A nerve growth-promoting bone organoid and a construction method and application thereof

Abstract

The application belongs to the technical field of bone organ preparation, and relates to a bone organ for promoting nerve growth and a construction method and application thereof. The construction method comprises the following steps: S1, immersing a hydrogel mineralization scaffold with conductive polymer induction in an EDC / NHS crosslinking agent, and sterilizing and crosslinking; S2, immersing the crosslinked product in a culture medium and culturing, inoculating bone organ seed cells, and replacing the culture medium with a complete culture medium for adaptive culture; and S3, transferring to an osteogenic induction culture medium for osteogenic induction, so as to obtain the bone organ for promoting nerve growth. Compared with the prior art, the bone organ constructed by the application has good biological activity, osteogenic activity and bone conduction, can effectively promote the growth of peripheral nerves, provides a reliable in-vitro model for bone-nerve interaction research, and provides a new technical path and theoretical basis for bone-nerve integrated regeneration and repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bone organ preparation, and in particular to a bone organ for promoting nerve growth, a construction method thereof and application. BACKGROUND

[0002] When the area of bone defect is small, most of the bone tissue has the ability of self-repair. However, when the bone defect exceeds the natural repair threshold (usually 2.5 cm), the tissue can be continuously necrotic and difficult to achieve functional regeneration. Various pathological factors can cause or aggravate bone tissue damage, such as tumor resection, severe trauma, infection, congenital deformity, bone dysplasia, rheumatoid arthritis and osteoporosis and the like.

[0003] At present, the treatment of bone defect in clinic mainly depends on bone transplantation technology, including autologous bone transplantation and allogeneic bone transplantation. Autologous bone transplantation is widely used due to its excellent biocompatibility, but has problems such as limited bone source and large surgical trauma; allogeneic bone transplantation can cause immune rejection and infection risk. In recent years, the development of bone tissue engineering technology provides a new idea for the treatment of bone defect. Typical materials such as bioglass and calcium phosphate cement can provide mechanical support and promote bone regeneration to a certain extent. However, these materials still have problems such as insufficient ability to simulate the natural bone microenvironment, limited regeneration effect, and risk of infection or rejection. Therefore, it has important clinical significance and application value to develop new bone repair materials with good biological activity, adjustable structure and excellent osteogenic performance.

[0004] Bone tissue shows certain electrical responsiveness in the process of damage repair and reconstruction, and can respond to and regulate exogenous or endogenous bioelectric signals. This electrical physiological characteristic is closely related to its structural components, among which the hydroxyapatite in the inorganic phase has piezoelectricity and surface charge accumulation ability, and the type I collagen in the organic phase has piezoelectricity and certain dielectric properties, which can respond to mechanical stimulation and produce local electrical signals at the microscale, thereby regulating cell behavior and mineralization process. In addition, various cells in bone tissue, such as osteoblasts, osteocytes and osteoclasts, all show good electrochemical response ability. Therefore, bone tissue has typical "bone-electric biology" characteristics, which has important influence on its osteogenesis, mineralization and cell behavior regulation.

[0005] However, the current research on bone organ still relatively ignores the electrical physiological characteristics of bone tissue, and lacks material design and systematic research aiming at enhancing "bone conductivity". The existing matrix material has obvious deficiencies in the functional construction of bone-nerve interface, which limits its application potential in nerve regulation of bone regeneration. SUMMARY

[0006] The purpose of this invention is to provide a bone organoid that promotes nerve ingrowth, a method for constructing it, and its application in bone repair.

[0007] To address the aforementioned issues, this invention proposes a method for culturing bone organoids using conductive polymer composite collagen as a base and conductive polymer-induced collagen mineralization scaffold materials. The prepared bone organoids exhibit excellent bioelectrical activity, significantly promoting osteoblast proliferation and differentiation, and accelerating bone formation and mineralization. Simultaneously, by combining the effects of Schwann cells and their secreted exosomes, the growth of nerve fibers into bone defect areas can be further induced, achieving integrated bone-nerve tissue repair. This provides a new technical pathway and theoretical basis for constructing functional bone organoids with neural pathway capabilities.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a method for constructing bone organoids that promote nerve ingrowth, comprising the following steps:

[0010] S1. The hydrogel mineralization scaffold with conductive polymer-induced properties is immersed in EDC (1-ethyl-3-(3-dimethylaminopropyl)) / NHS (N-hydroxysuccinimide) crosslinking agent and sterilized and crosslinked under ultraviolet light.

[0011] The method for preparing the hydrogel mineralization scaffold induced by the conductive polymer is as follows:

[0012] A hydrogel matrix material, conductive polymer, water-soluble calcium salt, and phosphine-containing biomolecules are mixed in an acidic solution and electrochemically deposited to obtain a hydrogel mineralization scaffold induced by conductive polymer.

[0013] S2. After cleaning the cross-linked product from step S1, soak it in complete culture medium and let it stand for culture. Inoculate bone organoid seed cells and continue to use complete culture medium for adaptive culture.

[0014] S3. Transfer the product obtained from step S2 to osteogenic induction medium for osteogenic induction to obtain bone organoids that promote nerve ingrowth.

[0015] In some specific embodiments, in step S1, the hydrogel matrix material is selected from any one of GelMA hydrogel, HAMA hydrogel, Matrigel matrix gel, and type I collagen;

[0016] The conductive polymer is selected from any one or more combinations of polyaniline, polypyrrole, and polydopamine;

[0017] The water-soluble calcium salt is CaCl2;

[0018] The phosphophosphoric biomolecule is selected from any one of the following: adenosine triphosphate disodium, 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.

[0019] The ratio of the water-soluble calcium salt, phosphine-containing biomolecule, conductive polymer, and hydrogel matrix material is (0.73 mg: 1.1 mg: 1.1 mg: 1 mL) to (3.65 mg: 11 mg: 11 mg: 1 mL), and the concentration of the hydrogel matrix material is 1 to 50 mg / mL.

[0020] The pH of the acidic solution is 2.0~6.0;

[0021] The electrochemical deposition reaction conditions 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.

[0022] In some specific embodiments, the crosslinking time in step S1 is 12~48 h.

[0023] In some specific embodiments, in step S2, the complete culture medium is composed of 10% volume of FBS (fetal bovine serum) and 1% volume of PS (penicillin-streptomycin mixture) added to the basal culture medium, wherein the basal culture medium is selected from any one of α-MEM, DMEM, and DMEM / F-12;

[0024] The conditions for static incubation are: incubation at 37℃ for 1~12 h.

[0025] In some specific embodiments, in step S2, the bone organoid seed cells are selected from any one of bone marrow mesenchymal stem cells (BMSC), mouse embryonic osteoblast precursor cells (MC3T3-E1), plasmacytoid dendritic cells (pDC), and osteoblasts.

[0026] In some specific embodiments, in step S2, the seeding density of the bone organoid seed cells is 1*102 3~ 1*10 9 / cm 3 Support material.

[0027] In some specific implementations, in step S2, the adaptive culture conditions are: cultured at 37°C for 1 to 3 days.

[0028] In some specific embodiments, in step S3, the osteogenic induction medium is composed of: 10% volume of FBS (fetal bovine serum), 1% volume of PS (penicillin-streptomycin mixture), 1% volume of 1M β-GP (β-glycerophosphate disodium hydrate), 0.25% volume of 20 mg / mL Vitamin C, and 0.01% volume of 100 μM Dex (dexamethasone) added to DMEM medium.

[0029] The conditions for osteogenic induction are: culture at 37℃ for 1 to 45 days, with fresh osteogenic induction medium replaced every 2 to 3 days during the culture period.

[0030] More preferably, the osteogenic induction medium is composed of DMEM medium with the addition of 10% volume of FBS (fetal bovine serum), 1% volume of PS (penicillin-streptomycin mixture), 1% volume of 1M β-GP (β-glycerophosphate disodium hydrate), 0.25% volume of 20 mg / mL Vitamin C, 0.01% volume of 100 μM Dex (dexamethasone), and an active factor, wherein the active factor is selected from any one of BMP-2, VEGF, EGF, and FGF, and the final concentration of the active factor is 1 μg / mL.

[0031] More preferably, the osteogenic induction culture medium can also be a commercially available osteogenic differentiation induction culture medium, depending on the requirements.

[0032] The second technical solution of the present invention is to provide a bone organoid that promotes nerve ingrowth, which is prepared by the construction method described in one of the above technical solutions.

[0033] The third technical solution of the present invention is to provide an application of bone organoids that promote nerve ingrowth as described in the second technical solution above in bone regeneration and repair.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention, based on the structure and function of bone tissue, biomimetically constructs bone organoids with electrical signal transduction capabilities. Through carefully designed matrix materials combined with a rational cell culture strategy, the osteogenic differentiation process of stem cells is effectively regulated, thereby enabling the material to guide cell behavior and the cells to provide feedback regulation of the material structure, thus promoting the tissue construction of bone organoids.

[0036] Based on a conductive polymer-induced hydrogel mineralization scaffold, stem cells are seeded in layers onto a freeze-dried scaffold and undergo processes such as proliferation, migration, self-organization, and osteogenic differentiation to form bone organoids. The cells used can be selected according to the needs of different stages of bone defect repair. The constructed bone organoids exhibit good bioactivity, osteogenic activity, and osteoconductivity, and can effectively promote the ingrowth of peripheral nerves. This provides a reliable in vitro model for the study of bone-nerve interactions and offers a new technical pathway and theoretical basis for integrated bone-nerve regeneration and repair.

[0037] This invention employs a biomimetic strategy to prepare bone organoids that promote nerve ingrowth for bone defect repair. Utilizing a hydrogel matrix material, it provides a foundation for the deposition of minerals such as hydroxyapatite. Furthermore, the hydrogel can form a highly ordered hierarchical structure, imparting strength and toughness to the bone. By mimicking this structure, the hydrogel matrix material exhibits excellent biocompatibility and mechanical properties. The interaction between conductive polymers and hydrogel fibers promotes the incorporation of calcium phosphate precursors into the hydrogel fibers, and electrochemical assembly technology enables the effective deposition of phosphine-containing biomolecules within the hydrogel fibers. Since nerves in bone tissue can release neurotransmitters and hormones, regulating bone metabolism and influencing the activity of osteoblasts and osteoclasts, thereby affecting bone formation and resorption, the bone organoids cultured using a conductive polymer-induced mineralized hydrogel scaffold as a matrix can, to some extent, promote the growth and regeneration of nerve cells, providing support for the recovery of nerve function after bone defect repair. Attached Figure Description

[0038] Figure 1 This is a physical image of the bone organoid of the present invention and the extension of cells within the organoid tissue.

[0039] Figure 2 This is a compressive stress-strain curve of the bone organoid of the present invention.

[0040] Figure 3 This is a three-dimensional live / dead staining image of cells and a three-dimensional cytoskeleton staining image of cells in bone organ tissues according to the present invention.

[0041] Figure 4 This image shows the ingrowth verification of Schwann cells (SC) and BMSCs in bone organoid tissues of the present invention.

[0042] Figure 5 This is a Micro-CT three-dimensional reconstruction image of the bone organoid of the present invention.

[0043] Figure 6 This is an H&E staining image of the bone organoids of the present invention.

[0044] Figure 7 This image shows the expression of the β3-Tubulin gene in Schwann cells after co-culturing bone organoids with Schwann cells for 3 days, according to the present invention.

[0045] Figure 8 Bright-field images and three-dimensional reconstructed cross-sectional and longitudinal sections of micro-CT scans taken 4 weeks after bone organoids were implanted subcutaneously in nude mice.

[0046] Figure 9 This is a Micro-CT three-dimensional reconstruction image and horizontal and coronal plane scan images of the bone organoid that promotes nerve ingrowth in this invention for the formation of new bone at the bone defect site after skull defect repair in C57 mice.

[0047] Figure 10 A flowchart illustrating a method for constructing bone organoids that promote nerve ingrowth, as provided by this invention. Detailed Implementation

[0048] 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.

[0049] In the following embodiments and comparative examples, the above-mentioned conductive polymer-induced hydrogel mineralization scaffold is used. Unless otherwise specified, the other raw materials or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0050] Example 1

[0051] This example provides a bone organoid that promotes nerve ingrowth and its construction method. Based on the electrochemical deposition method, a conductive polymer-induced collagen hydrogel mineralization scaffold, denoted as ECEP, is prepared in a type I collagen solution containing soluble calcium salt (CaCl2), phosphine-containing biomolecules (Na2ATP) and conductive polymer (polyaniline-polypyrrole dimer, ANPy). Subsequently, a large number of expanded BMSC cells are seeded in the sterilized ECEP scaffold material and adaptively cultured for 1-3 days and osteogenic induced differentiation for 30 days.

[0052] like Figure 10 As shown, the specific steps include:

[0053] S1: Preparation of freeze-dried hydrogel matrix material for bone organoids based on conductive polymer-induced collagen hydrogel mineralization scaffolds (i.e., ECEP):

[0054] S101. Preparation of Type I Collagen Solution: Dissolve Type I collagen with a molecular weight of approximately 300 kDa at a concentration of 10 mg / ml in a 0.1 M acetic acid solution with a pH of 3.5-4. Stir evenly at 37°C until all collagen sponges are completely dissolved. After the solution becomes transparent and viscous, a collagen hydrogel precursor solution is obtained. Take 50 mL of the solution and place it in an electrolytic cell of a three-electrode system.

[0055] S102. Preparation of Na2ATP solution: Weigh adenosine 5′-triphosphate disodium salt and dissolve it in deionized water to prepare a 0.11 g / mL Na2ATP solution.

[0056] S103. Preparation of CaCl2 solution: Weigh CaCl2 powder and dissolve it in deionized water to prepare a 0.073 g / mL CaCl2 solution.

[0057] S104. Preparation of polyaniline-polypyrrole polymer (ANPy):

[0058] 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.

[0059] 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.

[0060] 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.

[0061] First, pre-cool and dissolve aniline, add pyrrole immediately before use, and slowly add APS dropwise. The volume ratio of the three solutions is 1:1:1. React under ice bath conditions for 4-24 hours, then filter and vacuum dry for 12 hours.

[0062] S105. Add 0.025 mL of 0.073 g / mL Na2ATP solution, 0.0177 mL of 0.073 g / mL CaCl2 solution, and 2.75 mg ANPy to each 1 mL of type I collagen hydrogel precursor solution. Sonicate the mixture until homogeneous, keeping the pH within the range of 3.5 to 4 throughout the process. After preparing the hydrogel precursor solution, store it at -20°C.

[0063] S106. 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 S105 for electrochemical deposition. Using the method of controlling the current Et curve, the current was set to 10 mA and the reaction time to 900 s. After the reaction was completed, the hydrogel film was gently peeled off from the working electrode, rinsed three times with deionized water, frozen at -20℃ for 12 h, and then freeze-dried for 24 h to obtain a conductive polymer-induced collagen hydrogel mineralization scaffold, denoted as ECEP.

[0064] S2: Provide bone organoid seed cells (BMSC cells in this example) for culture and expand them in large quantities using complete culture medium.

[0065] S3: The freeze-dried hydrogel scaffold material (i.e., ECEP) was immersed in EDC (1-ethyl-3-(3-dimethylaminopropyl)) / NHS (N-hydroxysuccinimide) crosslinking agent and sterilized and crosslinked under ultraviolet light for 24 h.

[0066] S4: Adaptive culture of bone organoids to promote nerve ingrowth. After washing the samples obtained in S3 three times with PBS, they were immersed in complete culture medium and incubated at 37°C for 5 minutes, followed by washing. BMSC cells were then seeded at a density of 5 x 10⁻⁶ cells / year. 6 / cm 3 The scaffold material was then replaced with fresh complete culture medium and subjected to adaptive culture at 37°C for 1–3 days.

[0067] The complete culture medium consists of 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin mixture) added to α-MEM basal medium.

[0068] S5: Osteogenic induction differentiation culture of bone organoids that promote nerve ingrowth. The samples obtained in S4 were transferred to osteogenic induction medium and osteogenic induction was performed at 37°C for 30 days. Fresh medium was replaced every 2-3 days during the culture period to obtain bone organoids that promote nerve ingrowth.

[0069] The osteogenic induction medium consisted of: 10% FBS (fetal bovine serum), 1% PS (penicillin-streptomycin mixture), 1% 1M β-GP (β-glycerophosphate disodium hydrate), 0.25% 20 mg / mL Vitamin C, and 0.01% 100 μM Dex (dexamethasone) added to DMEM medium.

[0070] Comparative Example 1

[0071] Compared with Example 1, most of the components are the same, except that the conductive polymer (polyaniline-polypyrrole dimer, ANPy) is omitted. The resulting scaffold material is denoted as ECE.

[0072] In this Comparative Example 1, bone organoids were cultured using ECE as a substrate.

[0073] Comparative Example 2

[0074] Compared with Example 1, most of the components are the same, except that the soluble calcium salt (CaCl2), phosphine-containing biomolecule (Na2ATP), and conductive polymer (polyaniline-polypyrrole dimer, ANPy) are omitted. The resulting scaffold material is denoted as EC.

[0075] In this comparative example 2, bone organoids were cultured using EC as a substrate.

[0076] Comparative Example 3

[0077] This comparative example provides a bone organoid that promotes nerve ingrowth and its construction method. Based on a physical mixing method, a self-assembled conductive collagen mineralized hydrogel, denoted as SCEP, is obtained by adjusting the pH of the precursor solution to 7.2 in a type I collagen solution containing a conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecules (Na2ATP). A large number of expanded BMSCs are seeded in the UV-sterilized SCEP scaffold material and adaptively cultured for 1-3 days followed by osteogenic induction differentiation for 30 days. The method includes the following steps:

[0078] S1: Lyophilized matrix material for preparing SCEP hydrogels:

[0079] S101. Preparation of Type I Collagen Solution: Dissolve Type I collagen with a molecular weight of approximately 300 kDa at a concentration of 10 mg / ml in a 0.1 M acetic acid solution with a pH of 3.5-4. Stir evenly at 37°C until all collagen sponges are completely dissolved. After the solution becomes transparent and viscous, a collagen hydrogel precursor solution is obtained. Take 50 mL of the solution and place it in an electrolytic cell of a three-electrode system.

[0080] S102. Preparation of Na2ATP solution: Weigh adenosine 5′-triphosphate disodium salt and dissolve it in deionized water to prepare a 0.11 g / mL Na2ATP solution.

[0081] S103. Preparation of CaCl2 solution: Weigh CaCl2 powder and dissolve it in deionized water to prepare a 0.073 g / mL CaCl2 solution.

[0082] S104. Preparation of polyaniline-polypyrrole polymer (ANPy):

[0083] 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.

[0084] 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.

[0085] 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.

[0086] First, pre-cool and dissolve aniline, add pyrrole immediately before use, and slowly add APS dropwise. The volume ratio of the three solutions is 1:1:1. React under ice bath conditions for 4-24 hours, then filter and vacuum dry for 12 hours.

[0087] S105. Add 0.025 mL of 0.073 g / mL Na2ATP solution, 0.0177 mL of 0.073 g / mL CaCl2 solution, and 2.75 mg ANPy to each 1 mL of type I collagen hydrogel precursor solution. Sonicate the mixture until homogeneous, keeping the pH within the range of 3.5 to 4 throughout the process. After preparing the hydrogel precursor solution, store it at -20°C.

[0088] S106. Adjust the pH of the hydrogel precursor solution obtained in step S105 to 7.2, and rapidly inject it into a 48-well plate at a ratio of 500 μL per well. Incubate at 37°C for 12 hours to achieve complete gelation, thereby preparing a self-assembled conductive collagen hydrogel mineralization scaffold containing conductive polymer (polyaniline-polypyrrole dimer, ANPy), soluble calcium salt (CaCl2), and phosphine-containing biomolecule (Na2ATP), denoted as SCEP.

[0089] S2: Provide seed cells for culturing bone organoids (BMSC cells in this comparative example), and culture and expand them in large quantities using complete culture medium.

[0090] S3: Immerse the SCEP scaffold material in EDC (1-ethyl-3-(3-dimethylaminopropyl)) / NHS (N-hydroxysuccinimide) crosslinking agent and sterilize and crosslink under ultraviolet light for 24 h.

[0091] S4: Adaptive culture of bone organoids to promote nerve ingrowth. After washing the samples obtained in S3 three times with PBS, they were immersed in complete culture medium and incubated at 37°C for 5 minutes, followed by washing. BMSC cells were then seeded at a density of 5 x 10⁻⁶ cells / year. 6 / cm 3 The scaffold material was then replaced with complete culture medium and adaptively cultured at 37°C for 1-3 days.

[0092] The complete culture medium consists of 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin mixture) added to α-MEM basal medium.

[0093] S5: Osteogenic induction differentiation culture of bone organoids that promote nerve ingrowth. The samples obtained in S4 were transferred to osteogenic induction medium and osteogenic induction was performed at 37°C for 30 days. Fresh medium was used every 2-3 days during the culture period to obtain bone organoids.

[0094] The osteogenic induction medium consisted of: 10% FBS (fetal bovine serum), 1% PS (penicillin-streptomycin mixture), 1% 1M β-GP (β-glycerophosphate disodium hydrate), 0.25% 20 mg / mL Vitamin C, and 0.01% 100 μM Dex (dexamethasone) added to DMEM medium.

[0095] Comparative Example 4

[0096] Compared with Comparative Example 3, most of them are the same, the only difference being that the conductive polymer (polyaniline-polypyrrole dimer, ANPy) was omitted. The resulting scaffold material is denoted as SCE.

[0097] In this comparative example 4, bone organoids were cultured using SCE as a substrate.

[0098] Comparative Example 5

[0099] Compared with Comparative Example 3, most of them are the same, the only difference being that the soluble calcium salt (CaCl2), phosphine-containing biomolecule (Na2ATP), and conductive polymer (polyaniline-polypyrrole dimer, ANPy) are omitted. The scaffold material prepared is denoted as SC.

[0100] In this comparative example 5, bone organoids were cultured using SC as a substrate.

[0101] Test Example 1

[0102] The bone organoids prepared in Example 1 and Comparative Examples 1-3 were observed macroscopically and microscopically, such as... Figure 1As shown, the bone organoids cultured using ECEP scaffold material as the matrix in this culture method have relatively regular shapes. The cryo-scanning electron microscopy image shows that the bone organoids cultured using ECEP scaffold material as the matrix have an ordered structure (i.e., the horizontal line structure shown in the figure), and the cells can stretch and spread along the gaps in the material (the red part in the figure shows the cells).

[0103] The bone organoids cultured using ECE and EC scaffold materials as matrices still had relatively regular shapes. Cryo-scanning electron microscopy images of the organoids after 30 days of osteogenic culture showed that the bone organoids cultured using ECE and EC scaffold materials as matrices had relatively orderly structures, and the cells could stretch and spread along the gaps in the materials.

[0104] Bone organoids cultured using SCEP scaffold material as a matrix exhibit relatively irregular shapes. Cryo-scanning electron microscopy images show that bone organoids cultured using SCEP scaffold material as a matrix present a relatively disordered structure, affecting cell stretching and spreading.

[0105] Test Example 2

[0106] The mechanical properties of bone organoids cultured using ECEP, ECE, EC, and SCEP scaffold materials as substrates in Examples 1 and 1-3 were tested, respectively. Figure 2 The compressive stress-strain curve is shown.

[0107] It can be seen that no fracture occurred in the ECEP group when the strain reached 65%, and the curve continued to rise, indicating extremely strong energy absorption capacity. This proves that bone organoids cultured using ECEP scaffold material as a matrix possess excellent toughness and can be implanted into animals as a tough material with good mechanical strength for bone defect repair. Furthermore, the ECEP group's curve shows a sustained non-linear rise to high strain, and the good mechanical strength of the ECEP group may be due to the conductive polymer promoting the adsorption and deposition of mineralization precursors, and the electrical signal during the electrochemical-assisted catalysis process promoting the self-mineralization of the matrix material to some extent.

[0108] No fracture occurred in the SCEP group when the strain reached 61%, and the curve continued to rise, demonstrating extremely strong energy absorption capacity. This also proves that bone organoids cultured using SCEP scaffold material as a matrix possess excellent toughness. However, compared to the ECEP group, its slope is not as steep, possibly due to its less favorable orientation.

[0109] No fracture occurred in the ECE group when the strain reached 57%, and no fracture occurred in the EC group when the strain reached 50%.

[0110] Test Example 3

[0111] Biocompatibility tests were performed on bone organoids cultured using ECEP, ECE, EC, and SCEP scaffold materials as matrices in Examples 1 and 1-3, respectively. Figure 3 The three-dimensional live and dead cell staining (Figure A, red represents dead cells (PI staining, PI is propidium iodide dye), green represents live cells (AM staining, AM is calcein acetoxymethyl ester dye)) and skeleton diagram (Figure B, red represents the skeleton (TRITC staining, TRITC is tetramethylrhodamine dye), blue represents the cell nucleus (DAPI staining, DAPI is 4′,6-diamidinyl-2-phenylindole dye)).

[0112] It can be seen that cells in the ECEP, ECE and EC groups can survive and spread in the scaffold, forming a cytoskeleton network, and the cytoskeleton structure shows a certain degree of ordered structure.

[0113] In the SCEP group, cells can survive and spread within the scaffold, forming a cytoskeleton network. However, the scaffold does not exhibit an ordered structure, which affects further cell elongation.

[0114] Test Example 4

[0115] The performance of bone organoids (BMSC cells labeled with green fluorescence) obtained from Examples 1 and Comparative Examples 1-3 using ECEP, ECE, EC, and SCEP scaffold materials as substrates was tested. The specific steps are as follows:

[0116] Schwann cells (SC cells) were labeled with red fluorescence, and the fluorescently labeled SC cells were then used in a 1*10-1 ratio. 3 ~1*10 9 / cm 3 The scaffold material was seeded into each bone organoid at a density, and cultured for 24 h to adapt. The ingrowth of SC cells was then observed under laser confocal microscopy.

[0117] like Figure 4 The three-view diagram shows that (blue represents the cell nucleus (DAPI staining, DAPI is 4′,6-diamidindo-2-phenylindole dye), red represents Schwann cells (SC cells) stained with red fluorescence, and green represents bone marrow mesenchymal stem cells (BMSC cells) stained with green fluorescence). In the ECEP group, SC cells edged into a larger area and deeper depth.

[0118] In the ECE group, SC cells edged into a larger area and deeper depth, but compared with the ECEP group, the edging range and depth of SC cells were smaller, possibly due to the lack of electrophysiological signal transduction by conductive polymers.

[0119] Compared to the ECE and ECEP groups, the SC cells in the EC group had a smaller ingrowth range and shallower depth.

[0120] Compared with the ECEP group, the SCEP group showed that the SC cells grew into a smaller area and a shallower depth.

[0121] Test Example 5

[0122] Micro-CT three-dimensional reconstructions were performed on bone organoids cultured using ECEP, ECE, EC, and SCEP scaffold materials as matrices in Examples 1 and 1-3, respectively. Figure 5 The image shown is a 3D reconstruction image from Micro-CT.

[0123] It can be seen that electrochemical deposition induces varying degrees of mineralization within the collagen scaffold itself. Compared to other groups, the ECEP group exhibited a higher degree of collagen fiber mineralization in the bone organoids. This self-mineralization is characterized by a higher degree of mineralization outside the collagen fibers and larger mineralized molecules. After 14 days of osteogenic induction culture, the mineral distribution became more uniform, large mineral molecules disintegrated, and better intracellular mineralization of collagen fibers was achieved under cellular action. After 30 days of osteogenic induction culture, even greater intracellular mineralization of collagen fibers was achieved under cellular action.

[0124] Compared to other groups, the SCEP group did not exhibit significant mineralization in its collagen scaffold. However, after 14 days of osteogenic induction culture, some degree of intrafibrillary mineralization within the collagen fibers was observed. After 30 days of osteogenic induction culture, further good intrafibrillary mineralization was achieved under cellular influence. However, compared to the bone organoids formed after 30 days of ECEP culture, the mineralization capacity was weaker. This may be because the collagen assembly method of the SCEP matrix material lacks the electric field effect of electrochemical deposition, which subtly influences the mineralization capacity.

[0125] Compared to the ECEP group, the ECE group also exhibited slight self-mineralization of the collagen scaffold. After 14 days of osteogenic induction culture, the mineral distribution was more uniform, and large-molecule minerals disintegrated, achieving better intracellular mineralization of collagen fibers under cellular action. After 30 days of osteogenic induction culture, further good intracellular mineralization of collagen fibers was achieved under cellular action. However, compared to the organoids formed after 30 days of ECEP culture, the mineralization capacity was weaker. This may be because the ECEP group contained conductive polymers, which facilitated the interaction of microcurrents within the bone organoids, promoting the transport of internal mineralization precursors and resulting in superior mineral deposition.

[0126] Compared to other groups, the EC group did not exhibit significant mineralization in its collagen scaffold. However, after 14 days of osteogenic induction culture, some degree of intrafibrous mineralization within the collagen fibers was observed. After 30 days of osteogenic induction culture, further intrafibrous mineralization was achieved under cellular influence. However, compared to organoids formed after 30 days of ECEP culture, the mineralization capacity was weaker. This may be because, compared to the ECEP group, the EC group lacks both phosphine-containing biomolecules and conductive polymers, resulting in fewer mineralization precursors in the EC group organoids and reduced internal microcurrent interactions, thus decreasing mineralization capacity.

[0127] Test Example 6

[0128] H&E staining was performed on bone organoids cultured using ECEP, ECE, EC, and SCEP scaffold materials as substrates in Examples 1 and Comparative Examples 1-3, respectively. Figure 6 The H&E staining diagram shown.

[0129] As can be seen, after 30 days of osteogenic induction culture, the bone organoids in the ECEP, ECE, EC, and SCEP groups exhibited a relatively uniform network structure and showed collagen fiber production (red striped areas). This indicates that the bone organoids induced for 30 days possess good self-organizing ability and can self-secrete collagen matrix. All of the above demonstrates the successful culture of bone organoids in each group.

[0130] Test Example 7

[0131] Bone organoids cultured using ECEP, ECE, EC, SCEP, SCE, and SC scaffold materials as substrates, obtained in Example 1 and Comparative Examples 1-5 respectively, were placed in the lower layer of a Transwell chamber to form a co-culture system. Schwann cells (SC) were cultured at a concentration of 5 × 10⁻⁶ cells / mL. 3Cells were seeded at a density on 24-well plates and placed in the lower layer of 0.4 μm diameter Transwell chambers using a culture medium suitable for Schwann cells (SC). After the cells in the wells grew to a suitable density, immunofluorescence staining was performed using β3-Tubulin. The differences in Schwann cell growth caused by bone organoids that promote nerve ingrowth were verified using laser confocal microscopy, and the fluorescence intensity was statistically analyzed using image analysis software (such as ImageJ).

[0132] like Figure 7 As shown (green represents β-tubulin (β3-Tubulin staining, β3-Tubulin is a characteristic marker dye for neurons), and blue represents the cell nucleus (DAPI staining, DAPI is 4′,6-diamidinyl-2-phenylindole dye)), the ECEP group promoted the expression of the neuronal-related gene β3-Tubulin in SC cells. Semi-quantitative results also indicated that the expression level of β3-Tubulin was highest in this bone organoid group. This is attributed to the combined effects of the phosphine-containing biomolecule solution and conductive polymer component in the ECEP group's organoid components, as well as the electrodeposition effect in the synthesis method. The addition of the conductive polymer established the interaction of microcurrents within the organoid, inducing organoid maturation and thus promoting β3-Tubulin gene expression in SC cells. The addition of the electric field from the electrodeposition process promoted the conduction of microcurrents within the organoid, similarly promoting β3-Tubulin gene expression in Schwann cells.

[0133] Co-culturing bone organoids with SC cells in the SCEP group promoted the expression of the neural-related gene β3-Tubulin in SC cells. Semi-quantitative results showed that the SCEP group promoted β3-Tubulin gene expression to a higher degree. However, compared with the ECEP group, the effect on organoids was weaker. This may be because the electrodeposition electric field in the matrix material synthesis lacked the conduction effect of the microcurrent, thus weakening the expression of the neural-related gene β3-Tubulin in SC cells.

[0134] Co-culturing bone organoids with SC cells in the ECE group promoted the expression of the neural-related gene β3-Tubulin in SC cells, but the effect was weaker compared to the ECEP group. This may be because the lack of conductive polymers weakened the interaction of internal microcurrents, thus reducing the expression of the neural-related gene β3-Tubulin in SC cells.

[0135] Co-culturing bone organoids with SC cells in the SCE group promoted the expression of the nerve-related gene β3-Tubulin in SC cells, but the effect was weaker compared to the ECEP group. This may be because the lack of conductive polymers weakened the interaction of internal microcurrents, and the absence of electric field effects in material synthesis inhibited the conduction of internal microcurrents, thus weakening the expression of the nerve-related gene β3-Tubulin in SC cells.

[0136] Co-culturing bone organoids from the EC group with SC cells promoted the expression of the neurotransmitter gene β3-Tubulin in SC cells, but the effect was weaker than that of organoids from the ECEP group. This may be because the EC group lacked conductive polymers, which weakened the interaction of internal microcurrents, and also lacked phosphine-containing biomolecules, which reduced mineralization precursors in the EC group organoids and decreased the interaction of internal mineral microcurrents, thus weakening the expression of the neurotransmitter gene β3-Tubulin in SC cells.

[0137] Co-culturing bone organoids with SC cells in the SC group promoted the expression of the neurotransmitter gene β3-Tubulin in SC cells, but the effect was weaker than that in the ECEP group organoids. This may be because the lack of conductive polymers weakened the interaction of internal microcurrents; the lack of phosphine-containing biomolecules reduced mineralization precursors in the SC group organoids, thus reducing the interaction of internal mineral microcurrents; and the lack of electric field effects in material synthesis inhibited the conduction of internal microcurrents, thereby weakening the expression of the neurotransmitter gene β3-Tubulin in SC cells.

[0138] Test Example 8

[0139] Animal experiments were conducted on the bone organoids obtained from Example 1 and Comparative Examples 1-3, which were cultured using ECEP, ECE, EC, and SCEP scaffold materials as substrates.

[0140] The aforementioned bone organs (3 mm in diameter and 1 mm in thickness) were implanted into female BALB / c nude mice (4-6 weeks old), and the tissue was harvested 4 weeks later under the skin.

[0141] like Figure 8 As shown, the images are bright-field images (Figure A and Figure B) and cross-sectional and longitudinal sections of the three-dimensional reconstruction from Micro-CT (Figure C).

[0142] Figure 8Bright-field images and three-dimensional reconstructed cross-sectional and longitudinal sections of micro-CT scans were presented for bone organoids cultured using ECEP hydrogel as a matrix and implanted into BALB / c nude mice 4 weeks after subcutaneous implantation. It can be seen that the conductive polymers in the ECEP group's bone organoid matrix material were not completely degraded, exhibiting a high mineral content. Quantitative analysis results of bone density and bone volume fraction (Figure D) clearly demonstrate that the ECEP group had the strongest mineralization capacity compared to other groups.

[0143] The conductive polymers in the SCEP group were not completely degraded and also contained a large amount of mineral components. The quantitative analysis results of bone density and bone volume fraction showed that its mineralization ability was weaker than that of the ECEP group. This may be because the collagen assembly mode of the SCEP matrix material lacks the electric field effect of electrochemical deposition, which has a slight impact on the mineralization ability.

[0144] The quantitative analysis results of bone mineral density and bone volume fraction of bone organoids in the ECE group show that its mineralization capacity is weaker than that of the ECEP group. The reason may be that the addition of conductive polymers in the ECEP group has established the interaction of microcurrents inside the bone organoids, which promotes the transport of internal mineralization precursors and thus leads to better mineral deposition.

[0145] Quantitative analysis of bone mineral density and bone volume fraction in the EC group showed that its mineralization capacity was weaker than that in the ECEP group. This may be because the EC group lacks both phosphine-containing biomolecules and conductive polymers, which reduces the mineralization precursors in the EC group bone organoids and decreases the interaction of internal microcurrents, thus reducing the mineralization capacity.

[0146] Test Example 9

[0147] The bone organoids prepared in Example 1 above were applied to bone defect repair.

[0148] (1) Skull defect modeling: Healthy male C57 mice aged 4-6 weeks were selected and anesthetized with a small animal anesthesia machine. The gas flow rate was set to 500 mL / min. During induction anesthesia, the concentration of inhaled anesthetic isoflurane was 4%, and during maintenance anesthesia, the concentration of isoflurane was 2-3%. After the mice were fully anesthetized, the skin was prepared, disinfected, and draped. A longitudinal surgical incision of 1.5-3 cm was made along the sagittal line of the skull. The soft tissue was dissected layer by layer, and the periosteum was fully dissected. Two bone defects with a diameter of 3 mm were made on both sides of the sagittal line using a round drill. The wounds were rinsed with physiological saline.

[0149] (2) Material implantation and animal sampling: After modeling, conductive polymer-induced collagen hydrogel mineralization scaffolds (ECEP) with a diameter of 3 mm and a thickness of 1 mm without cell encapsulation, materials (ECEP&CELL) based on conductive polymer-induced collagen hydrogel mineralization scaffolds encapsulating BMSC cells cultured for 7 days, and organoids (ECEP&Organoid) cultured based on conductive polymer-induced collagen hydrogel mineralization scaffolds were implanted into the bone defect sites. Sampling and analysis were performed on mice in each group at 4 weeks (4w) and 12 weeks (12w) of treatment.

[0150] Figure 9 Micro-CT three-dimensional reconstruction images and horizontal and coronal scan images of the skull defects in mice after implantation of materials at different time sequences are presented. Figure 9 The colored areas represent the newly grown bone. In the control group (Con), the areas are marked in orange, while in the ECEP, ECEP&CELL, and ECEP&Organoid groups, the areas are marked in red.

[0151] From the 4w Micro-CT 3D reconstructed images and horizontal and coronal scans, it can be seen that the red new bone formation area in the ECEP, ECEP&CELL, and ECEP&Organoid groups is larger than that in the Con group. Compared to the ECEP group, the ECEP&CELL group has a larger red new bone formation area. Compared to the ECEP&CELL group, the ECEP&Organoid group shows significant callus formation in the central bone defect area. This indicates that ECEP, ECEP&CELL, and ECEP&Organoid can all promote new bone formation at the defect site, with the ECEP&Organoid group showing the best bone formation effect.

[0152] Similarly, analysis of the 12-week Micro-CT 3D reconstructed images and horizontal and coronal scans revealed that the red new bone area in the ECEP, ECEP&CELL, and ECEP&Organoid groups was larger than that in the Con group. The ECEP&CELL group also showed a larger red new bone area compared to the ECEP group. Furthermore, the ECEP&Organoid group exhibited significant callus formation in the central bone defect area compared to the ECEP&CELL group. After 12 weeks of defect repair, each group showed better new bone formation at the defect site compared to the corresponding 4-week defect site. The ECEP&Organoid group demonstrated the best integration with the original bone tissue after 12 weeks.

[0153] 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 constructing an osteoid organ in which nerve ingrowth is promoted, characterized by, The method comprises the following steps: S1, soaking the hydrogel mineralization scaffold with conductive polymer induction in EDC / NHS crosslinking agent and sterilizing and crosslinking under ultraviolet environment; The preparation method of the hydrogel mineralization scaffold with conductive polymer induction comprises the following steps: S1, soaking the hydrogel mineralization scaffold with conductive polymer induction in EDC / NHS crosslinking agent and sterilizing and crosslinking under ultraviolet environment; S2, soaking the product after crosslinking in step S1 in complete culture medium for static culture, inoculating bone organ seed cells, and continuing to use the complete culture medium for adaptive culture; S3, transferring the product obtained in step S2 to a bone induction culture medium for bone induction to obtain a bone organ promoting nerve growth; In step S1, the hydrogel matrix material is type I collagen; The conductive polymer is a combination of polyaniline and polypyrrole; The water-soluble calcium salt is CaCl2; The phosphorus-containing biomolecule is adenosine disodium triphosphate; The reaction conditions of the electrochemical deposition are as follows: a three-electrode system is adopted, a titanium sheet is used as a working electrode, Ag / AgCl is used as a reference electrode, a platinum sheet is used as a counter electrode, and the reaction is carried out at a current of 10 mA for 900 s; In step S2, the bone organ seed cells are selected from bone marrow mesenchymal stem cells.

2. The method of claim 1, wherein the method is performed in vitro. In step S1, the pH of the acidic solution is 2-6.

3. The method of claim 1, wherein the method is performed in vitro. In step S1, the crosslinking time is 12-48 h.

4. The method of claim 1, wherein the method is performed in vitro. In step S2, the complete culture medium comprises 10% FBS and 1% PS by volume added to a basic medium, and the basic medium is selected from any one of α-MEM, DMEM and DMEM / F-12; The static culture condition is 1-12 h of culture at 37℃.

5. The method of claim 1, wherein the method is performed in vitro. In S2 step, the seeding density of the bone organ seed cells is 1 x 10 3 -1 x 10 9 / cm 3 Scaffold material.

6. The method of claim 1, wherein the method is performed in vitro. In step S2, the adaptive culture condition is 1-3 days of culture at 37℃.

7. The method of claim 1, wherein the method is performed in vitro. In step S3, the bone induction culture medium comprises 10% FBS, 1% PS, 1% 1M β-GP, 0.25% 20 mg / mL Vitamin C and 0.01% 100 μM Dex by volume added to a DMEM culture medium; The bone induction condition is 1-45 days of culture at 37℃, and the fresh bone induction culture medium is replaced every 2-3 days during the culture.

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