Piezoelectric porous scaffold with high efficiency osteogenesis promoting effect and preparation method thereof

Abstract

The application discloses a piezoelectric porous scaffold with high efficient osteogenesis and a preparation method thereof. The piezoelectric porous scaffold is obtained by mixing a biological component and a piezoelectric component through an adhesive, sintering and then performing polarization treatment. The piezoelectric porous scaffold can efficiently load and adsorb bioactive protein factors, realizes effective release of the protein, and has piezoelectric performance and osteogenesis activity. Under the multiple effects of the piezoelectric effect, the porous structure and the bioactivity of the protein of the scaffold, the piezoelectric porous scaffold can quickly start early response of bone marrow stromal cells in a synergistic manner, and then promote cell adhesion, proliferation and osteogenic differentiation. The piezoelectric porous scaffold has important significance for efficient and rapid repair of large bone defects, bone nonunion and other bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a piezoelectric porous scaffold with highly efficient osteogenic activity, possessing tunable piezoelectric properties and excellent osteogenic activity. More specifically, it relates to a piezoelectric porous bone repair scaffold capable of carrying bioactive factors. Background Technology

[0002] With the continuous development of biomedical technology, people have begun to address the problems of bone tissue trauma and defects caused by disease, accidents, and aging. Unlike conventional fractures, large-segment bone injuries (bone defects longer than 1.5 to 2 times the bone diameter) remain a significant challenge in the biomedical field because human bone tissue lacks the ability to self-repair defects exceeding a critical size. For these conditions, extensive research and medical practice have shown that implanting porous scaffolds to promote bone tissue growth within the scaffold, thereby achieving rapid and high-quality reconstruction of bone defects, is currently a commonly used and highly effective solution.

[0003] Human bone tissue itself possesses a piezoelectric effect, generating a surface potential under mechanical stress. This piezoelectric response microenvironment can regulate osteogenic differentiation and stimulate cell remodeling of bone structure. Currently, clinical applications utilizing bone electrical properties (such as electrostimulation osteogenic technology and electromagnetic field stimulation osteogenic technology) have achieved good therapeutic effects in treating orthopedic diseases such as fractures, bone defects, osteoarthritis, and osteonecrosis. However, electrostimulation devices are complex, difficult to implant, and prone to infection and secondary disease. Furthermore, the superposition of electric and magnetic fields also poses a risk of causing pathological changes in normal tissues. Therefore, developing bone substitutes with piezoelectric properties similar to human bone is a hot research topic in the field of bio-tissue engineering.

[0004] Research has found that the polar asymmetric structure of lead-free piezoelectric ceramic materials such as barium titanate endows them with excellent ferroelectric and piezoelectric properties. Under external stress, the internal electric axis of the material deflects, generating piezoelectric charges, thereby mimicking the piezoelectric microenvironment generated in human bone tissue under stress during movement, effectively promoting bone cell regeneration. By incorporating non-piezoelectric bioactive components, the aim is to obtain composite materials that combine piezoelectricity and biocompatibility, and to some extent solve the problem of the non-biodegradability of piezoelectric ceramics, so as to promote bone integration, osteogenic formation, and ossification more quickly and effectively.

[0005] Bone tissue regeneration is a complex biological process involving multiple cytokines. Therefore, immobilizing bioactive factors in tissue engineering scaffolds is an effective way to enhance their bioactivity. However, few studies have combined the piezoelectric effect of materials with bioactive factors to investigate the combined effects of electrical signals and bioactive factors on cell biocompatibility and osteogenic activity.

[0006] Therefore, there is an urgent need to study a piezoelectric porous scaffold. Its three-dimensional interconnected pore structure not only ensures the effective transmission of mechanical stimuli, but its high specific surface area also provides ample space for the immobilization of bioactive factors. The piezoelectric porous scaffold can leverage the synergistic effect of the scaffold's piezoelectricity, its porous structure, and the bioactivity of the factors to promote efficient and rapid repair of bone defects. Summary of the Invention

[0007] The purpose of this invention is to provide a piezoelectric porous scaffold for rapid repair and treatment of bone defects.

[0008] In a first aspect, the present invention provides a piezoelectric porous scaffold, wherein the piezoelectric porous scaffold is obtained by mixing biological components and piezoelectric components with a binder, sintering, and then polarizing the mixture, wherein...

[0009] The biological component is one or a mixture of two or more of the following: hydroxyapatite (HA), tricalcium phosphate (β-TCP), bioglass (MBG), calcium polyphosphate, tetracalcium phosphate, octacalcium phosphate, biphasic calcium phosphate, and calcium polyphosphate.

[0010] The piezoelectric components are calcium titanate (CaTiO3) and potassium sodium niobate (K). 0.5 Na 0.5 One or a mixture of two or more of NbO3 and barium titanate (BaTiO3);

[0011] The binder is one or a mixture of two or more of sodium alginate, polyvinyl alcohol, and cellulose.

[0012] In another preferred embodiment, the adhesive is used in the form of an aqueous solution. In yet another preferred embodiment, the adhesive is used in the form of an aqueous solution with a mass concentration of 5%-20%, 8%-15%, or 10%.

[0013] In another preferred embodiment, the biological component is β-TCP, the piezoelectric component is CaTiO3, and the binder used is an aqueous solution of polyvinyl alcohol with a mass concentration of 5%-20%, 8%-15%, or 10%. In another preferred embodiment, the mass fraction of the biological component β-TCP is 5%-50%, 8%-35%, or 10%-20%. In another preferred embodiment, the mass fraction of the piezoelectric component BaTiO3 is 95%-50%, 92%-65%, or 90%-80%. In another preferred embodiment, the binder is a 5%-20%, 8%-15%, or 10% polyvinyl alcohol solution, accounting for 5%-50%, 8%-35%, or 10%-20% of the total mass of the biological component and the piezoelectric component.

[0014] In another preferred embodiment, the bio-component accounts for 5% to 95% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 95% to 5% of the total mass of the bio-component and the piezoelectric component; and / or

[0015] The binder accounts for 5% to 30% of the total mass of the biological and piezoelectric components.

[0016] In another preferred embodiment, the bio-component accounts for 10% to 80% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 90% to 20% of the total mass of the bio-component and the piezoelectric component.

[0017] In another preferred embodiment, the bio-component accounts for 20%–50%, 5%–50%, 8%–35%, or 10%–20% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 80%–50%, 95%–50%, 92%–65%, or 90%–80% of the total mass of the bio-component and the piezoelectric component.

[0018] In another preferred embodiment, the mass of the binder accounts for 5% to 10%, 5% to 50%, or 8% to 35% of the total mass of the biological component and the piezoelectric component.

[0019] In another preferred embodiment, the piezoelectric constant d of the resulting piezoelectric porous support is... 33 The ratio is 8 pC / N to 13 pC / N. This is similar to the dn of natural human bone. 33 (7.5pC / N~10pC / N) equivalent.

[0020] A second aspect of the present invention provides an active piezoelectric porous scaffold, comprising the piezoelectric porous scaffold described in the first aspect and a bioactive factor loaded on the piezoelectric porous scaffold, wherein the bioactive factor is one or a mixture of two or more of VEGF, BMP-2, TGF-β1, and FGF-9.

[0021] In another preferred embodiment, the bioactive factor is BMP-2.

[0022] In another preferred embodiment, the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.1-0.5:1.

[0023] In another preferred embodiment, the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.2-0.3:1.

[0024] In another preferred embodiment, the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.26:1.

[0025] In another preferred embodiment, the load is physical adsorption or coupling agent coupling, preferably freeze-dried physical adsorption.

[0026] In another preferred embodiment, bioactive factors are physically adsorbed on the surface of the piezoelectric porous scaffold described in the first aspect, thereby endowing it with bioactivity.

[0027] Rat bone marrow stromal stem cells (BMSCs) were cultured on the surface of an active piezoelectric porous scaffold. After a specific culture time, the cell count and bone morphogenetic protein mass density of the scaffold and a conventional scaffold were measured to determine the efficient bone-promoting piezoelectric porous scaffold surface.

[0028] This invention leverages the synergistic effect of osteogenic regulatory factors and porous piezoelectric scaffolds in promoting bone repair, achieving rapid bone repair and treating bone defects. By loading bioactive factors, this invention exhibits superior biocompatibility compared to conventional piezoelectric porous scaffolds, facilitating further medical applications.

[0029] The electrostatic interaction between the surface electrical signals and bioactive factors of the piezoelectric porous scaffold that promotes efficient osteogenic action can firmly adsorb the factors onto the material surface and achieve long-term sustained release of the factors. This not only improves the efficiency of the active factors but also avoids the potential side effects caused by excessive use of active factors. At the same time, the combination of rapid initiation of cell response by electrical signals and osteogenic induction by active factors enables rapid and efficient repair and functional reconstruction of defect sites.

[0030] A third aspect of the present invention provides a method for preparing the piezoelectric porous support described in the first aspect, the method comprising the following steps:

[0031] (i) The piezoelectric component, biological component and binder are mixed evenly to obtain a composite slurry;

[0032] (ii) The composite slurry is mixed evenly with the porous template to obtain a composite support;

[0033] (iii) The composite scaffold is sintered at high temperature to obtain a porous scaffold;

[0034] (iv) The porous support is subjected to high voltage polarization treatment to obtain a piezoelectric porous support.

[0035] This invention employs a polyurethane sponge template method, providing a novel approach for the fabrication of porous scaffolds. This method involves pre-mixing a slurry with a polyurethane sponge, followed by co-sintering, and then removing the polyurethane sponge to obtain the piezoelectric porous scaffold. Compared to traditional methods, this approach offers lower costs, simpler operation, and shorter material forming time. Furthermore, due to the diverse selectivity of the slurry, the various physical properties of the resulting scaffold can be improved by altering the composition and proportion of the slurry.

[0036] This invention combines piezoelectric ceramic powder, inorganic bioactive components, and a binder in a specific ratio, and mixes them uniformly with a pre-treated porous template. After drying, molding, and high-temperature sintering, a piezoelectric porous scaffold with highly efficient osteogenic properties is prepared. The scaffold is simple and convenient to prepare, possessing a three-dimensional interconnected pore structure that ensures effective transmission of mechanical stress within the scaffold. Simultaneously, the prepared piezoelectric porous scaffold can efficiently carry and adsorb bioactive protein factors, achieving effective sustained protein release. Through the combined effects of the scaffold's piezoelectric properties, porous structure, and bioactive proteins, it can synergistically and rapidly initiate the early response of bone marrow stromal stem cells (BMSCs), significantly promoting BMSC adhesion, proliferation, and osteogenic differentiation. This piezoelectric porous scaffold combines excellent piezoelectric properties and osteogenic activity, making it significant for the efficient and rapid repair of large bone defects, nonunion, and other bone defects.

[0037] In another preferred embodiment, the bio-component accounts for 5% to 95% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 95% to 5% of the total mass of the bio-component and the piezoelectric component. In another preferred embodiment, the bio-component accounts for 20% to 80% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 80% to 20% of the total mass of the bio-component and the piezoelectric component.

[0038] In another preferred embodiment, the bio-component accounts for 20%–50%, 5%–50%, 8%–35%, or 10%–20% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 80%–50%, 95%–50%, 92%–65%, or 90%–80% of the total mass of the bio-component and the piezoelectric component.

[0039] In another preferred embodiment, the binder accounts for 5% to 30% of the total mass of the biological component and the piezoelectric component. In yet another preferred embodiment, the binder accounts for 5% to 10%, 5% to 50%, or 8% to 35% of the total mass of the biological component and the piezoelectric component.

[0040] In another preferred embodiment, the porous template is a polymethyl methacrylate (PMMA) colloidal template, a polyurethane (PU) sponge template, a polycarbonate (PC) film template, or a cellulose template.

[0041] In another preferred embodiment, the sintering temperature is 800-2000℃, more preferably 1100-1500℃ or 1400℃.

[0042] In another preferred embodiment, the sintering time is 1-5 hours, more preferably 2-4 hours.

[0043] In another preferred embodiment, the polarization voltage during polarization is 2-15 kV / mm, more preferably 5-10 kV / mm.

[0044] This invention uses a novel preparation method to obtain a porous scaffold material with a certain porosity and pore size distribution. The resulting piezoelectric scaffold can be arbitrarily changed in shape, providing a suitable environment for the next step of physical adsorption.

[0045] A fourth aspect of the present invention provides the use of the piezoelectric porous scaffold described in the first aspect or the active piezoelectric porous scaffold described in the second aspect for preparing materials for repairing and treating bone defects.

[0046] The beneficial effects of this invention are as follows:

[0047] (1) The preparation method used in this invention is the polyurethane foam method. In terms of preparation scheme, this method has simple preparation steps, low cost, and short time consumption, making it convenient for large-scale production and use. In terms of product performance, the piezoelectric properties of the product obtained by this invention are superior to those of the traditionally prepared finished products. This method can solve the problems of preparation difficulties and high costs existing in the original preparation methods, and provides possibilities for its broad clinical application.

[0048] (2) The piezoelectric porous scaffold prepared by the present invention has a three-dimensional interconnected pore structure, which ensures the effective transmission of mechanical signals inside the scaffold; at the same time, the high specific surface area of ​​the scaffold provides sufficient space for the immobilization of bioactive factors and is conducive to the ingrowth of new bone tissue and the transport of substances; in addition, the unique electrical signal on the surface of the piezoelectric scaffold can realize the strong adsorption and effective sustained release of bioactive factors, improve the utilization rate of factors, and avoid the potential side effects caused by sudden release and overuse of factors.

[0049] (3) This invention combines the piezoelectric effect with bioactive factors. The electrical signal can rapidly activate early cellular behavioral responses and osteogenic differentiation. Combined with the highly efficient osteoinductive activity of the bioactive factors, it synergistically promotes the rapid recruitment, adhesion, and osteogenic differentiation of cells at the bone defect site on the scaffold surface. Compared with traditional bone repair scaffolds, this invention, through the resulting highly efficient osteogenic piezoelectric porous scaffold, combines the piezoelectric effect with bioactive factors, resulting in a faster and more significant bone repair effect.

[0050] By combining piezoelectric effects and bioactive factors, two bone-promoting factors, bioactive factors are loaded onto a porous piezoelectric scaffold, resulting in a bone repair piezoelectric porous scaffold with both piezoelectric properties and bioactivity. Compared with traditional bone repair scaffolds, this invention combines piezoelectric effects with bioactive factors through the resulting highly efficient bone-promoting piezoelectric porous scaffold, rapidly initiating early adhesion, spreading, and migration of bone mesenchymal stem cells (BMSCs), enhancing BMPR expression, and synergistically promoting rapid repair of bone defects with rhBMP-2. This approach is expected to significantly reduce the amount of factors used, avoiding the potential risks associated with high-dose administration.

[0051] It should be understood that in this invention patent, the above-described technical features of the present invention and the various technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative that provides the same, equivalent, or similar purpose; due to space limitations, these will not be elaborated upon here. Attached Figure Description

[0052] Figure 1 This is a flowchart illustrating the fabrication process of a piezoelectric porous scaffold.

[0053] Figure 2 Digital photographs and scanning electron microscope images of the piezoelectric porous support.

[0054] Figure 3 The results of the cytotoxicity evaluation of BMSCs on the piezoelectric porous scaffold (A) and the scanning electron microscope images of BMSCs adhesion and spreading on the scaffold surface (B).

[0055] Figure 4 The results of ALP activity expression in vitro are shown.

[0056] Figure 5 The results show the expression of osteogenic-related genes. Detailed Implementation

[0057] This invention, through extensive experimental research, addresses the problems of difficult fabrication, high cost, and limited efficacy of traditional bone repair scaffolds. It employs a novel polyurethane sponge method to prepare porous piezoelectric scaffolds, while simultaneously loading bioactive factors to significantly enhance their bone repair effect. The two complement each other: the porous scaffold provides an attachment substrate for bioactive factors, indirectly increasing their loading capacity and osteogenic ability; correspondingly, compared to ordinary porous active scaffolds, the piezoelectric scaffold of this invention allows its surface electrical signals to firmly immobilize active factors on the scaffold surface through electrostatic interactions. This not only enables effective sustained release of active factors, improving their utilization efficiency, but also reduces the amount of active factors used, to some extent avoiding potential side effects from overuse; furthermore, the surface electrical signals of the piezoelectric scaffold effectively activate early behavioral responses such as cell adhesion, spreading, and migration, which is beneficial for the subsequent active factors to fully exert their osteoinductive effect. The synergistic effect of the piezoelectric effect and bioactive factors enables rapid, high-quality repair and functional reconstruction of the defect site.

[0058] piezoelectric porous support

[0059] The piezoelectric porous scaffold described in this invention is a porous scaffold material with simple preparation steps, short preparation time, and excellent piezoelectric properties. In a preferred embodiment, the biological component used in the preparation process of this porous scaffold material is tricalcium phosphate (β-TCP), the piezoelectric component is calcium titanate (BaTiO3), and the binder is polyvinyl alcohol (PVA) solution.

[0060] Bioactive factors

[0061] The bioactive factor loaded in this experiment is bone morphogenetic protein-2 (BMP-2). This invention achieves a highly efficient osteogenic effect by loading BMP-2 onto the surface of a piezoelectric porous scaffold through a freeze-drying physical adsorption method.

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specified in the following embodiments are generally performed using conventional methods.

[0063] To better illustrate the present invention, the definitions of groups and components appearing in the embodiments are explained as shown in Table 1.

[0064] Table 1 lists the names and component definitions of each group of materials.

[0065]

[0066] Example 1

[0067] Preparation of β-TCP

[0068] Prepare 500 mL solutions of Ca(NO3)2 and (NH4)2HPO4, respectively. Under a water bath at 37°C, add 0.6 mol / L Ca(NO3)2 solution dropwise to 0.4 mol / L (NH4)2HPO4 solution with thorough stirring. Adjust the pH value with ammonia solution during the addition, strictly controlling it between 7.0 and 7.5. After the addition is complete, the pH value should be 7.2. Continue the reaction for 5 hours. After vacuum filtration and washing, obtain the precursor. Finally, sinter at 800°C for 2 hours in a high-temperature resistance furnace to obtain β-TCP, which is then passed through a 400-mesh sieve for later use.

[0069] Example 2

[0070] 2.1 Fabrication of porous scaffolds

[0071] After cutting the polyurethane foam template into cylinders of the required size, it was soaked in a 10% NaOH solution for 1 hour, rinsed with clean water, and then dried for later use. A mixed slurry was prepared according to the ratio of β-TCP:BaTiO3:10% polyvinyl alcohol solution = 1:4:5. The mixed slurry was then impregnated into the polyurethane foam, and repeatedly squeezed until the slurry was fully coated in the foam. After drying in a 60℃ oven for 72 hours, it was sintered in a high-temperature resistance furnace at 1400℃ for 180 minutes. After furnace cooling, O-BTCP was obtained. The preparation flow chart is shown below. Figure 1 As shown.

[0072] 2.2 Polarization of O-BTCP

[0073] At room temperature, O-BTCP is placed on the negative plate of the high-voltage polarization device, the distance between the polarization needle and the support surface is adjusted to 1 mm, the high-voltage power supply is turned on and the voltage is slowly increased to 6 kV. After polarization for 20 minutes, P-BTCP is obtained.

[0074] Example 3

[0075] 3.1 Preparation of O-BTCP

[0076] After cutting the polyurethane foam template into cylinders of the required size, it was immersed in a 10% NaOH solution for 1 hour, rinsed with clean water, and dried for later use. A mixed slurry was prepared according to the ratio of β-TCP:BaTiO3:10% polyvinyl alcohol solution = 1:4:5, and the polyurethane foam template was immersed in it, repeatedly squeezed to ensure the slurry fully adhered to the template. After drying in a 60℃ oven for 72 hours, it was sintered in a high-temperature resistance furnace at 1200℃ for 120 minutes, and cooled with the furnace to obtain O-BTCP. The preparation flow chart is shown below. Figure 1 As shown.

[0077] 3.2 Polarization of O-BTCP

[0078] At room temperature, O-BTCP is placed on the negative plate of the high-voltage polarization device, the distance between the polarization needle and the support surface is adjusted to 1 mm, the high-voltage power supply is turned on and the voltage is slowly increased to 3 kV. After polarization for 20 minutes, P-BTCP is obtained.

[0079] The piezoelectric coefficient d of the P-BTCP sample obtained by 6kV polarization 33 The data are shown in Table 2. Although the lower density of the P-BTCP sample due to its porous structure affects its piezoelectric properties, its average piezoelectric coefficient dp remains relatively stable. 33 It still reached 10 pC / N, which is similar to the piezoelectric coefficient d of natural bone. 33(7.5pC / N~10pC / N) is equivalent to this, which can significantly improve the electrical activity of the implantation site, meet the requirements of the material piezoelectric response during bone repair, and better accelerate the process of bone regeneration.

[0080] Table 2 shows the piezoelectric coefficient d of the P-BTCP sample with a polarization intensity of 6 kV. 33

[0081] sample 1 2 3 4 5 6 <![CDATA[d 33 (pC / N)]]> 10.1 9.5 11.0 8.4 13.3 10.6

[0082] Example 4

[0083] Morphology and performance characterization of P-BTCP

[0084] After sputtering gold onto the surface of P-BTCP for 45 seconds, its structural morphology and domain structure were observed using a digital camera and a scanning electron microscope. The results are as follows: Figure 2 As shown, P-BTCP effectively replicates the interconnected pore structure of the template, with pore sizes ranging from 100 to 500 μm. This allows for the efficient transport of nutrients or metabolic waste, as well as the adhesion and growth of bone tissue. The magnified view shows that the BaTiO3 crystals in the scaffold form an electric domain structure (black dashed box), which positively contributes to the piezoelectric properties of P-BTCP.

[0085] Although the density and domain structure of piezoelectric porous supports affect their piezoelectric properties, their average piezoelectric coefficient d 33 It still reached 10 pC / N, which is comparable to the d of natural bone. 33 (7.5pC / N~10pC / N) is equivalent to this, which can significantly improve the electrical activity of the implantation site, meet the requirements of the material piezoelectric response during bone repair, and better accelerate the process of bone regeneration.

[0086] Example 5

[0087] Cytotoxicity test of BTCP scaffold

[0088] CCK-8 (Beyotime, Shanghai) was used. O-BTCP and P-BTCP were sterilized at 121℃ and 0.12 kPa for 30 minutes, and then placed in 48-well plates according to a 1×10⁻⁶ ratio. 4 BMSCs were seeded onto the scaffold surface at cell / well density and cultured in a cell culture incubator for 1, 3, and 5 days, respectively.

[0089] Detection procedure: Discard the old culture medium, fix cells with glutaraldehyde for 15 minutes at room temperature, and gently wash three times with PBS buffer. Add 40 μL of CCK-8 to each well (protect from light), incubate at 37°C for 2 hours, and measure the absorbance at 450 nm using a continuous-wavelength microplate reader (with the absorbance at 650 nm as the background value). Results are as follows. Figure 3 As shown in Figure A; each well was treated with a gradient of ethanol for dehydration, and the scaffold surface was covered with isoamyl acetate. After drying overnight in a 37°C oven, the adhesion and spreading morphology of cells were observed using a scanning electron microscope. The results are as follows. Figure 3 As shown in B.

[0090] pass Figure 3 As shown in Figure A, the cell proliferation on the surfaces of O-BTCP and P-BTCP did not differ significantly from that of the blank control group, indicating that they have good cell compatibility, which is beneficial for cell adhesion, proliferation and differentiation on the material surface, as well as for activating the expression of osteogenic-related genes. Figure 3 As shown in Figure B, cells adhere extensively and maintain good morphology on the surface of the communicating pores (especially P-BTCP), and pseudopodia extend fully and are tightly integrated with the material, further demonstrating the potential and advantages of P-BTCP as an implant material in the field of bone tissue repair and regeneration.

[0091] Example 6

[0092] Effect of polarization operation on the saturation adsorption capacity of proteins on porous scaffolds

[0093] Bovine serum albumin (BSA) solutions with concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, and 16 mg / mL were prepared. Autoclaved O-BTCP and P-BTCP scaffolds were placed in 24-well plates, with 1 mL of protein solution of different concentrations added to each well. The plates were incubated at 37°C for 2 hours. The scaffolds were gently rinsed with PBS buffer, and then protein lysis buffer was added to dislodge all firmly adsorbed proteins. The total protein content was determined using the BCA standard curve method, and the protein adsorption capacity of the O-BTCP and P-BTCP scaffolds was calculated using the difference method. The results are shown in Table 3.

[0094] Table 3 shows the adsorption amount of bioactive proteins on the surface of the piezoelectric porous scaffold.

[0095]

[0096] Table 3 shows that the scaffold samples with three-dimensional interconnected pore structures provide ample space for protein loading due to their high specific surface area. As the protein solution concentration increases, the protein adsorption capacity of the porous scaffolds first increases rapidly, then stabilizes, indicating saturation adsorption. The saturated protein adsorption capacity on the surface of the P-BTCP scaffold is approximately twice that of the O-BTCP scaffold, demonstrating that polarization can significantly enhance the protein adsorption capacity of porous ceramics, improve the bioactivity at the implantation site, and better promote bone repair and regeneration.

[0097] O-BTCP and P-BTCP scaffolds were immersed in 8 mg / mL BSA protein solution for 2 h, respectively. After removal, they were gently rinsed twice with PBS to remove unadsorbed protein. After freeze-drying, porous scaffolds immobilized with BSA were obtained. 1 mL of PBS was added to the BSA-immobilized scaffolds, and the scaffolds were placed in a 37°C incubator with shaking at 20 rpm. 20 μL of supernatant was collected periodically and replenished with an equal volume of fresh PBS. After the sustained release was complete, the BSA concentration at each time point was determined using the BCA method, and the cumulative percentage of sustained release was calculated.

[0098] The O-BTCP sample released a large amount of protein on day 1, with a protein release of approximately 50% in the first 7 days; while the P-BTCP sample released only 20% of its protein in the first 7 days. This indicates that the electrostatic interaction between the surface charge of P-BTCP and protein molecules can achieve long-term sustained release of protein. When loaded with bioactive factors, it can greatly improve the utilization rate of bioactive factors and avoid the negative effects caused by burst release or overuse of bioactive factors.

[0099] Example 7

[0100] 7.1 Loading methods of bioactive factors

[0101] In this invention, the main methods for loading bioactive factors are physical adsorption or coupling with coupling agents (such as silane coupling agents). Since bioactive factors are easily deactivated at high temperatures and lose their biological activity, and the high thermal weight loss rate of coupling agents leads to a low loading content of bioactive factors, in order to increase the total amount of bioactive factors adsorbed while reducing the impact on protein activity, this invention preferably adopts the freeze-drying physical adsorption method.

[0102] 7.2 Loading bioactive factors

[0103] After high-temperature and high-pressure sterilization, O-BTCP and P-BTCP were immersed in 0.25 mg / mL solutions of vascular endothelial growth factor (VEGF), bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and human fibroblast growth factor 9 (FGF-9), respectively, with 200 μL of each sample, fully covering the scaffold. The samples were incubated at 37℃ with a shaking incubator for 24 h to allow for full absorption of bioactive factors. The scaffolds were then removed and aseptically freeze-dried to obtain scaffold samples loaded with bioactive factors, as shown in Table 4.

[0104] Table 4 shows scaffold samples loaded with different bioactive factors.

[0105]

[0106] Example 8

[0107] BMSCs alkaline phosphatase (ALP) activity assay

[0108] After autoclaving O-BTCP and P-BTCP scaffolds, 200 μL of BMP-2 (0.25 mg / mL) protein solution was added to each group. After incubation at 37°C for 24 h, the scaffolds were removed and aseptically freeze-dried to obtain BMP-2-loaded O-BTCP / BMP-2 and P-BTCP / BMP-2 scaffolds.

[0109] The autoclaved O-BTCP, P-BTCP, O-BTCP / BMP-2, and P-BTCP / BMP-2 stents were placed in 24-well plates at a density of 1×10⁻⁶. 5 Cells / wells were seeded onto the scaffold surface at a density of BMSCs and cultured in a cell culture incubator for 24 hours.

[0110] Detection steps: Replace the culture medium with osteogenic induction medium and culture in a cell culture incubator for 7 days (changing the medium every two days); gently wash three times with PBS buffer, add 500 μL of NP-40 cell lysis buffer to each well, and incubate at 37°C for 90 minutes; determine the total protein content using the BCA standard curve method; pipette 50 μL of lysis buffer into each well and place it in a 96-well plate, add 100 μL of ALP working solution (PNPP-Na concentration 1 mg / mL) to each well, incubate at 37°C for 2 hours, and measure the absorbance at 405 nm using a continuous spectrum microplate reader. The results are as follows: Figure 4 As shown.

[0111] The ALP activity of P-BTCP was significantly higher than that of O-BTCP, indicating that the surface electroactivity of P-BTCP samples can induce osteogenic differentiation of BMSCs by activating early cellular behavioral responses. The ALP activity of O-BTCP / BMP-2 and P-BTCP / BMP-2 scaffolds loaded with growth factors was significantly higher than that of O-BTCP and P-BTCP porous scaffolds without growth factors, indicating that high expression of growth factors can also improve the maturity of osteogenic differentiation and osteogenic mineralization capacity of BMSCs.

[0112] Example 9

[0113] Expression analysis of growth factor receptors and osteogenic-related genes

[0114] The autoclaved O-BTCP, P-BTCP, O-BTCP / BMP-2, and P-BTCP / BMP-2 stents were placed in 24-well plates at a density of 1×10⁻⁶. 5 Cells / wells were seeded onto the scaffold surface at a density of BMSCs and cultured in a cell culture incubator for 3 days.

[0115] Detection steps: Add cell lysis buffer to each well, extract mRNA, prepare reverse transcription reagent, and use PrimeScript. TM The RT reagent kit reverse transcribes mRNA into cDNA; the resulting cDNA is used as a template, and SYBR reagent and upstream and downstream primers are added for amplification using an RT-qPCR instrument; finally, the expression of the target receptor and gene is measured, and the results are as follows. Figure 5 As shown.

[0116] Compared with O-BTCP, BMP receptor expression on the P-BTCP scaffold was significantly enhanced. However, the expression of BMP receptors did not change significantly after loading the bioactive factor BMP-2, indicating that the electro-signal on the surface of the P-BTCP scaffold can significantly promote the expression of BMP receptors on the surface of BMSCs. Compared with O-BTCP, both P-BTCP and O-BTCP / BMP-2 enhanced the expression of downstream osteogenic-related genes, indicating that the electroactive and bioactive factor BMP-2 can improve the osteogenic differentiation capacity of BMSCs by stimulating osteogenic-related signaling pathways. Among them, the expression of osteogenic-related genes of BMSCs on the surface of the P-BTCP / BMP-2 scaffold was the most significant, demonstrating that the electro-signal on the scaffold surface and the bioactive factor BMP-2 can synergistically promote the rapid recruitment, adsorption, and osteogenic differentiation of cells in bone defect sites on the scaffold surface, exhibiting a faster and more significant bone repair effect.

[0117] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An active piezoelectric porous scaffold, characterized in that, The active piezoelectric porous scaffold comprises a piezoelectric porous scaffold and bioactive factors loaded on the piezoelectric porous scaffold, wherein the piezoelectric porous scaffold has a three-dimensional interconnected pore structure and is prepared by a method comprising the following steps: (i) The piezoelectric component, biological component and binder are mixed evenly to obtain a composite slurry; (ii) The composite slurry is thoroughly mixed with the porous template to obtain a composite support; (iii) The composite scaffold is sintered at high temperature to obtain a porous scaffold; (iv) The porous support is subjected to high-voltage polarization treatment to obtain the piezoelectric porous support. in, The biological component is one or a mixture of two or more of the following: hydroxyapatite, β-TCP, bioglass, calcium polyphosphate, tetracalcium phosphate, octacalcium phosphate, and biphasic calcium phosphate. The piezoelectric component is one or a mixture of two or more of calcium titanate, potassium sodium niobate, and barium titanate. The binder is one or a mixture of two or more of sodium alginate, polyvinyl alcohol, and cellulose; The adhesive is used in the form of an aqueous solution with a mass concentration of 5%-20%; The bio-component accounts for 5% to 95% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 5% to 95% of the total mass of the bio-component and the piezoelectric component; the binder accounts for 5% to 30% of the total mass of the bio-component and the piezoelectric component. The porous template is a polyurethane foam template; The sintering temperature is 800-2000℃; The polarization voltage of the polarization is 2-15 kV / mm; The piezoelectric constant d of the obtained piezoelectric porous support 33 The value is 8 pC / N ~ 13 pC / N.

2. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The adhesive is used in the form of an aqueous solution with a mass concentration of 8%-15%.

3. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The adhesive is used in the form of a 10% aqueous solution.

4. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bio-component accounts for 10% to 80% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 20% to 90% of the total mass of the bio-component and the piezoelectric component.

5. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bio-component accounts for 20% to 50% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 50% to 80% of the total mass of the bio-component and the piezoelectric component.

6. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bio-component accounts for 5%-50% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 50%-95% of the total mass of the bio-component and the piezoelectric component.

7. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bio-component accounts for 8%-35% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 65%-92% of the total mass of the bio-component and the piezoelectric component.

8. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bio-component accounts for 10%-20% of the total mass of the bio-component and the piezoelectric component, and the piezoelectric component accounts for 80%-90% of the total mass of the bio-component and the piezoelectric component.

9. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The mass of the binder accounts for 8% to 10% of the total mass of the biological component and the piezoelectric component.

10. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The biological component is β-TCP, the piezoelectric component is CaTiO3, and the binder used is polyvinyl alcohol, which is used in the form of an aqueous solution of polyvinyl alcohol with a mass concentration of 5%-20%.

11. The active piezoelectric porous scaffold as described in claim 10, characterized in that, The adhesive used is polyvinyl alcohol, and it is used in the form of an aqueous solution of polyvinyl alcohol with a mass concentration of 8%-15%.

12. The active piezoelectric porous scaffold as described in claim 10, characterized in that, The adhesive used is polyvinyl alcohol, and it is used in the form of a 10% (w / w) aqueous solution of polyvinyl alcohol.

13. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The biological component is β-TCP, and the piezoelectric component is BaTiO3. The mass of the biological component β-TCP accounts for 5%-50% of the total mass of the biological component and the piezoelectric component; the mass of the piezoelectric component BaTiO3 accounts for 50%-95% of the total mass of the biological component and the piezoelectric component.

14. The active piezoelectric porous scaffold as described in claim 13, characterized in that, The bio-component β-TCP accounts for 8%-35% of the total mass of the bio-component and piezoelectric component; the piezoelectric component BaTiO3 accounts for 65%-92% of the total mass of the bio-component and piezoelectric component.

15. The active piezoelectric porous scaffold as described in claim 13, characterized in that, The piezoelectric component BaTiO3 accounts for 80%-90% of the total mass of the biological component and the piezoelectric component; the biological component β-TCP accounts for 10%-20% of the total mass of the biological component and the piezoelectric component.

16. The active piezoelectric porous scaffold according to any one of claims 10-15, characterized in that, The mass of the binder accounts for 10%-20% of the total mass of the biological and piezoelectric components.

17. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The sintering temperature is 1100-1500℃.

18. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The sintering temperature is 1400℃.

19. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The bioactive factor is one or a mixture of two or more of VEGF, BMP-2, TGF-β1, and FGF-9.

20. The active piezoelectric porous scaffold as described in claim 19, characterized in that, The mass ratio of the bioactive factor to the piezoelectric porous scaffold is (0.1-0.5):

1.

21. The active piezoelectric porous scaffold as described in claim 19, characterized in that, The mass ratio of the bioactive factor to the piezoelectric porous scaffold is (0.2-0.3):

1.

22. The active piezoelectric porous scaffold as described in claim 19, characterized in that, The mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.26:

1.

23. The active piezoelectric porous scaffold as described in claim 1, characterized in that, The load is either physically adsorbed or coupled with a coupling agent.

24. The active piezoelectric porous scaffold according to any one of claims 13-15, characterized in that, The adhesive used is polyvinyl alcohol, and it is used in the form of an aqueous solution of polyvinyl alcohol with a mass concentration of 5%-20%.

25. The active piezoelectric porous scaffold as described in claim 24, characterized in that, The adhesive used is polyvinyl alcohol, and it is used in the form of an aqueous solution of polyvinyl alcohol with a mass concentration of 8%-15%.

26. The active piezoelectric porous scaffold as described in claim 24, characterized in that, The adhesive used is polyvinyl alcohol, and it is used in the form of a 10% (w / w) aqueous solution of polyvinyl alcohol.

27. The method for preparing the active piezoelectric porous scaffold as described in claim 1, characterized in that, The preparation method includes the preparation of a piezoelectric porous scaffold, comprising the following steps: (i) The piezoelectric component, biological component and binder are mixed evenly to obtain a composite slurry; (ii) The composite slurry is thoroughly mixed with the porous template to obtain a composite support; (iii) The composite scaffold is sintered at high temperature to obtain a porous scaffold; (iv) The porous support is subjected to high voltage polarization treatment to obtain a piezoelectric porous support; The sintering temperature is 800-2000℃; The polarization voltage in the polarization treatment is 2-15 kV / mm. The porous template is a polyurethane foam template.

28. The preparation method according to claim 27, characterized in that, The sintering temperature is 1100-1500℃.

29. The preparation method according to claim 27, characterized in that, The sintering temperature is 1400℃.

30. The preparation method according to claim 27, characterized in that, The sintering time is 1-5 hours.

31. The preparation method according to claim 27, characterized in that, The polarization voltage during polarization treatment is 5-10 kV / mm.

32. The use of the active piezoelectric porous scaffold according to claim 1, characterized in that, Materials used to prepare for the repair and treatment of bone defects.

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