defect area 4 weeks after scaffold implantation. Scale bar = 100 μm. Figure 16B is a representative image of CD31 immunofluorescence staining in the bone defect area 4 and 8 weeks after scaffold implantation. Scale bar = 100 μm. Figure 16C is a representative image of VEGF immunofluorescence staining in the bone defect area 4 and 8 weeks after scaffold implantation. Scale bar = 100 μm. *P<0.05 and **P<0.01 indicate statistically significant differences compared to the CON group.

[0257] Results of research on the molecular mechanism by which piezoelectric scaffolds promote bone regeneration

[0258] In vitro and in vivo experiments were conducted using RNA sequencing to investigate the molecular mechanism of piezoelectric scaffold-induced macrophage polarization. A total of 11,312 differentially expressed genes (DEGs) were obtained from the 10% PBT piezoelectric scaffold group and the CON group, of which 130 upregulated genes and 393 downregulated genes were detected (Figure 17A). The clustering results of the differentially expressed genes are shown in Figure 17B. Enrichment analysis of biological processes showed strong associations with genes related to responses to macrophage colony-stimulating factors (Figure 17D). RNA sequencing results indicated that the NF-κB signaling pathway may be involved in regulating macrophage M2 polarization (Figure 17C). NF-κB activation is thought to occur in three key stages. First, pro-inflammatory stimuli bind to adaptors such as MyD88, TAK1 / TAB1, or RIP1, thereby activating the IKK complex composed of IKKα, IKKβ, and IKKγ subunits. This activation, in turn, triggers phosphorylation and subsequent degradation of IkB (especially Ikbα), thereby promoting the rapid release of NF-κB dimers (e.g., p65 / p50) into the nucleus and stimulating the transcription of pro-inflammatory genes. Based on GO analysis, this application hypothesizes that the piezoelectric scaffold can regulate the immune response of macrophages. Since phosphorylated proteins are active and regulate cellular function, this application detected the expression of p-P65 in RAW264.7 cells using immunofluorescence. After piezoelectric stimulation, the expression of p-P65 in the nucleus of RAW264.7 cells was significantly downregulated, while p-P65 was expressed in the cytoplasm. In unstimulated cells, p-P65 translocated to the nucleus (Fig. 17E). Immunohistofluorescence assays of p-P65 levels in regenerated bone tissue revealed a significant decrease in p-P65 expression after implantation of the piezoelectric scaffold (Fig. 17F). Membrane proteins, as signal integrators, can respond to external stimuli and changes in membrane potential. The cascade response induced by electric field stimulation (ES) may be mediated by the redistribution of cell surface receptors. Fibronectin receptor (α5β1 integrin) is a transmembrane heterodimeric protein capable of sensing the cellular microenvironment; calcium ions are an essential cation for its binding to fibronectin. Integrin aggregation or redistribution induces autophosphorylation of focal adhesion kinase (FAK). FAK-mediated extracellular matrix (ECM) signaling stimulates the exchange of guanosine diphosphate (GDP) and guanosine triphosphate (GTP), activating Ras and its downstream signaling pathways, including the p38 kinase pathway, the PI3K / Akt pathway, the JNK pathway, and the NF-κB pathway. Therefore, it can be inferred that the local electric field generated by the ultrasound-assisted piezoelectric scaffold effectively blocks the integrin-induced NF-κB inflammatory signaling pathway, modulates macrophage M2 polarization, and thus inhibits the inflammatory response (Fig. 17G). Fig. 17A shows the quantitative analysis of DEG in macrophages in the CON group and the 10% PBT group. Fig. 17B is a DEG thermogram. Figure 17C shows the KEGG pathway analysis of macrophages in the CON group and the 10% PBT group. Figure 17D shows the GO enrichment analysis of the 20 most differentially upregulated and downregulated biological processes.Figure 17E shows immunofluorescence images of p-P65 (green) and F-actin (red), scale bar = 50 μm. Figure 17F shows representative immunofluorescence staining images of p-P65 in the defect area at 4 and 8 weeks post-implantation, scale bar = 100 μm. Figure 17G is a schematic diagram illustrating the possible molecular mechanism by which the piezoelectric scaffold promotes bone repair by modulating macrophage M2 polarization and inhibiting the NF-κB axis.

[0259] in conclusion

[0260] In at least one embodiment, a novel synthetic piezoelectric composite material (BTP) prepared by the method of the present invention is described. This composite material is made of barium titanate nanoparticles and coated with polydipamine to aid dispersion and prevent aggregation. BTP has been successfully mixed with β-TCP to form the bioactive piezoelectric composite material PCLBTP / PCL. The microstructure of BTP was confirmed using TEM. BTP can be thermally melt-mixed into PCL without a solvent, demonstrating that the BTP / polymer can be used for 3D printing. Under ultrasound stimulation, macrophages secrete BMP2 and VEGF, and skull experiments in 24 rats showed significant bone growth after 8 weeks compared to pure PCL.

[0261] Therefore, after describing several embodiments, those skilled in the art will recognize that different modifications, alternative structures, and equivalents can be used without departing from the essence of the invention. Accordingly, the above description should not be considered as a limitation on the scope of the invention as defined by the claims.

[0262] Exemplary embodiments of the present invention have thus been fully described. Although the description refers to specific embodiments, those skilled in the art will understand that the invention can be practiced with variations in these specific details. Therefore, the invention should not be construed as being limited to the embodiments set forth herein.

Claims

1. A method for fabricating a piezoelectric composite biomaterial, comprising: Multiple BT / PDA particles are obtained by forming a poly(xylene)-coating on the surface of multiple barium titanate (BT) nanoparticles. The plurality of BT / PDA particles are distributed onto the surface of a plurality of β-tricalcium phosphate β-TCP particles to form bioactive composite BTP particles; The composite BTP particles are thermally melt-mixed with a polymer matrix to form a BTP polymer composite biomaterial.

2. The manufacturing method according to claim 1, characterized in that: The quality ratio of BT, PDA, and β-TCP is (1-30):(0.05-2):(0.1-2).

3. The manufacturing method according to claim 1, characterized in that: The quality ratio of BT, PDA, and β-TCP is 6:0.1:0.

64.

4. The manufacturing method according to claim 1, characterized in that: The polymer is polycaprolactone (PCL) or a bioabsorbable polymer.

5. The manufacturing method according to claim 3, characterized in that: The step of forming a poly(xyleneamide) PDA coating on the surface of BT particles includes: PDA was added to deionized water to obtain a 2.0 g / L PDA solution; Adjust the pH of the PDA solution to 8.5; BT particles were added to the PDA solution and stirred for 24 hours to obtain BT / PDA particles; The BT / PDA particles were washed with distilled water and dried in a vacuum oven at 45°C.

6. The manufacturing method according to claim 1, characterized in that: The steps of distributing the plurality of BT / PDA particles onto the surface of β-TCP particles include: adding β-TCP and BT / PDA particles to deionized water at 50°C, mechanically stirring to obtain composite BTP particles; washing the composite BTP particles with distilled water and drying them in a vacuum oven at 60°C.

7. The manufacturing method according to claim 1, characterized in that: The thickness of the PDA coating on the surface of the BT particles is 5-40 nm.

8. The manufacturing method according to claim 1, characterized in that: The average particle size of the BT particles is 60-100 nm.

9. The manufacturing method according to claim 1, characterized in that: The mass of the composite BTP particles is 5-20% relative to the polymer matrix.

10. The manufacturing method according to claim 4, characterized in that: The steps of hot-melt mixing composite BTP particles with polycaprolactone (PCL) include: PCL is placed in an open-type heated twin-roll mill with the heated rolls at 100°C. When the PCL melts and covers the twin-roll mill, BTP particles are mixed into the hot melt, and the composite melt is turned over for at least 30 minutes. After cooling to room temperature, a solvent-free PCL / BTP polymer composite biomaterial is obtained for melt extrusion 3D printing.

11. A piezoelectric composite biomaterial, comprising: Multiple composite BTP particles, wherein each composite BTP particle comprises: Multiple β-tricalcium phosphate β-TCP particles have multiple BT / PDA particles distributed on their surface. The BT / PDA particles are formed by coating the surface of nano-barium titanate BT particles with a polydimethylbenzamide PDA coating.

12. The piezoelectric composite biomaterial according to claim 11 further includes a polymer matrix, wherein a plurality of composite BTP particles are thermally melt-mixed with the polymer matrix to form a BTP polymer composite biomaterial.

13. An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods described in claims 1-10 or the piezoelectric composite biomaterial described in any of claims 10-12 is used to manufacture bone tissue, including any one of oral and maxillofacial tissue, bone plate, and cartilage tissue.

14. An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods of claims 1-10 or the piezoelectric composite biomaterial of any of claims 10-12 is used to manufacture wires and / or sutures.

15. An application of a piezoelectric composite biomaterial, wherein the piezoelectric composite biomaterial obtained by any of the manufacturing methods of claims 1-10 or the piezoelectric composite biomaterial of any of claims 10-12 is fabricated into a sheet.

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