Conductive polymer-induced collagen hydrogel mineralized scaffold, its preparation method and application in bone organoid construction

Abstract

This invention belongs to the technical field of biomaterial preparation methods, and relates to a conductive polymer-induced collagen hydrogel mineralization scaffold, its preparation method, and its application in bone organoid construction. The preparation method includes the following steps: dissolving type I collagen in an acidic solution with a pH of 2.0-6.0 and stirring to obtain a transparent, viscous hydrogel precursor solution; adding a phosphine-containing biomolecule solution, a calcium salt solution, and conductive polymer powder to the hydrogel precursor solution, maintaining the pH at 2.0-6.0, and performing an electrochemical deposition reaction to prepare the conductive polymer-induced collagen hydrogel mineralization scaffold. Compared with existing technologies, this invention achieves the composite of conductive polymers and in-situ mineralization of collagen hydrogel through an electrochemical deposition process. The constructed collagen hydrogel mineralization scaffold possesses suitable mechanical properties, good biocompatibility, and exhibits excellent osteogenic capacity, making it suitable as a three-dimensional culture substrate material for bone organoids, with good cell support and osteogenic induction capabilities.

Description

Technical Field

[0001] This invention relates to the field of biomaterial preparation methods, and in particular to a conductive polymer-induced collagen hydrogel mineralization scaffold, its preparation method, and its application in bone organoid construction. Background Technology

[0002] To overcome the shortcomings of traditional bone tissue engineering materials in microenvironment simulation and functional reconstruction, bone organoid technology has emerged. Organoids are miniature organoid structures formed through self-organization by inducing stem cells to differentiate into specific lineages using 3D culture technology. They can reconstruct the complex cellular composition and function of target tissues in vitro. Compared to traditional two-dimensional culture systems, bone organoids can more realistically simulate the physiological environment of bone tissue in vivo, encompassing microstructures such as vascularization and neural innervation, thus supporting the reconstruction of complete bone function.

[0003] Bone biomineralization is a highly complex and precise process involving the interaction of multiple cell types, molecules, and signaling pathways. This process is not only crucial for maintaining bone structure and function but also provides valuable insights for biomaterials engineering. However, bone organoid culture, i.e., in vitro experiments simulating bone formation and mineralization, still faces several challenges, one of which is the lack of application of the electrophysiological characteristics of bone.

[0004] Collagen is the main organic component of natural bone tissue and has good biocompatibility. Due to its similar composition to the extracellular matrix (ECM), it provides a microenvironment for osteocyte attachment and growth. In addition, mineralized collagen fibers are the main source of bone mechanical strength, but achieving intramural mineralization of collagen fibers in vitro remains a challenge that urgently needs to be solved.

[0005] Studies have shown that osteoblasts (such as osteocytes) respond to electrical stimulation, and appropriate electric fields or conductive materials can promote the proliferation, differentiation, and mineralization of osteoblasts. Conductive polymers (such as polypyrrole, polyaniline, polyethylenedioxythiophene, and their derivatives) have shown great potential in tissue engineering due to their unique conductivity, good biocompatibility, and tunable physicochemical properties. Bone organoids cultured using conductive polymers as matrix materials are expected to provide bioelectrical signals to osteoblasts, thereby regulating their behavior and promoting bone regeneration.

[0006] However, effectively combining conductive polymers with collagen hydrogels and mineralization processes to construct composite scaffolds that possess conductivity, good biocompatibility, controllable mineralization capabilities, and excellent mechanical properties still faces challenges.

[0007] In existing technologies, the preparation of collagen-based mineralized scaffolds typically requires complex post-processing steps to introduce inorganic minerals, and the introduction of conductive materials is often independent of the mineralization process or suffers from compatibility issues. This results in scaffolds that are insufficient in mimicking the multi-scale structure and function of natural bone tissue (especially the synergy of conductivity, biocompatibility, and mineralization capacity), making it difficult to meet the needs of constructing complex bone organoids. The construction of bone organoids requires scaffolds that not only support cell growth but also mimic the microenvironment of bone tissue, including the composition of the extracellular matrix, the mineralization process, and potential bioelectrical signals. Therefore, developing a novel scaffold that organically combines conductive polymers, collagen matrix, and the mineralization process is of significant scientific and practical value for constructing bone organoids that more closely resemble physiological states. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing scaffolds in terms of the synergistic effect of conductivity, biocompatibility and mineralization, and to provide a conductive polymer-induced collagen hydrogel mineralization scaffold, its preparation method and its application in bone organoid construction.

[0009] This invention assembles collagen hydrogel with conductive polymer hydrogel using electrochemical deposition technology to prepare hydrogel precursor materials with good mechanical properties and osteoinductive properties, and explores their application potential in constructing functionalized bone organoids, in order to overcome the shortcomings of existing technologies.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] One of the technical solutions of the present invention is to provide a method for preparing a conductive polymer-induced collagen hydrogel mineralization scaffold, comprising the following steps:

[0012] S1. Dissolve type I collagen in an acidic solution with a pH of 2.0~6.0 and stir to obtain a transparent and viscous type I collagen hydrogel precursor solution;

[0013] S2. Add the phosphine-containing biomolecule solution, calcium salt solution, and conductive polymer powder to the type I collagen hydrogel precursor solution in step S1, maintain the pH at 2.0~6.0, and carry out an electrochemical deposition reaction to prepare a conductive polymer-induced collagen hydrogel mineralization scaffold.

[0014] In some specific embodiments, in step S1, the acid solution is selected from any one of acetic acid, sulfuric acid, and hydrochloric acid; the type I collagen is selected from type I collagen with a molecular weight of 100-300 kDa.

[0015] The final molar concentration of the acid solution in the type I collagen hydrogel precursor solution is 0.1~5M, and the final concentration range of the type I collagen solution in the type I collagen hydrogel precursor solution is 1~50 mg / mL.

[0016] In some specific embodiments, in step S1, the stirring temperature is 30-50°C.

[0017] In some specific embodiments, in step S2, the phosphine-containing biomolecules in the phosphine-containing biomolecule solution are selected from any one of the following: adenosine triphosphate disodium (Na2ATP), creatine phosphate, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, thymidine monophosphate, thymidine diphosphate, and thymidine triphosphate.

[0018] The calcium salt in the calcium salt solution is CaCl2;

[0019] The conductive polymer is selected from any one or a combination of polyaniline, polypyrrole, and polydopamine.

[0020] In some specific embodiments, in step S2, the concentration range of the phosphine-containing biomolecule solution is 0.05~0.5 g / mL, and the concentration range of the calcium salt solution is 0.01~0.50 g / mL.

[0021] In some specific embodiments, in step S2, the ratio of calcium salt in the calcium salt solution, phosphine-containing biomolecules in the phosphine-containing biomolecule solution, conductive polymer powder, and type I collagen hydrogel precursor solution is (0.73 mg~3.65 mg): (1.1 mg~11 mg): (1.1 mg~11 mg): 1 mL.

[0022] In some specific embodiments, in step S2, the conditions for the electrochemical deposition reaction are as follows: a three-electrode system is used, with a titanium sheet as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet as the counter electrode, and the reaction is carried out for 1 to 600 min at a current of 1 to 20 mA or a voltage of 1 to 10 V.

[0023] The second technical solution of the present invention is to provide a conductive polymer-induced collagen hydrogel mineralization scaffold, which is obtained by the preparation method described in one of the above technical solutions.

[0024] The third technical solution of the present invention is to provide an application of the conductive polymer-induced collagen hydrogel mineralization scaffold as described in the second technical solution above in the construction of bone organoids.

[0025] In some specific embodiments, the method for constructing bone organoids is as follows: using the conductive polymer-induced collagen hydrogel mineralization scaffold as a matrix material, bone organoid seed cells are inoculated and cultured to obtain bone organoids.

[0026] In this invention, each component possesses a different matrix that promotes the growth and mineralization of bone marrow mesenchymal stem cells:

[0027] (1) The fibrous structure of type I collagen mimics the natural structure of human bones, which is conducive to cell attachment and growth. At the same time, type I collagen can serve as a template for bone mineralization, guiding the deposition of biomimetic natural biological minerals such as calcium and phosphorus in the gaps between collagen fibers, providing a basis for the formation of bone organs with a natural structure that mimics bones.

[0028] (2) Conductive polymers such as polyaniline and polypyrrole are polycations prepared by oxidizing aniline and pyrrole molecules. At the same time, combined with the electrophysiological properties of bone, the conductivity of conductive polymers can maximize the osteoconductivity of the constructed organoids.

[0029] The collagen hydrogel mineralization scaffold material prepared by combining the three has suitable physicochemical properties, good mineralization performance and osteoconductive properties, and is suitable for culturing bone organs that promote nerve ingrowth.

[0030] In this invention, type I collagen can become charged by binding hydrogen or hydroxide ions in the solution, thereby affecting the entry of calcium ions into the fiber interior and further influencing the collagen mineralization process. The charge of conductive polymers can interact with the charge of mineralization precursors to control the mineralization process. For example, polyaniline (PANI) and polypyrrole (PPy), and their polymeric complexes, can form positively charged polycations in an acidic environment, which electrostatically adsorb onto anionic groups (such as carboxyl and phosphate groups) on the surface of collagen fibers, promoting the adsorption and deposition of mineralization precursors; they can also exhibit charge repulsion with phosphate ions, preventing the aggregation and nucleation of mineralization precursors, thus controlling the mineralization process. Furthermore, in terms of ion interactions, conductive polymers such as polyaniline and polypyrrole can act as ion channels, promoting the transport of ions (such as calcium and phosphate ions) in the mineralization precursors, thereby accelerating the mineralization process; they can also adsorb ions from the mineralization precursors, altering the ion concentration in the solution and thus affecting the mineralization process. In terms of electrophysiological characteristics, the cationic chains of conductive polymers such as polyaniline and polypyrrole can induce phase separation of ions in mineralization precursor solutions, forming charged aggregates, thereby promoting mineralization.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Based on the physicochemical properties of type I collagen and conductive polymers, this invention achieves the composite of conductive polymers and in-situ mineralization of collagen hydrogels through an electrochemical deposition process, thereby constructing a collagen hydrogel mineralization scaffold with suitable mechanical properties, good biocompatibility, and excellent osteogenic capacity. It can be used as a three-dimensional culture substrate material for bone organoids, with good cell support and osteogenic induction capabilities.

[0033] Based on the inherent properties of the hydrogel raw materials, it can serve as a template for mineral deposition, facilitating the orderly deposition of minerals such as calcium and phosphorus to form osteoid structures, thereby promoting bone tissue mineralization. The soluble calcium salts and phosphine-containing biomolecules in the hydrogel enable the rapid and controlled release of calcium and phosphorus ions, promoting cell proliferation and osteogenic differentiation in bone organoids; the conductive polymer materials are compatible with the electrophysiological characteristics of bone.

[0034] In this hydrogel system, the molecular weight of collagen and the content of conductive polymers can be controlled to meet different experimental needs and endow the scaffold with specific functional properties. Since the magnitude of the current and the time during electrochemical deposition can lead to variations in the porosity of the scaffold material, thus affecting the elongation of stem cells within the scaffold, the degree of mineralization, mechanical properties, and regulatory ability on stem cell proliferation and differentiation can be controlled by optimizing the electrochemical deposition parameters. The scaffold prepared in this invention exhibits excellent biocompatibility and osteoconductivity, strong osteogenic differentiation capacity, and strong mid-to-late-stage osteogenic ability, making it suitable for bone organoid construction and bone defect repair, and showing broad application prospects in the fields of tissue engineering and regenerative medicine. Attached Figure Description

[0035] Figure 1 These are photographs of the gel-forming materials ECEP from Example 1 of the present invention and ECE, EC, SCEP, SCE, and SC from Comparative Examples 1 to 5.

[0036] Figure 2 These are cryo-scanning electron microscope images of ECEP, the scaffold material in Example 1 of the present invention, and ECE, EC, SCEP, SCE, and SC, the scaffold materials in Comparative Examples 1 to 5.

[0037] Figure 3 The rheological curves of ECEP, the stent material in Example 1 of the present invention, and ECE, EC, SCEP, SCE, and SC, the stent materials in Comparative Examples 1 to 5 are shown.

[0038] Figure 4 CCK-8 results of the effect of ECEP scaffold material in Example 1 of this invention and scaffold materials ECE, EC, SCEP, SCE and SC in comparative examples 1 to 5 on the proliferation of bone marrow mesenchymal stem cells in two-dimensional culture;

[0039] Figure 5 Alkaline phosphatase staining images showing the effect of ECEP scaffold material in Example 1 of the present invention and scaffold materials ECE, EC, SCEP, SCE, and SC in comparative examples 1-5 on osteogenic differentiation of bone marrow mesenchymal stem cells in two-dimensional co-culture.

[0040] Figure 6 Alizarin red staining image showing the effect of ECEP scaffold material of Example 1 and scaffold materials ECE, EC, SCEP, SCE, and SC on the formation of calcium nodules in bone marrow mesenchymal stem cells in two-dimensional co-culture.

[0041] Figure 7 These are macroscopic images of the scaffold material ECEP from Embodiment 1 of the present invention and the scaffold materials ECE, EC, and SCEP from Comparative Examples 1 to 3, applied to the construction of bone organoids.

[0042] Figure 8 The images show cryo-electron microscopy images of ECEP scaffold material in Example 1 of the present invention and scaffold materials ECE, EC, and SCEP in Comparative Examples 1-3, applied to bone organs, as well as cell stretching and elongation.

[0043] Figure 9 This is a flowchart illustrating the preparation steps of ECEP, the stent material, in Embodiment 1 of the present invention. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0045] Unless otherwise specified, the raw materials or processing techniques used in the following embodiments and comparative examples are all conventional commercially available raw materials or conventional processing techniques in the art.

[0046] Example 1

[0047] Based on type I collagen (Suzhou Pinglan Biotechnology Co., Ltd., PLJ25080901), a conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecule (Na2ATP) were introduced into the type I collagen solution. A conductive polymer-induced collagen hydrogel mineralization scaffold, denoted as ECEP, was prepared by electrochemical deposition.

[0048] like Figure 9 As shown, the specific steps include:

[0049] S1: Prepare type I collagen solution by dissolving type I collagen in an acidic solution with a pH of 2.0~6.0 and stirring evenly at 30-50℃ until all collagen sponges are dissolved into a transparent and viscous hydrogel precursor solution.

[0050] In this embodiment, type I collagen with a molecular weight of approximately 300 kDa was dissolved at a concentration of 10 mg / ml in a 0.1 M acetic acid solution with a pH of 3.5-4. The solution was stirred uniformly at 37°C until all the collagen sponges were completely dissolved and the solution became transparent and viscous, thus obtaining a collagen hydrogel precursor solution. 50 mL of the precursor solution was then placed in an electrolytic cell of a three-electrode system.

[0051] S2: Prepare Na2ATP solution by weighing adenosine 5′-triphosphate disodium salt hydrate and dissolving it in deionized water, and store it at -20℃ protected from light.

[0052] In this embodiment, adenosine 5′-triphosphate disodium salt was weighed and dissolved in deionized water to prepare a 0.11 g / mL Na2ATP solution.

[0053] S3: Prepare a CaCl2 solution by weighing CaCl2 powder and dissolving it in deionized water, then storing it at room temperature.

[0054] In this embodiment, CaCl2 powder was weighed and dissolved in deionized water to prepare a CaCl2 solution with a concentration of 0.073 g / mL.

[0055] S4: Mix the above S2 and S3 solutions in a certain mass ratio and add them to the collagen solution dissolved in S1. Add conductive polymer powder and stir thoroughly in a magnetic stirrer. During this process, maintain the pH at 2.0~6.0. After preparing the hydrogel precursor solution, store it at -20℃.

[0056] This embodiment also provides a method for preparing polyaniline-polypyrrole polymer (ANPy), which is as follows:

[0057] To prepare a 0.1 M aniline hydrochloric acid solution: Add 0.456 mL of aniline (approximately 0.466 g, 0.005 mol) to 50 mL of 1.0 M HCl and incubate on an ice bath at 0–5 °C.

[0058] To prepare a 0.1 M pyrrole hydrochloric acid solution: Take 0.347 mL of pyrrole (approximately 0.335 g, 0.005 mol) and add it to 50 mL of 1.0 M HCl. Incubate on an ice bath at 0–5 °C.

[0059] To prepare 0.1 M APS: Weigh 1.141 g APS (0.005 mol) and dissolve it in 50 mL of 1.0 M HCl.

[0060] Aniline was first pre-cooled and dissolved, pyrrole was added immediately before use, and APS was added slowly dropwise. The volume ratio of the three solutions was 1:1:1. After reacting for 8 h in an ice bath, the mixture was filtered and dried under vacuum for 12 h to obtain ANPy.

[0061] In this embodiment, 0.025 mL of Na2ATP solution with a concentration of 0.073 g / mL, 0.0177 mL of CaCl2 solution with a concentration of 0.073 g / mL, and 2.75 mg of ANPy were added to each 1 mL of type I collagen hydrogel precursor solution. The mixture was ultrasonically vibrated until homogeneous, and the pH was maintained within the range of 3.5 to 4 throughout the process. After preparing the hydrogel precursor solution, it was stored at -20°C.

[0062] S5: The hydrogels prepared by electrochemical deposition were freeze-dried for 24 h and then sterilized by ultraviolet light before being used for organoid culture.

[0063] In this embodiment, a three-electrode system (CHI660E) for electrodeposition was used, with a titanium sheet (2 cm × 3 cm) as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet (2 cm × 3 cm) as the counter electrode. The three electrodes were immersed in the hydrogel precursor solution described in S4 for electrochemical deposition. Using a controlled current-Et curve method, the current was set to 10 mA and the reaction time to 900 s. After the reaction was complete, the hydrogel film was gently peeled off from the working electrode, rinsed three times with deionized water, frozen at -20°C for 12 h, and then freeze-dried for 24 h to obtain a conductive polymer-induced collagen hydrogel mineralization scaffold, denoted as ECEP.

[0064] Comparative Example 1

[0065] The majority of the components are the same as in Example 1, except that the conductive polymer (polyaniline-polypyrrole bispolymer, ANPy) is omitted.

[0066] Specifically, based on type I collagen, a collagen hydrogel mineralization scaffold, denoted as ECE, is prepared by adding CaCl2 and Na2ATP to a type I collagen solution and then using an electrochemical deposition method.

[0067] Comparative Example 2

[0068] Compared with Example 1, most of them are the same, except that the conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecule (Na2ATP) are omitted.

[0069] Based on type I collagen, a pure collagen hydrogel mineralization scaffold, denoted as EC, was prepared by electrochemical deposition.

[0070] Comparative Example 3

[0071] Compared to Example 1, most aspects are the same, except that the electrochemical deposition in step S6 is replaced with self-assembly. The steps are as follows: the hydrogel precursor solution obtained in step S5 is adjusted to pH 7.2 and rapidly injected into a 48-well plate at a ratio of 500 μL per well. It is then incubated at 37°C for 12 hours to achieve complete gelation, thus preparing a self-assembled conductive collagen hydrogel mineralization scaffold containing a conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecules (Na2ATP), denoted as SCEP.

[0072] Comparative Example 4

[0073] Compared with Comparative Example 3, most of the results are the same, except that the conductive polymer (polyaniline-polypyrrole dimer, ANPy) is omitted, and a self-assembled collagen hydrogel mineralization scaffold, denoted as SCE, is prepared.

[0074] Comparative Example 5

[0075] Compared with Comparative Example 3, most of them are the same, the only difference being that the conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecule (Na2ATP) are omitted to prepare a self-assembled collagen hydrogel mineralization scaffold, denoted as SC.

[0076] Test Example 1

[0077] Macroscopic, microscopic, and elastic property analyses were performed on the scaffold materials prepared in Example 1 and Comparative Examples 1-5:

[0078] like Figure 1 The image shows physical images of ECEP, ECE, EC, and self-assembled SCEP, SCE, and SC prepared by electrochemical deposition. It can be seen that:

[0079] ECEP, ECE, and EC all have good gelling properties, while SCEP, SCE, and SC all have gelling properties.

[0080] ECEP and SCEP are black due to the loading of conductive polymer materials. However, compared to ECEP, SCEP gels have a more irregular shape.

[0081] ECE appears white due to the deposition of CaP during electrochemical deposition caused by the reaction of CaCl2 and Na2ATP. However, compared to ECE, SCE gels have more irregular shapes.

[0082] EC is transparent because it is not loaded with any mineralized precursors and exists under acidic conditions. In contrast, SC gels have more irregular shapes.

[0083] like Figure 2 The image shown is a cryo-scanning electron microscope (SEM) image of ECEP, ECE, EC prepared by electrochemical deposition, and self-assembled SCEP, SCE, SC. ​​It can be seen that:

[0084] ECEP, ECE, and EC all have good porosity and suitable pore size. ECEP has a certain degree of ordered structure (that is, the horizontal line structure shown in the figure) and a compact structure, which is very similar to the collagen morphology in natural bone tissue, making it suitable for cell ingrowth and elongation.

[0085] SCEP, SCE, and SC all have good porosity and suitable pore size, but compared with ECEP, ECE, and EC, they do not have a certain degree of ordered structure. This may be because they lack the arrangement of charged ions under the influence of an electric field caused by electrodeposition.

[0086] like Figure 3 The figure shows the rheological curves of ECEP, ECE, EC prepared by electrochemical deposition, and self-assembled SCEP, SCE, SC, indicating that:

[0087] The energy storage modulus (G') of ECEP, ECE, EC, SCEP, SCE, and SC is greater than the loss modulus (G''), which is consistent with the characteristics of an elastic solid.

[0088] Test Example 2

[0089] The effects of scaffold materials prepared in Examples 1 and Comparative Examples 1-5 on the proliferation of bone marrow mesenchymal stem cells were investigated in a two-dimensional co-culture with the scaffold materials. The specific experimental methods are as follows:

[0090] (1) Extraction of the extract: The prepared scaffold material was soaked in 75% alcohol and sterilized overnight under ultraviolet light; the scaffold material was soaked in the culture medium for 24 h according to the ratio of scaffold material to culture medium of 0.01 g: 1 mL, and then filtered through a 0.22 μm filter membrane for sterilization and stored in a refrigerator at 4°C.

[0091] The components and their volume percentages in the culture medium are: 10% FBS, 1% PS, and 89% α-MEM medium.

[0092] (2) BMSCs cells were loaded at 5×104 BMSCs were seeded at a density of 5 × 10⁶ wells into 96-well plates, and 100 μL of culture medium containing the extract (extraction medium to culture medium ratio 1:1) was added to each well for induction. After 24 h, 48 h, and 5 days of co-culture, cell viability was assessed using the CCK-8 cell viability kit. Similarly, BMSCs were seeded at a density of 5 × 10⁶ wells into each well. 4 The cells were seeded at a density of / wells into 96-well plates. After adding 100 μL of extraction solution to each well and culturing for 24 h, the cells were stained for liveness and deadness and photographed to record cell viability.

[0093] like Figure 4 The figure shows the CCK-8 and live / dead staining results of the effects of the extracts of ECEP, ECE, EC and self-assembled SCEP, SCE and SC scaffold materials prepared by electrochemical deposition on the proliferation of bone marrow mesenchymal stem cells in two-dimensional culture. BMSCs cells without extracts were used as blank control (Con group). The results show that the ECEP group, ECE group, EC group, SCEP group and SCE group all have good biocompatibility.

[0094] Test Example 3

[0095] The effect of scaffold materials prepared in Examples 1 and Comparative Examples 1-5 on the osteogenic differentiation of bone marrow mesenchymal stem cells in two-dimensional co-culture with bone marrow mesenchymal stem cells was investigated. The specific experimental methods are as follows:

[0096] (1) Extraction of the extract: The prepared scaffold material was soaked in 75% alcohol and sterilized overnight under ultraviolet light; the scaffold material was soaked in the culture medium for 24 h according to the ratio of scaffold material to culture medium of 0.01 g: 1 mL, and then filtered through a 0.22 μm filter membrane for sterilization and stored in a refrigerator at 4°C.

[0097] The components and their volume percentages in the culture medium are: 10% FBS, 1% PS, and 89% α-MEM medium.

[0098] (2) BMSCs cells were loaded at a rate of 1×10 5 The samples were seeded at a density in 24-well plates, and 500 μL of osteogenic induction complete medium containing the extract was added to each well for induction (the ratio of extract to osteogenic induction complete medium was 1:1). After culturing for 7 days, ALP (alkaline phosphatase) staining was performed using the BCIP / NBT ALP kit, and the results were photographed and recorded.

[0099] The osteogenic induction medium consisted of 10% FBS, 1% PS, 10 nM β-GP, 50 μg / mL Vitamin C, and 10 nM Dex added to DMEM basal medium.

[0100] like Figure 5 The image shows alkaline phosphatase staining patterns affecting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in two-dimensional co-culture, using ECEP, ECE, EC, and self-assembled SCEP, SCE, and SC cells prepared by electrochemical deposition. BMSCs without extract were used as a blank control (Con group). The results show that:

[0101] Compared with other groups, the ECEP group showed a deeper color in alkaline phosphatase staining and the best positive results in quantitative analysis. This may be because the conductive polymer has conductivity, and the presence of the conductive polymer enhances the electrostatic adsorption of collagen. At the same time, electrocatalysis decomposes ATP in Na2ATP to obtain P, which promotes the in-situ assembly of P and Ca in CaCl2 solution.

[0102] The color of alkaline phosphatase staining in the ECE group was not as good as that in the ECEP group. This may be because the removal of conductive polymers greatly reduced the electrostatic and ionic effects on the mineralization precursors, thereby weakening the early osteogenic capacity.

[0103] The EC group showed a deeper color from alkaline phosphatase staining compared to the control group, and the positive result from quantitative analysis was also higher than that of the control group. This demonstrates that ECs prepared by electrochemical deposition have the ability to stimulate osteogenic differentiation. However, the ECs were not as effective as the ECEP group, possibly because the removal of conductive polymers significantly reduced the electrostatic and ionic effects on the mineralization precursors, thus weakening their early osteogenic ability. The ECs were also not as effective as the ECE group, possibly because they lacked the calcium phosphate component formed by CaCl2 and Na2ATP under electrochemical assisted catalysis, which weakened the osteogenic induction ability of the scaffold material for BMSCs.

[0104] Compared to the ECEP group, the SCEP group showed a lighter alkaline phosphatase staining color, possibly due to the lack of osteogenic ability from the electrochemical-assisted deposition method; compared to other groups, the SCEP group showed a darker alkaline phosphatase staining color, indicating that the mineralization-promoting ability of conductive polymers can offset some of the effects of the self-assembly synthesis method.

[0105] Compared to the ECE group, the SCE group showed a lighter alkaline phosphatase staining, possibly due to the lack of osteogenic capacity from the electrochemical-assisted deposition method; compared to the EC group, the SCE group showed a darker alkaline phosphatase staining, indicating that the mineralization precursors formed by soluble calcium salts and phosphine-containing biomolecules can offset some of the effects of the self-assembly synthesis method.

[0106] Compared with the SCE group, the alkaline phosphatase staining color in the SC group was lighter, which may be due to the lack of mineralization precursors and the lack of ion transport in mineralization precursors, thus weakening the osteogenic capacity.

[0107] Test Example 4

[0108] The effect of scaffold materials prepared in Examples 1 and Comparative Examples 1-5 on calcium nodule formation in bone marrow mesenchymal stem cells during two-dimensional co-culture was investigated. The specific experimental methods are as follows:

[0109] (1) Extraction of the extract: The prepared scaffold material was soaked in 75% alcohol and sterilized overnight under ultraviolet light; the scaffold material was soaked in the culture medium for 24 h according to the ratio of scaffold material to culture medium of 0.01 g: 1 mL, and then filtered through a 0.22 μm filter membrane for sterilization and stored in a refrigerator at 4°C.

[0110] The components and their volume percentages in the culture medium are: 10% FBS, 1% PS, and 89% α-MEM medium.

[0111] (2) BMSCs cells were loaded at a rate of 1×10 5 The samples were seeded at a density in 24-well plates, and 500 μL of osteogenic induction complete medium containing the extract was added to each well for induction (the ratio of extract to osteogenic induction complete medium was 1:1). After culturing for 14 days, alizarin red staining was performed using alizarin red solution, and photographs were taken for recording.

[0112] The osteogenic induction complete medium consisted of 10% FBS, 1% PS, 10 nM β-GP, 50 μg / mL Vitamin C, and 10 nM Dex added to DMEM basal medium.

[0113] like Figure 6 The image shows the effect of Alizarin Red staining on the formation of calcium nodules in bone marrow mesenchymal stem cells (BMSCs) prepared by electrochemical deposition, including ECEP, ECE, EC, and self-assembled SCEP, SCE, and SC, in two-dimensional co-culture. BMSCs without the extraction solution were used as a blank control (Con group). The results show that:

[0114] Compared to other groups, the ECEP group showed a deeper and larger area of ​​red color, indicating a greater quantity and extent of calcium nodules. The quantitative analysis also yielded the best positive results, demonstrating ECEP's excellent ability to promote bone formation in the mid-to-late stages. This may be because polyaniline and polypyrrole can act as ion channels, promoting the transport of ions that are mineralization precursors, thereby accelerating the mineralization process and promoting the formation of calcium nodules.

[0115] Compared to the ECEP group, the ECE group had a lighter color, indicating a smaller number of calcium nodules. This may be because the removal of conductive polymers inhibited the formation of calcium nodules in the later stages of osteoblast development.

[0116] Compared to the ECEP group, the EC group had a lighter color, indicating a lower number of calcium nodules. This may be because the removal of conductive polymers inhibited the formation of calcium nodules in the later stages of osteogenic development. The lack of calcium phosphate, formed from CaCl2 and Na2ATP under electrochemical catalytic conditions, also weakened the osteogenic induction ability of BMSCs. Compared to the ECE group, the EC group also showed weakened osteogenic induction ability of BMSCs due to the absence of calcium phosphate.

[0117] Compared with the ECEP group, the SCEP group had less alizarin red stained calcium nodules, which may be due to the lack of late-stage osteogenic capacity of the electrochemical-assisted deposition method. Compared with other groups, the SCEP group had more alizarin red stained calcium nodules, indicating that conductive polymers can act as ion channels to promote the transport of ions of mineralization precursors, thereby accelerating the mineralization process.

[0118] Compared with the ECE group, the SCE group had less alizarin red staining calcium nodules, possibly due to a lack of late-stage osteogenic capacity from electrochemically assisted deposition methods. Compared with the EC group, the SCE group had more alizarin red staining calcium nodules, indicating that the various anions and cations decomposed in the mineralization precursors and the zwitterions from collagen decomposition under neutral and alkaline conditions promote the transport of ions in the mineralization precursors, thereby accelerating the mineralization process.

[0119] Compared with the SCE group, the SC group had less alizarin red staining calcium nodules, which may be due to the lack of mineralization precursors and the lack of ion transport in the mineralization precursors, thus weakening the late-stage osteogenic capacity.

[0120] Test Example 5

[0121] The scaffold materials prepared in Example 1 and Comparative Examples 1-3 were subjected to bone organoid culture, including the following steps:

[0122] (1) A large number of BMSCs cells were expanded. After collecting the cells by digestion with trypsin, the cells were centrifuged, the supernatant was discarded, and the cells were resuspended in culture medium at 37°C.

[0123] (2) Cut the scaffold material into a cylindrical shape with a diameter of 3 mm and a thickness of 1 mm, soak it in EDC / NHS crosslinking agent, and sterilize and crosslink it under ultraviolet light for 24 h.

[0124] (3) The cross-linked scaffold material was immersed in MEM complete culture medium and then subjected to 5*10 6 / cm 3 Cells are seeded at a density of scaffold material and cultured adaptively for 1-3 days.

[0125] The composition of the MEM complete medium is: 10% volume of FBS and 1% volume of PS added to the DMEM basal medium.

[0126] (4) Osteogenic induction of bone organoids: Replace the MEM complete medium in step (3) with osteogenic induction medium. After 30 days of culture, bone organoids are formed and can be used for subsequent analysis.

[0127] The osteogenic induction medium consisted of 10% FBS, 1% PS, 10 nM β-GP, 50 μg / mL Vitamin C, and 10 nM Dex added to DMEM basal medium.

[0128] like Figure 7 The image shown is a macroscopic photograph of ECEP, ECE, EC, and self-assembled SCEP prepared by electrochemical deposition in cultured BMSCs cells to form bone organoids. It can be seen that:

[0129] No significant structural collapse was observed in ECEP, ECE, EC, and SCEP after 30 days of culture.

[0130] like Figure 8 The image shows scanning electron microscopy (SEM) images of ECEP, ECE, EC, and self-assembled SCEP prepared by electrochemical deposition in cultured BMSCs cells to form bone organoids. As can be seen:

[0131] The ECEP group has an ordered structure, and ECE and EC have a relatively ordered structure (i.e., the horizontal line structure shown in the figure), and the BMSCs cells contained therein (shown in red in the figure) can stretch and spread well. In contrast, SCEP lacks a relatively ordered structure, thus limiting the stretching and spreading of BMSCs cells.

[0132] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a conductive polymer-induced collagen hydrogel mineralization scaffold, characterized in that, Includes the following steps: S1. Dissolve type I collagen in an acidic solution with a pH of 2.0~6.0 and stir to obtain a transparent and viscous type I collagen hydrogel precursor solution; S2. Add the phosphine-containing biomolecule solution, calcium salt solution, and conductive polymer powder to the type I collagen hydrogel precursor solution in step S1, maintain the pH at 2.0~6.0, and carry out an electrochemical deposition reaction. After the reaction is completed, peel off the hydrogel film from the working electrode to prepare a conductive polymer-induced collagen hydrogel mineralization scaffold. The phosphine-containing biomolecule in the phosphine-containing biomolecule solution is adenosine 5′-triphosphate disodium salt hydrate; The calcium salt in the calcium salt solution is CaCl2; The conductive polymer is a polyaniline-polypyrrole polymer ANPy, which is obtained by the following preparation method: To prepare a 0.1 M aniline hydrochloric acid solution: Take 0.456 mL of aniline, i.e., 0.466 g, 0.005 mol, and add it to 50 mL of 1.0 M HCl. Incubate on an ice bath at 0–5 °C. To prepare a 0.1 M pyrrole hydrochloric acid solution: Take 0.347 mL of pyrrole (0.335 g, 0.005 mol), add 50 mL of 1.0 M HCl, and incubate in an ice bath at 0–5℃. To prepare 0.1 M APS: Weigh 1.141 g APS, or 0.005 mol, and dissolve it in 50 mL of 1.0 M HCl; Aniline was first pre-cooled and dissolved, pyrrole was added immediately before use, and APS was added slowly dropwise. The volume ratio of the three solutions was 1:1:

1. After reacting for 8 h under ice bath conditions, the mixture was filtered and dried under vacuum for 12 h to obtain ANPy. In step S2, the ratio of calcium salt in the calcium salt solution, phosphine-containing biomolecules in the phosphine-containing biomolecule solution, conductive polymer powder, and type I collagen hydrogel precursor solution is (0.73 mg~3.65 mg): (1.1 mg~11 mg): (1.1 mg~11 mg): 1 mL; In step S2, the conditions for the electrochemical deposition reaction are as follows: a three-electrode system is used, with a titanium sheet as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet as the counter electrode, and the reaction is carried out at a current of 1~20 mA or a voltage of 1~10 V for 1~600 min.

2. The method for preparing the conductive polymer-induced collagen hydrogel mineralization scaffold according to claim 1, characterized in that, In step S1, the acid solution is selected from any one of acetic acid, sulfuric acid, and hydrochloric acid; the type I collagen is selected from type I collagen with a molecular weight of 100-300 kDa. The final molar concentration of the acid solution in the type I collagen hydrogel precursor solution is 0.1~5M, and the final concentration range of the type I collagen solution in the type I collagen hydrogel precursor solution is 1~50 mg / mL.

3. The method for preparing the conductive polymer-induced collagen hydrogel mineralization scaffold according to claim 1, characterized in that, In step S1, the stirring temperature is 30-50℃.

4. A conductive polymer-induced collagen hydrogel mineralization scaffold, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 3.

5. The application of the conductive polymer-induced collagen hydrogel mineralization scaffold as described in claim 4 in the construction of bone organoids.

6. The application of the conductive polymer-induced collagen hydrogel mineralization scaffold according to claim 5 in the construction of bone organoids, characterized in that, The method for constructing bone organoids is as follows: using the conductive polymer-induced collagen hydrogel mineralization scaffold as the matrix material, bone organoid seed cells are inoculated and cultured to obtain bone organoids.

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