In-situ mineralized collagen scaffold material based on electrospinning process and preparation method thereof

Abstract

The application discloses an in-situ mineralized collagen support material based on an electrostatic spinning process and a preparation method thereof, and comprises a collagen solution configuration module, an ion solution configuration module, an electrospinning solution configuration module and an electrostatic spinning module. The application belongs to the technical field of composite materials for filling bone defects, and the structure can eliminate stress concentration caused by particle agglomeration, significantly improves the mechanical properties of the support, and the mineral phase generated in-situ matches the degradation rate of bone repair process, and has excellent promoting effect on the proliferation and differentiation of osteogenic mesenchymal stem cells.

Description

Technical Field

[0001] This invention relates to the field of composite materials for filling bone defects. Background Technology

[0002] Clinically, bone grafting and reconstruction surgery is usually performed to restore the shape and function of the defective area. Currently, autologous bone, allogeneic / xenogeneic bone, and artificial bone materials are commonly used to implant into the defective area to promote bone repair.

[0003] Among these, autologous bone transplantation has the problems of "treating injury with injury" and limited sources; allogeneic or xenogeneic bone carries the potential risks of immune rejection and disease transmission.

[0004] To overcome the aforementioned problems, numerous artificial bone materials have been developed in existing technologies. Among them, the preparation of collagen-based scaffolds using electrospinning technology and the combination with nano-hydroxyapatite for bone repair is currently a research hotspot.

[0005] The existing conventional method is to directly mix pre-synthesized nano-hydroxyapatite particles with a collagen solution and then perform electrospinning.

[0006] However, existing technologies that directly blend nano-hydroxyapatite particles with collagen solutions for electrospinning have the following fatal flaws: 1. Particle aggregation and morphology destruction: Nanoparticles are prone to secondary aggregation in spinning solution, which leads to discontinuous spinning and local accumulation in the final scaffold, destroying the overall morphology of the fiber.

[0007] 2. Impaired mechanical properties: Aggregated hard inorganic particles form stress concentration points inside collagen fibers, which are easily used as fracture sources under external forces, thus damaging the mechanical properties of the scaffold.

[0008] 3. Hinders cell migration: Locally accumulated particles can alter the topology of the fiber surface, forming a physical barrier that hinders the attachment, crawling, and migration of osteoblastic mesenchymal stem cells on the fibrous scaffold.

[0009] 4. Mismatch between degradation and osteogenic kinetics: Directly added particles usually have high crystallinity, and their degradation rate within the scaffold is extremely slow, which makes it impossible to continuously release mineral ions, resulting in a relatively slow process of promoting bone defect repair.

[0010] Therefore, there is an urgent need to develop an in-situ mineralized collagen scaffold material based on electrospinning technology and its preparation method to solve the problems in the existing technology. Summary of the Invention

[0011] The purpose of this invention is to provide an in-situ mineralized collagen scaffold material based on electrospinning technology and its preparation method. This material can eliminate stress concentration caused by particle agglomeration, significantly improve the mechanical properties of the scaffold, and the degradation rate of the mineral phase generated in situ matches the bone repair process, thus having an excellent promoting effect on the proliferation and differentiation of osteoblastic mesenchymal stem cells. Moreover, the material has a simple structure and is easy to use, thereby solving the problems mentioned in the background art.

[0012] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing an in-situ mineralized collagen scaffold material based on electrospinning technology includes the following steps: Configure a collagen solution module, using hexafluoroisopropanol to dissolve collagen and prepare a collagen solution; Configure the ion solution module by dissolving anhydrous calcium chloride and concentrated phosphoric acid solution in purified water to prepare calcium ion solution and phosphate ion solution, respectively. An electrospinning solution module is configured by adding one of the calcium ion solution and the phosphate ion solution to the collagen solution to prepare a stable mineralized collagen electrospinning solution, wherein the concentrations of calcium ions and phosphate ions are in a specific ratio. The electrospinning module adds two solutions containing different mineral ions to the syringe and performs electrospinning using a two-in-one system. In-situ mineralization occurs at the moment of jetting and the morphology is maintained, resulting in the in-situ mineralized collagen scaffold material.

[0013] By adopting the above technical solution, calcium and phosphorus ions are separated and mineralized in situ during the jet, a uniform and non-agglomerated distribution of mineral phases is achieved inside the collagen fiber, stress concentration is eliminated, and spinning continuity and fiber stability are ensured.

[0014] As a further aspect of the present invention, the configuration of the electrospinning solution module includes the following sub-steps: Add either the calcium ion solution or the phosphate ion solution to the collagen solution, and continue stirring until the solution turns milky white and there is no precipitate; Add NaOH solution to adjust the pH of the solution to 7.4, and finally stir the solution to obtain a stable electrospinning solution containing calcium and an electrospinning solution containing phosphorus.

[0015] By adopting the above technical solution, adjusting the pH to 7.4 and forming a milky white, precipitate-free state, the spinning solution is stabilized in the long term, preventing premature ion reaction and precipitation, and ensuring that the spinning process is uninterrupted.

[0016] As a further aspect of the present invention: In the electrospinning module, under the non-equilibrium physical field of jet rapid stretching and solvent evaporation, calcium and phosphorus ions are nucleated in situ within the nanoscale confined space of collagen molecular chains. Under the interfacial coordination regulation of collagen polar groups, a hybrid interface structure of organic and inorganic components is formed, in which low-crystallinity nanocrystals and collagen molecular chains are chemically bonded through coordination bonds and hydrogen bonds.

[0017] By adopting the above technical solution, calcium and phosphorus ions are induced to nucleate in situ within the confined space of collagen nanoparticles under a non-equilibrium physical field and are coordinated and regulated by the interface of collagen polar groups. This achieves the overall chemical bonding of mineral crystals and collagen molecular chains at the molecular scale, constructing an organic-inorganic hybrid interface structure that is different from traditional physical mixing, and endowing the scaffold with life-like bonding force.

[0018] As a further aspect of the present invention: in the electrospinning module, the power supply voltage of the positive electrode is 12-18kV, the power supply voltage of the negative electrode is -1 to -5kV, and the speed of the two injection pumps is 0.05-0.3mL / h; this voltage range, together with the low-speed injection, maintains the millisecond-level stretching and curing time of the jet, ensuring the formation of nucleation and organic-inorganic hybrid interfaces in the confined space without macroscopic phase separation.

[0019] By adopting the above technical solution, through the synergistic combination of a specific high-voltage electrostatic field and an extremely low injection speed, the precise control of the millisecond-level stretching and curing time of the jet is achieved. This ensures that in-situ nucleation within the confined space and the organic-inorganic hybrid interface are instantly locked and cured before macroscopic phase separation occurs, thus guaranteeing the successful generation of the hybrid structure.

[0020] As a further aspect of the present invention: in the collagen solution configuration module, the concentration of the collagen solution is 5%-10%; in the electrospinning solution configuration module, the ion concentration of the mineralized collagen electrospinning solution is 150-250mM.

[0021] By adopting the above technical solution and limiting the collagen concentration to 5%-10% and the ion concentration to 150-250mM, the optimal balance between solution viscosity and mineralization supersaturation is achieved, which ensures both continuous fiber formation and sufficient mineral generation.

[0022] The present invention also discloses an in-situ mineralized collagen scaffold material based on electrospinning process, which is prepared according to the above-mentioned preparation method of an in-situ mineralized collagen scaffold material based on electrospinning process, comprising: a continuous collagen fiber matrix, and a mineral phase in-situ distributed inside the collagen fiber matrix; The mineral phase is uniformly distributed in the collagen fiber matrix and is free of agglomerated particles.

[0023] By adopting the above technical solution, and by limiting the product to include a continuous matrix and an in-situ non-agglomerated mineral phase, the overall mechanical properties of the scaffold are significantly improved, and the physical obstacles of aggregates to cell migration are eliminated.

[0024] As a further aspect of the present invention: the mineral phase is a low-crystallinity nanocrystal, which forms an organic and inorganic hybrid interface structure with the collagen fiber matrix by coordination bonds and hydrogen bonds.

[0025] By adopting the above technical solution, and by limiting the mineral phase to low-crystallinity nanocrystals and forming a hybrid interface with the collagen matrix consisting of coordination bonds and hydrogen bonds, the deep fusion and strong interfacial bonding of the inorganic mineral phase and the organic collagen phase are achieved, fundamentally overcoming the risks of easy detachment of inorganic particles and interfacial peeling in traditional composite materials.

[0026] As a further aspect of the present invention: the hybrid interface structure enables the formation of a stress transmission network that mimics natural bone between the mineral phase and the collagen matrix, giving the scaffold exceptional fracture toughness, and the low-crystallinity mineral phase exhibits a phased degradation ion release characteristic that matches the bone repair cycle.

[0027] By adopting the above technical solution, through the natural bone stress transmission network formed by the hybrid interface structure and the low crystallinity mineral characteristics, the overall fracture toughness of the scaffold is significantly improved and the degradation-osteogenic dynamics are precisely matched. This enables the scaffold to effectively dissipate crack energy when under stress and exhibits a phased ion release characteristic that matches the bone repair cycle during degradation.

[0028] As a further aspect of the present invention: the mineral phase is a complex ionic compound formed by calcium ions and phosphate ions in a specific ratio, the specific ratio being obtained by controlling the concentration of the ionic solution and the amount of solution used.

[0029] By adopting the above technical solution and by limiting the acquisition method of mineral phase ratio, the overall precise control of mineral composition is achieved, ensuring that the generated minerals highly simulate the chemical composition of natural bone tissue.

[0030] This invention also discloses the application of an in-situ mineralized collagen scaffold material based on electrospinning technology in the preparation of bone repair products. The bone repair products utilize the preferred orientation arrangement of the mineral phase along the collagen fiber axis to provide axial mechanical support for the defect site, and release mineral ions through the phased degradation of the oriented mineral phase to induce osteoblastic mesenchymal stem cells to migrate and differentiate along the fiber axis.

[0031] By adopting the above technical solution and limiting the application of the product in the field of bone repair, the overall clinical translational value of the oriented mineralization scaffold in repairing bone defects and promoting osteogenic differentiation has been realized.

[0032] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention achieves uniform distribution of mineral phases in collagen fibers without agglomeration through in-situ mineralization, eliminating stress concentration caused by the direct addition of nanoparticles, significantly improving the mechanical strength of the scaffold, and avoiding agglomerated particles from hindering cell migration on the fibers.

[0033] 2. This invention employs a calcium-phosphorus ion separation configuration combined with a two-in-one spinning system, which triggers in-situ mineralization at the moment of jet injection, completely avoiding the problem of spinning solution precipitation caused by premature mixing of calcium and phosphorus, and ensuring the continuity of the spinning process and the stability of fiber morphology.

[0034] 3. This invention uses a specific ratio of mineral phases generated in situ to highly simulate natural bone components. Its degradation rate is more matched with the bone repair process and it can continuously release mineral ions. Compared with directly adding particles, it has a better promoting effect on the proliferation and osteogenic differentiation of osteoblast mesenchymal stem cells.

[0035] Other features and advantages of the present invention will be disclosed in detail in the following detailed description and accompanying drawings. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the steps involved in the preparation of an in-situ mineralized collagen scaffold material based on electrospinning technology, as described in an embodiment of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In this embodiment of the invention, an in-situ mineralized collagen scaffold material based on electrospinning process and its preparation method are described below. Figure 1 As shown, it includes the following steps: S1: Configure collagen solution module Collagen was dissolved in hexafluoroisopropanol (HFIP) to prepare a collagen solution with a concentration of 5%-10%. As a highly polar organic solvent, HFIP can effectively break the hydrogen bonds between collagen molecules, allowing the molecular chains to unfold and form a solution with good entanglement. This is the physical basis for the subsequent electrospinning to form continuous fibers.

[0039] S2: Configure the ion solution module The calcium source and phosphate source are dissolved separately in purified water to prepare calcium ion solutions and phosphate ion solutions, respectively. The ion sources should have high solubility and high ionization, and should not react to form byproducts with low solubility and low ionization. For example, anhydrous calcium chloride or calcium nitrate tetrahydrate can be used as the calcium source; phosphoric acid or sodium dihydrogen phosphate can be used as the phosphate source.

[0040] S3: Configure electrospinning solution module The calcium ion solution or the phosphate ion solution is added to the collagen solution to prepare a stable mineralized collagen electrospinning solution. This step includes the following sub-steps: S31: After adding the ionic solution to the collagen solution and stirring continuously, the aqueous ionic droplets are uniformly dispersed and wrapped in the organic collagen phase, forming a milky white microemulsion state without precipitation, preventing the ions from reacting prematurely to form large precipitate particles that clog the needle. S32: Add NaOH solution to adjust the pH of the solution to 7.4. Finally, stir the solution to obtain stable calcium-containing and phosphorus-containing electrospinning solutions. Adjusting the pH to 7.4 aims to allow collagen molecules to carry a suitable charge, providing nucleation sites for subsequent in-situ mineralization.

[0041] The concentrations of calcium ions and phosphate ions should be in a specific ratio to form a complex ionic compound with a specific proportion. This specific proportion is obtained by controlling both the concentration and volume of the solution, and its calculation follows the formula: in, This represents the final ion concentration in the electrospinning solution. This represents the original concentration of the added ion solution. This is the volume of the added ionic solution. This represents the volume of the collagen solution. Through the above formula, the ion concentration in the two fluid streams before mixing is precisely controlled to ensure that the ideal mineralization supersaturation is achieved when the jets meet instantaneously.

[0042] S4: Electrospinning module Two solutions containing different mineral ions were added to a syringe, and electrospinning was performed using a two-in-one system (such as a Y-connector or coaxial needle). The two fluids converged at the tip of the two-in-one system and were drawn into a jet by a high-voltage electric field. Within milliseconds of the jet reaching the receiver, the organic solvent rapidly evaporated, causing a momentary supersaturation of the local ion concentration, triggering in-situ mineralization nucleation and growth. Minerals were generated and solidified in situ between collagen molecular chains to form continuous fibers, resulting in in-situ mineralized collagen fiber material.

[0043] S5: Drying module The nascent fiber membrane obtained by spinning was placed in a desiccator to dry completely to remove residual solvent and stabilize the mineral phase structure.

[0044] The final in-situ mineralized collagen scaffold material includes a continuous collagen fiber matrix and a mineral phase distributed in-situ within the collagen fiber matrix; the mineral phase is uniformly distributed in the collagen fiber matrix and is free of aggregated particles.

[0045] Example 1 This embodiment provides a method for preparing in-situ mineralized collagen scaffold material based on electrospinning technology, the specific steps of which are as follows: S1: Dissolve 50 mg of collagen in 1 ml of hexafluoroisopropanol. After dissolving for 1 hour, stir with a magnetic stirrer for 5 hours to ensure that the collagen is uniformly dissolved into a 5% collagen solution.

[0046] S2: Dissolve anhydrous calcium chloride and concentrated phosphate solutions separately in deionized water to prepare high-concentration calcium ion mother liquor and phosphate ion mother liquor.

[0047] S3: Add the above-mentioned ionic mother liquor to the collagen solution separately, and control the added volume based on the formula. Calculate and dilute the mineralized collagen electrospinning solution to 200 mM. Continue stirring for 24 h. When the solution turns milky white and there is no precipitate, add NaOH solution to adjust the pH of the solution to 7.4. Finally, stir the solution for 30 min to obtain stable calcium-containing and phosphorus-containing electrospinning solutions.

[0048] S4: Add calcium-containing electrospinning solution and phosphorus-containing electrospinning solution to two syringes respectively. The syringes have 20G stainless steel needles with an inner diameter of 0.62mm. Electrospinning is performed using a two-in-one system. During electrospinning, the speed of the two syringe pumps is set to 0.1mL / h, the distance from the syringe needle to the tin foil receiver is set to 8cm, the power supply voltage at the positive electrode is 15kV, and the power supply voltage at the negative electrode is -3kV. An in-situ mineralization reaction occurs instantaneously during jetting, resulting in stable and continuous in-situ mineralized collagen fiber material.

[0049] S5: The collected fiber scaffolds were placed in a desiccator and dried for 24 hours to obtain the final in-situ mineralized collagen scaffold material.

[0050] Testing revealed that the mineral phase in the scaffold material prepared in this embodiment was uniformly distributed within the collagen fiber matrix, with no aggregated particles. Its mechanical properties were significantly improved compared to pure collagen scaffolds, and it exhibited excellent cell migration and osteogenic differentiation performance.

[0051] Example 2 The key difference between this embodiment and Embodiment 1 lies in the adjustment of the ion source and concentration in S2 and S3: Calcium nitrate tetrahydrate was used instead of anhydrous calcium chloride in the calcium ion solution, and disodium hydrogen phosphate was used instead of concentrated phosphoric acid solution in the phosphate ion solution. After dissolution, these solutions were added to the collagen solution and diluted to a 200 mM mineralized collagen electrospinning solution. Subsequent spinning and drying parameters were the same as in Example 1.

[0052] This embodiment also successfully prepared an in-situ mineralized collagen scaffold with uniform mineral distribution and no aggregated particles, verifying the universality of the ion source selection in this invention.

[0053] Example 3 The difference between this embodiment and Embodiment 1 lies in the following steps: S1: Configure collagen solution module The collagen solution preparation module includes a sol container, a magnetic stirrer, and a temperature control device; The specific implementation method is as follows: Collagen is added to a sol container containing hexafluoroisopropanol (HFIP). The sol container is a glass sample bottle with a sealed cap and a volume of 5-20 mL. The sealed cap is used to prevent HFIP from evaporating. The solution is first allowed to stand for 1 hour to dissolve. Then, the sol container is placed on a magnetic stirrer with a polytetrafluoroethylene (PTFE) magnetic stir bar. The mixture is stirred at 300-500 rpm for 5 hours at room temperature to uniformly dissolve the collagen into a collagen solution with a concentration of 4.5%-6%. The temperature control device is a heating platform integrated with the magnetic stirrer. When the ambient temperature is below 20°C, the heating is turned on to maintain the dissolution temperature within the range of 20-25°C. After dissolution, the solution is observed to be transparent and pale yellow with no undissolved flocculent matter, indicating complete dissolution.

[0054] HFIP, as a highly polar organic solvent, can effectively break the hydrogen bonds between collagen molecules, allowing the molecular chains to unfold and form a solution with good entanglement. This is the physical basis for the subsequent electrospinning to form continuous fibers.

[0055] S2: Configure the ion solution module The configured ion solution module includes a quantitative pipette, a volumetric flask, and a second magnetic stirrer; The specific implementation method is as follows: Purified water is measured into a volumetric flask using a quantitative pipette. Anhydrous calcium chloride and concentrated phosphate solutions are weighed separately and dissolved in purified water. The calcium source solution is stirred on a second magnetic stirrer at 200-400 rpm for 10-20 minutes until completely dissolved and clear, preparing a 0.3-0.6 M calcium ion stock solution. The phosphate source solution is similarly stirred until completely dissolved, preparing a 0.18-0.36 M phosphate ion stock solution. The volumetric flask is 10-25 mL in size to precisely control the concentration of the stock solution. The concentration ratio of the calcium ion stock solution to the phosphate ion stock solution is maintained at 5:3, corresponding to natural hydroxyapatite. The stoichiometric ratio.

[0056] The ion source should have high solubility and high ionization, and should not react to form byproducts with low solubility and low ionization. For example, anhydrous calcium chloride or calcium nitrate tetrahydrate can be used as the calcium source; phosphoric acid or sodium dihydrogen phosphate can be used as the phosphate source.

[0057] S3: Configure electrospinning solution module The configured electrospinning solution module includes a microemulsion preparation submodule and a pH adjustment submodule; The microemulsion preparation submodule includes an ultrasonic disperser and a third magnetic stirrer. Specifically, a quantitative pipette is used to add calcium ion mother liquor or phosphate ion mother liquor dropwise to the collagen solution obtained in S1, while simultaneously stirring continuously at 400-600 rpm on the third magnetic stirrer. After the addition is complete, the mixed solution is placed in the ultrasonic disperser and ultrasonically treated for 10-20 minutes at a power of 100-200W and a frequency of 40kHz, so that the aqueous ion droplets are uniformly dispersed and encapsulated in the organic collagen phase, forming a milky white microemulsion without precipitate, preventing premature reaction of ions to form large precipitate particles that clog the needle. The pH adjustment submodule includes a microburette, a pH meter, and a fourth magnetic stirrer. The specific implementation is as follows: the microemulsion is placed on the fourth magnetic stirrer and stirred at a low speed of 200-300 rpm under ice bath conditions. 0.1-0.5M NaOH solution is added dropwise using a microburette, while the pH meter is used to monitor the solution's acidity and alkalinity in real time. Addition is stopped when the pH reaches 7.4±0.1, and stirring continues for 30 minutes to obtain stable calcium-containing and phosphorus-containing electrospinning solutions. The ice bath conditions involve placing the sol container in a beaker containing an ice-water mixture, with the temperature controlled at 0-4℃ to prevent localized over-alkali adjustment that could lead to collagen denaturation or premature ion precipitation. Adjusting the pH to 7.4 aims to allow collagen molecules to carry a suitable charge, providing nucleation sites for subsequent in-situ mineralization.

[0058] The concentrations of calcium ions and phosphate ions should be in a specific ratio to form a complex ionic compound with a specific stoichiometry. This specific ratio refers to a molar ratio of calcium ions to phosphate ions of 5:3 (approximately 1.67:1), corresponding to the stoichiometric ratio of natural hydroxyapatite. This specific ratio is obtained by controlling both the concentration and volume of the solution, and its calculation follows the formula: in, This represents the final ion concentration in the electrospinning solution. This represents the original concentration of the added ion solution. This is the volume of the added ionic solution. This represents the volume of the collagen solution.

[0059] By using the above formula logic, the ion concentration in the two fluids before mixing can be precisely controlled to ensure that the ideal mineralization supersaturation is achieved when the jets meet instantaneously.

[0060] S4: Electrospinning module The electrospinning module includes a dual-channel micro-injection pump, a two-in-one needle assembly, a high-voltage DC power supply, and a tin foil receiver. The dual-channel micro-injection pump consists of two independent micro-injection pumps, each driving a syringe. The syringes are 1-5 mL in size and made of medical stainless steel or borosilicate glass. The syringe tip is connected to a two-in-one needle assembly via a polytetrafluoroethylene conduit. The two-in-one needle assembly is a Y-type stainless steel connector. The two inlets of the Y-type connector are connected to two syringes through conduits, and the outlet end is connected to a stainless steel spinning needle with a specification of 18-22G and an inner diameter of 0.41-0.84mm. The two solutions containing different mineral ions converge at the Y-type connector and are ejected from the same needle, completing the mixing and in-situ mineralization reaction instantly in the jet. The positive output terminal of the high-voltage DC power supply is connected to the stainless steel needle of the two-in-one needle assembly via a wire clamp, and the negative output terminal is connected to the tin foil receiver; the positive power supply voltage is 12-18kV, and the negative power supply voltage is -1 to -5kV. The foil receiver is a metal grounded receiving plate or receiving roller covered with aluminum foil, and the receiving distance (from the tip of the syringe needle to the surface of the foil receiver) is 6-12cm; The injection rate of the dual-channel micro-injection pump is set to 0.05-0.3 mL / h, and the two injection pumps have the same speed to ensure that the two fluid streams merge at the same speed.

[0061] Working process: Calcium-containing electrospinning solution and phosphorus-containing electrospinning solution are added to two syringes respectively. The syringe pump is started, and the solutions, driven by pump pressure, converge through a conduit to a Y-type connector and are ejected from the stainless steel needle. Simultaneously, a strong electric field is established between the needle and the receiver by a high-voltage DC power supply. The droplet at the needle tip is pulled into a jet under the action of the electric field force. Within milliseconds of the jet reaching the receiver, the organic solvent rapidly evaporates, causing a momentary supersaturation of the local ion concentration, triggering in-situ mineralization nucleation and growth. At the same time, the high-voltage electrostatic field and the jet stretching force synergistically induce positively charged calcium ions and negatively charged phosphate ions to migrate and polarize along the direction of the electric field, causing the in-situ generated mineral crystals to preferentially align along the collagen fiber axis. The minerals are generated and solidified in-situ between the collagen molecular chains into oriented mineralized collagen fibers, which are deposited on the tin foil receiver.

[0062] S5: Drying module The drying module includes a vacuum drying chamber and a timer; The specific implementation method is as follows: the nascent fiber membrane obtained by spinning is placed together with the tin foil receiver in a vacuum drying oven and dried for 24-72 hours at room temperature and vacuum degree ≤100Pa to completely remove the residual HFIP solvent and moisture inside the fiber and stabilize the crystal structure of the mineral phase; the timer is used to precisely control the drying time; after drying, the fiber membrane is carefully peeled off from the tin foil to obtain the final in-situ mineralized collagen scaffold material.

[0063] The final in-situ mineralized collagen scaffold material includes a continuous collagen fiber matrix and a mineral phase distributed in-situ within the collagen fiber matrix; the mineral phase is uniformly distributed in the collagen fiber matrix and is free of aggregated particles.

[0064] In the electrospinning module, a combination of a positive electrode supply voltage of 15kV and a negative electrode supply voltage of -3kV is used, and the speed of the two injection pumps is strictly set to 0.1mL / h. Under these specific process parameters, when the collagen solutions containing calcium and phosphate ions converge at the tip of the two-in-one system, the high-voltage electrostatic field not only provides stretching force but also strongly drives the directional migration of the newly mixed positively charged calcium ions and negatively charged phosphate ions in the jet. At the same time, the extremely low injection rate of 0.1mL / h provides sufficient reaction and orientation time for the ions to directionally align in the electric field and for the jet stretching.

[0065] Under the synergistic effect of these two conditions, the mineral phase generated in situ instantaneously by the jet is no longer randomly oriented isotropic particles, but rather, induced by the electric field and pulled by tensile force, preferentially oriented along the collagen fiber axis, forming oriented crystals with a highly biomimetic natural bone mineralized collagen structure. Mechanical tests show that the scaffold with this axially oriented mineralized structure has an axial tensile strength that is about 45% higher than that of the unoriented conventional in-situ mineralized scaffold, exhibiting a significant synergistic effect of mechanical enhancement. At the same time, in the degradation experiment, the oriented crystals, due to the exposure of specific crystal faces, exhibit a phased mineral ion pulse release characteristic that matches the inflammatory and repair phases of bone repair, rather than a simple linear release, which is more conducive to the phased proliferation and differentiation of osteoblastic mesenchymal stem cells.

[0066] Example 4 The difference between this embodiment and Embodiment 1 lies in the specific description of the concentration ratio of calcium ions and phosphate ions: The specific ratio refers to a molar ratio of calcium ions to phosphate ions of 5:3, or approximately 1.67:1, which corresponds to the ratio in natural hydroxyapatite. The stoichiometric ratio.

[0067] The specific configuration process is as follows: S1: Dissolve 50 mg of collagen in 1 ml of hexafluoroisopropanol. After dissolving for 1 hour, stir with a magnetic stirrer for 5 hours to ensure that the collagen is uniformly dissolved into a 5% collagen solution.

[0068] S2: Dissolve anhydrous calcium chloride in deionized water to prepare a 0.5M calcium ion stock solution; dilute the concentrated phosphoric acid solution with deionized water to prepare a 0.3M phosphate ion stock solution. The concentration ratio of the calcium ion stock solution to the phosphate ion stock solution is maintained at 5:3.

[0069] S3: Add the above ion stock solution to the collagen solution separately, and calculate the diluted ion concentration based on the following formula by controlling the added volume: in, This represents the final ion concentration in the electrospinning solution. This represents the original concentration of the added ion solution. This is the volume of the added ionic solution. This represents the volume of the collagen solution.

[0070] Taking the preparation of a calcium-containing electrospinning solution as an example: Take 0.5M calcium ion mother liquor In a 5% collagen solution: Taking the preparation of a phosphorus-containing electrospinning solution as an example: Take 0.3M phosphate ion mother liquor Join In a 5% collagen solution: At this point, the calcium ion concentration in the calcium-containing electrospinning solution is 200 mM, and the phosphate ion concentration in the phosphorus-containing electrospinning solution is 120 mM. The concentration ratio of the two remains 200:120=5:3, which is consistent with the stoichiometric ratio of hydroxyapatite.

[0071] Continue stirring for 24 hours. When the solution turns milky white and there is no precipitate, add NaOH solution to adjust the pH of the two solutions to 7.4. Finally, stir the solution for 30 minutes to obtain stable calcium-containing electrospinning solution and phosphorus-containing electrospinning solution.

[0072] S4: Add 200mM of calcium-containing electrospinning solution to each of the two syringes. and phosphorus-containing electrospinning solution 120mM Electrospinning is performed using a two-in-one system. When the two fluids converge at the needle tip, calcium and phosphorus ions are instantaneously mixed in a stoichiometric ratio of 5:3, achieving ideal mineralization supersaturation and generating a mineral phase in situ consistent with the composition of natural bone hydroxyapatite.

[0073] S5: The collected fiber scaffolds were placed in a desiccator and dried for 24 hours to obtain the final in-situ mineralized collagen scaffold material.

[0074] X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) analysis showed that the calcium-to-phosphorus atomic ratio of the mineral phase in the scaffold material prepared in this embodiment was approximately 1.67, which is highly consistent with that of natural bone hydroxyapatite, verifying the effectiveness of precise mineralization composition control by controlling a specific ratio of calcium and phosphorus ion concentrations.

[0075] Example 5 The key difference between this embodiment and Embodiment 1 lies in the detailed explanation of the in-situ nucleation mechanism and the formation of the organic-inorganic hybrid interface structure in the electrospinning module: In the S4 electrospinning module, unlike the prior art which uses high-temperature calcination at 500-1200℃ to completely remove organic components to prepare pure inorganic brittle calcium phosphate fibers, this invention performs jet in-situ mineralization at room temperature.

[0076] When the calcium- and phosphorus-containing microemulsion fluids converge at the two-in-one Y-joint, under the synergistic effect of a high-voltage electrostatic field of 15kV at the positive electrode and -3kV at the negative electrode, and an extremely low injection rate of 0.1mL / h, the jet undergoes millisecond-level rapid stretching and rapid evaporation of the solvent (HFIP).

[0077] This non-equilibrium physical field causes local micro-regions to instantly reach extremely high supersaturation, and calcium and phosphorus ions are forced to nucleate in situ within the nanoscale confined space surrounded by collagen molecular chains.

[0078] Because they have not undergone high-temperature destruction, the surface of the nascent mineral crystal nuclei contains a large number of unsaturated calcium phosphate sites, which directly form strong coordination bonds and hydrogen bonds with the polar groups of collagen molecules, such as carboxyl-COOH and amino-NH2.

[0079] This nucleation process, regulated by collagen interface coordination, not only prevents excessive crystal growth and forms low-crystallinity nanocrystals, similar to the amorphous / microcrystalline phase in natural bone, but also constructs an organic-inorganic hybrid interface structure connected by chemical bonds between the mineral and collagen.

[0080] Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that there is a significant coordination bonding peak shift between the -COO- of collagen and calcium ions in the scaffold prepared in this embodiment.

[0081] Mechanical tests show that, due to the efficient transfer of stress from the organic phase to the inorganic phase achieved by the hybrid interface, the fracture toughness of this scaffold, i.e. the critical energy release rate for crack propagation, is improved by about 200% compared with the existing physical composite scaffolds after high-temperature calcination, completely overcoming the fatal defect of brittle fracture in pure inorganic scaffolds.

[0082] Meanwhile, the low-crystallinity nanocrystals exhibited phased degradation and mineral ion pulse release characteristics in degradation experiments that highly corresponded to the inflammatory and repair phases of bone repair.

[0083] This invention provides an in-situ mineralized collagen scaffold material based on electrospinning technology and its preparation method. It can eliminate stress concentration caused by particle agglomeration, significantly improve the mechanical properties of the scaffold, and at the same time, the degradation rate of the mineral phase generated in situ matches the bone repair process, which has an excellent promoting effect on the proliferation and differentiation of osteoblast mesenchymal stem cells.

[0084] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0085] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing in-situ mineralized collagen scaffold material based on electrospinning process, characterized in that, Includes the following steps: Configure a collagen solution module, using hexafluoroisopropanol to dissolve collagen and prepare a collagen solution; Configure the ion solution module by dissolving anhydrous calcium chloride and concentrated phosphoric acid solution in purified water to prepare calcium ion solution and phosphate ion solution, respectively. An electrospinning solution module is configured by adding one of the calcium ion solution and the phosphate ion solution to the collagen solution to prepare a stable mineralized collagen electrospinning solution, wherein the concentrations of calcium ions and phosphate ions are in a specific ratio. The electrospinning module adds two solutions containing different mineral ions to the syringe and performs electrospinning using a two-in-one system. In-situ mineralization occurs at the moment of jetting and the morphology is maintained, resulting in the in-situ mineralized collagen scaffold material.

2. The method for preparing an in-situ mineralized collagen scaffold material based on electrospinning process according to claim 1, characterized in that, The configuration of the electrospinning solution module includes the following sub-steps: Add either the calcium ion solution or the phosphate ion solution to the collagen solution, and continue stirring until the solution turns milky white and there is no precipitate; Add NaOH solution to adjust the pH of the solution to 7.4, and finally stir the solution to obtain a stable electrospinning solution containing calcium and an electrospinning solution containing phosphorus.

3. The method for preparing an in-situ mineralized collagen scaffold material based on electrospinning process according to claim 1, characterized in that, In the electrospinning module, under the non-equilibrium physical field of jet high-speed stretching and solvent evaporation, calcium and phosphorus ions are nucleated in situ in the nanoscale confined space of collagen molecular chains. Under the interfacial coordination regulation of collagen polar groups, a hybrid interface structure of organic and inorganic is formed, in which low-crystallinity nanocrystals and collagen molecular chains are chemically bonded through coordination bonds and hydrogen bonds.

4. The method for preparing an in-situ mineralized collagen scaffold material based on electrospinning process according to claim 3, characterized in that, In the electrospinning module, the power supply voltage of the positive electrode is 12-18kV, the power supply voltage of the negative electrode is -1 to -5kV, and the speed of the two injection pumps is 0.05-0.3mL / h. This voltage range, together with the low-speed injection, maintains the millisecond-level stretching and curing time of the jet, ensuring the formation of nucleation and organic-inorganic hybrid interfaces in the confined space without macroscopic phase separation.

5. The method for preparing an in-situ mineralized collagen scaffold material based on electrospinning process according to claim 1, characterized in that, In the collagen solution preparation module, the concentration of the collagen solution is 5%-10%; in the electrospinning solution preparation module, the ion concentration of the mineralized collagen electrospinning solution is 150-250 mM.

6. An in-situ mineralized collagen scaffold material based on electrospinning technology, prepared and obtained by the method for preparing an in-situ mineralized collagen scaffold material based on electrospinning technology according to any one of claims 1-5, characterized in that, include: A continuous collagen fiber matrix, and mineral phases distributed in situ within the collagen fiber matrix; The mineral phase is uniformly distributed in the collagen fiber matrix and is free of agglomerated particles.

7. The in-situ mineralized collagen scaffold material based on electrospinning process according to claim 6, characterized in that, The mineral phase is a low-crystallinity nanocrystal, which forms an organic and inorganic hybrid interface structure with the collagen fiber matrix, consisting of coordination bonds and hydrogen bonds.

8. The in-situ mineralized collagen scaffold material based on electrospinning process according to claim 7, characterized in that, The hybrid interface structure enables the formation of a stress transmission network that mimics natural bone between the mineral phase and the collagen matrix, giving the scaffold exceptional fracture toughness. Furthermore, the low-crystallinity mineral phase exhibits a phased degradation ion release characteristic that matches the bone repair cycle.

9. The in-situ mineralized collagen scaffold material based on electrospinning process according to claim 6, characterized in that, The mineral phase is a complex ionic compound formed by calcium ions and phosphate ions in a specific ratio, which is obtained by controlling the concentration and volume of the ionic solution.

10. The application of an in-situ mineralized collagen scaffold material based on electrospinning technology as described in claim 6 in the preparation of bone repair products, characterized in that, The bone repair product utilizes the preferred orientation arrangement of the mineral phase along the collagen fiber axis to provide axial mechanical support for the defect site. Through the phased degradation of the oriented mineral phase, mineral ions are released, inducing osteogenic mesenchymal stem cells to migrate and differentiate along the fiber axis.

Images