Multi-spectral response oxygen-carrying artificial periosteum and preparation method thereof

Abstract

This invention discloses a multispectral responsive oxygen-carrying artificial bone membrane, comprising an inner oxygen-carrying module and an outer control module controlled by responsive light. The preparation method of this artificial bone membrane includes: 1. preparing an inner core solution and an outer shell solution; 2. injecting the solution into a coaxial electrospinning device to prepare the inner oxygen-carrying module; 3. preparing a dopamine solution; 4. immersing the inner oxygen-carrying module in the dopamine solution to obtain the artificial bone membrane described in this invention. This invention solves the problem of local oxygen supply in existing tissue-engineered bone membranes; it achieves the goal of remotely regulating the oxygen release rate through non-invasive intervention, providing oxygen based on the hypoxia tolerance characteristics of stem cells, and improving early stem cell survival rate; it proposes a "sequential oxygen supply" strategy, that is, through early vascularization, providing endogenous oxygen to the damaged tissue microenvironment after exogenous oxygen depletion to maintain the oxygen level of the microenvironment. Ultimately, it regulates stem cell fate, accelerating tissue regeneration and functional recovery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology and relates to a multispectral responsive oxygen-carrying artificial bone membrane. This invention also relates to a method for preparing the multispectral responsive oxygen-carrying artificial bone membrane. Background Technology

[0002] Oxygen-releasing biomaterials have been recognized as an innovative and unique technology for regulating local oxygen supply during bone regeneration. This technology significantly improves the survival rate of transplanted and host cells by overcoming cytotoxic hypoxia. Perfluorotributylamine (PFTBA), a fluorocarbon compound, has attracted widespread attention in the industry due to its excellent oxygen-dissolving capacity, stable chemical properties, biocompatibility, and easy sterilization, and is considered a potential alternative to artificial blood. PFTBA can effectively store and release oxygen according to the metabolic needs and oxygen saturation of specific tissues, thereby optimizing oxygen transport and supply to ischemic tissues.

[0003] Early research has shown that encapsulating PFTBA within a reservoir structure can prolong oxygen release time at specific injury sites, promoting oxygen supply to cell grafts and thus accelerating the regeneration of peripheral nerves and intervertebral disc tissues. However, existing oxygen-carrying strategies fail to adequately consider the time-dependent response of specific stem cells to hypoxia, which may limit tissue regeneration efficacy. Furthermore, current artificial periosteum tissue engineering methods struggle to achieve oxygen regulation. Summary of the Invention

[0004] The purpose of this invention is to provide a multispectral responsive oxygen-carrying artificial periosteum, solving the problem of local oxygen supply in existing tissue-engineered periosteums. By responding to multispectral stimulation, it achieves the goal of remotely regulating the oxygen release rate through non-invasive intervention. Personalized intervention is carried out based on the different hypoxia tolerance characteristics of stem cells, improving the early survival rate of stem cells. A "sequential oxygen supply" strategy is proposed, which provides endogenous oxygen to the damaged tissue microenvironment after exogenous oxygen depletion through early vascularization, thereby maintaining the oxygen level of the tissue microenvironment, promoting long-term functional integration of stem cells, regulating stem cell fate, and accelerating tissue repair and regeneration.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned multispectral response oxygen-carrying artificial bone membrane.

[0006] The technical solution adopted in this invention includes an inner oxygen-carrying module and an outer control module for responding to light. The inner oxygen-carrying module is a core-shell fiber membrane, the inner core of which is perfluorotributylamine carrying oxygen, and the outer shell is polycaprolactone carrying pravastatin. The outer control module is polydopamine nanoparticles.

[0007] Another technical solution adopted in this invention is to prepare a multispectral responsive oxygen-carrying artificial bone membrane by following these steps:

[0008] Step 1: Prepare the core solution and the outer shell solution;

[0009] Step 1.1: Add lecithin to the tabletop liquid, perform the first ultrasonic treatment, then add perfluorotributylamine, and after a second ultrasonic treatment, obtain perfluorotributylamine emulsion.

[0010] The proportion of lecithin added to the styrosol solution is 180-200 mg / mL; the specific parameters for the first ultrasonic oscillation treatment are: 2-4 oscillations at 250-350 W power at 0-4℃, each lasting 15-20 seconds; the proportion of perfluorotributylamine solvent is 1.8-2 g / mL; the specific parameters for the second ultrasonic oscillation treatment are: 10-12 oscillations at 250-350 W power at 0-4℃, each lasting 15-20 seconds.

[0011] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, producing an inner core solution;

[0012] Among them, after adding carboxylated chitosan to form a gel, the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v;

[0013] The specific steps for preparing the shell solution are as follows:

[0014] Step 1.3: Dissolve polycaprolactone in a mixture of methanol and chloroform to obtain a polycaprolactone solution;

[0015] Polycaprolactone is dissolved in a mixture of methanol and chloroform at a ratio of 0.2-0.3 g / mL, where the ratio of methanol to chloroform is 1:4-5.

[0016] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3;

[0017] Pravastatin was dissolved in polycaprolactone solution at a ratio of 90-100 μmol / mL.

[0018] Step 1.5: Stir the mixed solution obtained in step 1.4 at room temperature for 12-16 hours to obtain the shell solution.

[0019] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner and outer needles of the coaxial electrospinning needle. The injection pumps pump out the liquid to obtain the inner oxygen-carrying module with a core-shell structure.

[0020] Among them, the inner needle of the coaxial electrospinning needle is 14G, the outer needle is 18G, the distance from the needle tip to the metal plate collector is 10-12cm, the voltage of the coaxial electrospinning needle is 9-10kV, the pumping rate of the inner core solution is 0.25-0.3 mL / h, and the pumping rate of the outer shell solution is 1.0-1.2 mL / h.

[0021] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution with phosphate buffer to adjust the pH of the solution to 8.5-8-8, and add dopamine powder to the solution to obtain a dopamine solution;

[0022] The tris(hydroxymethyl)aminomethane hydrochloride solution was diluted with phosphate buffer to 10-12 mmol / L, and the dopamine solution concentration was 2.0-2.5 mg / mL.

[0023] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and continuously shake at room temperature for 24-48 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0024] The artificial periosteum of this invention non-invasively regulates local oxygen release to match the time-dependent effect of periosteal stem cells' tolerance to hypoxia, promoting the long-term survival of transplanted periosteal stem cells in bone defect areas. Pravastatin simultaneously promotes rapid vascularization to bridge exogenous oxygen, providing a long-term, stable endogenous oxygen supply (sequential oxygenation) for tissue repair, promoting osteogenic differentiation and functional integration of periosteal stem cells, and accelerating bone regeneration and repair. Furthermore, the oxygen release of this product can be controlled in response to near-infrared, ultraviolet, and visible light spectra. Attached Figure Description

[0025] Figure 1(a) is a physical diagram of the present invention;

[0026] Figure 1(b) is a scanning electron microscope image and a partial magnified view of the present invention;

[0027] Figure 1(c) is a scanning electron microscope image and a partial magnified view of the coaxial electrospinning structure of the present invention;

[0028] Figure 2(a) is a temperature change curve of the present invention under continuous near-infrared stimulation;

[0029] Figure 2(b) is a graph showing the oxygen release curve of the present invention under continuous near-infrared stimulation;

[0030] Figure 2(c) is a temperature change curve of the present invention under periodic near-infrared stimulation;

[0031] Figure 2(d) is a graph showing the oxygen release curve of the present invention under periodic near-infrared stimulation;

[0032] Figure 3(a) is a detection curve of the polydopamine coating promoting the sustained release of pravastatin in this invention;

[0033] Figure 3(b) is a detection curve of pravastatin release in this invention under the dual influence of pH / NIR;

[0034] Figure 4(a) shows the immunofluorescence pattern of periosteal stem cells under hypoxia in a hypoxic environment;

[0035] Figure 4(b) shows the immunofluorescence pattern of the effect of hypoxic environment on the proliferation of periosteal stem cells (Ki67+ cells);

[0036] Figure 4(c) is a photograph of the early osteogenic differentiation of periosteal stem cells under hypoxic conditions (alkaline phosphatase staining).

[0037] Figure 4(d) is a photograph of late-stage calcium deposition (Alizarin Red staining) in periosteal stem cells under hypoxic conditions;

[0038] Figure 5(a) shows a gross and light microscopic image of the migration ability of HUVEC cells;

[0039] Figure 5(b) is an immunofluorescence image of HUVEC cells forming tubes;

[0040] Figure 6(a) is a real-life photograph of the surgical procedure for implantation when repairing skull defects in rats using the present invention;

[0041] Figure 6(b) is a three-dimensional reconstruction image of bone repair in the skull defect area of ​​a rat after implantation of the product of the present invention and related treatment;

[0042] Figure 6(c) is a three-dimensional reconstruction image of vascular perfusion in the skull defect area of ​​a rat after implantation of the product of the present invention and related treatment.

[0043] Figure 7 These are actual photographs of HE histological staining of the heart, liver, spleen, lungs, and kidneys after implantation of the product of this invention. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0045] The multispectral response oxygen-carrying artificial bone membrane of the present invention consists of an inner oxygen-carrying module and an outer control module controlled by an outer light response module. The inner oxygen-carrying module is a fiber membrane woven from core-shell structured fibers. The core of the core-shell structured fibers is oxygen-carrying perfluorotributylamine, and the outer shell is polycaprolactone loaded with pravastatin. The outer control module consists of polydopamine nanoparticles, which are linked to the polycaprolactone of the outer shell by hydrogen bonds.

[0046] The preparation method of the multispectral response oxygen-carrying artificial bone membrane of the present invention is as follows:

[0047] Step 1: Prepare the inner core solution and the outer shell solution.

[0048] Step 1.1: Add lecithin to the benchtop solution at a mass concentration of 180-200 mg / mL, and treat with ultrasound at 250-350 W power for 2-4 times at 0-4℃, each time for 15-20 seconds. Then add perfluorotributylamine solvent at a mass concentration of 1.8-2 g / mL, and treat with ultrasound at 250-350 W power for 10-12 times at 0-4℃, each time for 15-20 seconds, to obtain perfluorotributylamine emulsion.

[0049] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v, to produce the inner core solution.

[0050] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.2-0.3 g / mL in a mixture of methanol and chloroform at a volume ratio of 1:4-5 to obtain a polycaprolactone solution;

[0051] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 90-100 μmol / mL;

[0052] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 12-16 hours to obtain the shell solution;

[0053] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 10-12cm. The voltage of the coaxial electrospinning needle is 9-10kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.25-0.3 mL / h for the inner needle and 1.0-1.2 mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0054] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 10-12 mmol / L with phosphate buffer, adjust the pH of the solution to 8.5-8.8, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0-2.5 mg / mL.

[0055] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at a speed of 80-100 rpm at room temperature for 24-48 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0056] Steps 1.1-1.5 involve constructing oxygen-carrying and drug-eluting membrane materials. The advantage of using perfluorotributylamine is that it has excellent oxygen solubility, approximately 20 times that of water, and was first used as a blood substitute, exhibiting good biocompatibility. The advantage of using pravastatin is that it can achieve a hydrophilic-hydrophobic transition at different pH values, binding to hydrophobic polycaprolactone to varying degrees, thereby achieving different drug release rates at different pH values. This is beneficial for accelerating the release rate of pravastatin based on the characteristic of gradually increasing pH after tissue damage.

[0057] Steps 3-4 involve preparing the oxygen supply membrane system. The advantages of using polydopamine are: First, it allows for self-assembly with the membrane material surface. This is because polydopamine can form hydrogen bonds with the outer shell component, polycaprolactone. Second, polydopamine allows for control of the oxygen release rate. This is achieved through photothermal conversion under NIR, influencing the conformational transformation of the polycaprolactone semi-crystal to fiber structure, increasing porosity. Simultaneously, temperature changes affect the solubility of oxygen in perfluorotributylamine solution, accelerating the oxygen release rate. Third, polydopamine also slows down the release rate of pravastatin. This is because pravastatin and polydopamine can form hydrogen bonds and π-π bonds; this non-covalent bonding reduces drug burst release. Fourth, polydopamine has excellent hydrophilicity and intracellular ROS scavenging capabilities. Decorating the material surface with polydopamine can significantly increase the material's biocompatibility and hydrophilicity.

[0058] Meanwhile, polydopamine colloidal particles of different sizes exhibit light absorption in the ultraviolet, visible, and near-infrared regions, with absorbance gradually decreasing as the wavelength increases, perfectly matching the spectroscopic characteristics of sunlight. Throughout the ultraviolet, visible, and near-infrared bands, polydopamine particles are high-performance light-absorbing materials, with negligible interference from light scattering. Therefore, polydopamine is a highly efficient broadband photothermal conversion material, capable of photothermal reactions in all three bands, causing polycaprolactone to undergo a semi-crystalline to fibrous transformation under heat. The oxygen release rate from the internal oxygen-carrying module changes synchronously with the temperature and conformational changes of polycaprolactone, thus controlling the oxygen release rate of the artificial bone membrane of this invention.

[0059] Example 1

[0060] The following steps were taken to prepare a spectrally responsive oxygen-carrying artificial bone membrane:

[0061] Step 1: Add lecithin to the benchtop solution at a mass concentration of 190 mg / mL, and treat it twice with ultrasound at 300 W power for 17.5 seconds each time at 4℃. Then add perfluorotributylamine at a mass concentration of 1.883 g / mL, and treat it 11 times with ultrasound at 300 W power for 17.5 seconds each time at 4℃ to obtain perfluorotributylamine emulsion.

[0062] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 12.5% ​​w / v, to produce the inner core solution.

[0063] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.25 g / mL in a 1:4 mixture of methanol and chloroform to obtain a polycaprolactone solution;

[0064] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 3 at a molar concentration of 100 μmol / mL;

[0065] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 14 hours to obtain the shell solution;

[0066] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11cm. The voltage of the coaxial electrospinning needle is 9.5kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.3mL / h for the inner needle and 1.2mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0067] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 10 mmol / L with phosphate buffer, adjust the pH of the solution to 8.65, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0 mg / mL;

[0068] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and continuously shake it at room temperature at a speed of 90 rpm for 36 hours on a shaker to form an outer control module covering the outside of the inner oxygen-carrying module.

[0069] The multispectral response oxygen-carrying artificial bone membrane manufactured according to Example 1 was tested.

[0070] As shown in Figure 1, Figure 1(a) is a physical image of the present invention; Figure 1(b) is a scanning electron microscope image and a partial magnified image of the present invention, with the yellow arrow indicating the deposition of polydopamine nanoparticles; Figure 1(c) is a scanning electron microscope image and a partial magnified image of the coaxial electrospinning structure of the present invention.

[0071] As shown in Figure 2, Figure 2 illustrates the photothermal conversion performance and oxygen release performance of the product of this invention. In Figure 2(a), the horizontal axis represents time and the vertical axis represents temperature. The test was conducted starting at time 0 with a wavelength of 808 nm and a power of 1.0 W / cm². 2 The temperature change curve of the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent) after NIR (near-infrared irradiation) treatment; Figure 2(b) shows the horizontal axis as time and the vertical axis as dissolved oxygen content in water, with the irradiation starting at time 0 at a wavelength of 808 nm and a power of 1.0 W / cm. 2 After NIR (near-infrared) irradiation treatment, the dissolved oxygen content change curve of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) in water is marked with a yellow triangle. As a control, the dissolved oxygen content change curve of deoxygenated water is marked with a green circle. In Figure 2(c), the horizontal axis represents time and the vertical axis represents temperature. The red area represents the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) subjected to NIR irradiation at a wavelength of 808 nm and a power of 1.0 W / cm². 2 The blue portion represents the time period of NIR (near-infrared) irradiation treatment, where the polydopamine@oxygen / pravastatin membrane (i.e., this patented product) was not subjected to NIR treatment at a wavelength of 808nm and a power of 1.0W / cm². 2 The time period of NIR (near-infrared) irradiation treatment is used to determine the temperature change curve of the final polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Figure 2(d) shows the horizontal axis as time and the vertical axis as dissolved oxygen content in water. The red area represents the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) subjected to NIR treatment at a wavelength of 808 nm and a power of 1.0 W / cm². 2 The blue portion represents the time period of NIR (near-infrared) irradiation treatment, where the polydopamine@oxygen / pravastatin membrane (i.e., this patented product) was not subjected to 808nm wavelength and power 1.0W / cm². 2 The time period of NIR (near-infrared light irradiation) treatment, and the final oxygen release curve of the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent); based on the above detection results, it can be seen that the product of this invention can rapidly change temperature under the influence of near-infrared light, and thus the temperature and oxygen release rate change with periodic near-infrared light stimulation.

[0072] As shown in Figure 3, Figure 3 illustrates the drug release of the product of this invention. In Figure 3(a), the horizontal axis represents time, and the vertical axis represents the cumulative pravastatin release percentage. The curve showing the change in the cumulative pravastatin release percentage of the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent) is marked with a yellow square. As a control, the curve showing the change in the cumulative pravastatin release percentage without polydopamine deposition of oxygen-containing material is marked with a black circle. In Figure 3(b), the horizontal axis represents time, and the vertical axis represents the cumulative pravastatin release percentage. The red area represents the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent) subjected to a wavelength of 808 nm and a power of 1.0 W / cm². 2 The blue portion represents the time period of NIR (near-infrared) irradiation treatment, where the polydopamine@oxygen / pravastatin membrane (i.e., this patented product) was not subjected to NIR treatment at a wavelength of 808nm and a power of 1.0W / cm². 2 During the NIR (near-infrared light irradiation) treatment period, the percentage change curve of accumulated pravastatin release of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) at pH 7.4 is marked with a yellow square. As a control, the percentage change curve of accumulated pravastatin release of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) at pH 6.0 is marked with a blue circle. Based on the above test results, it can be seen that the polydopamine nanoparticle coating can significantly delay the drug release of pravastatin. At the same time, it can be seen that the product of the present invention responds to near-infrared light and pH value, and the release rate of pravastatin is accelerated in a higher pH value environment, and it can respond to periodic near-infrared light stimulation.

[0073] As shown in Figure 4, Figure 4 illustrates the effect of the product of the present invention on the fate of periosteal stem cells under in vitro hypoxic conditions. The specific experimental method is as follows: periosteal stem cells were cultured on the product of the present invention and placed in a hypoxic environment. After 48 hours, they were subjected to 808nm NIR (1.0W / cm²). 2The product of this invention was used to stimulate the periosteal stem cells in a hypoxic environment. Figure 4(a) shows the effect of the product of this invention on the hypoxia of periosteal stem cells in a hypoxic environment, where green represents the fluorescence of the hypoxia probe. The left panel of Figure 4(a) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right panel of Figure 4(a) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). Figure 4(b) shows the effect of the product of this invention on the proliferation of periosteal stem cells (Ki67+ cells) in a hypoxic environment. The left panel of Figure 4(b) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right panel of Figure 4(b) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). Figure 4(c) The images show the early osteogenic differentiation (alkaline phosphatase staining) of periosteal stem cells under hypoxic conditions after using the product of this invention. The left image of Figure 4(c) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 4(c) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). Figure 4(d) shows the late calcium deposition (Alizarin Red staining) of periosteal stem cells under hypoxic conditions after using the product of this invention. The left image of Figure 4(d) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 4(d) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). Based on the above actual images, immunofluorescence staining shows that the product of this invention can significantly promote the improvement of hypoxia and the proliferation rate of periosteal stem cells. The actual images stained with alkaline phosphatase and Alizarin Red show that this invention significantly promotes the early and late osteogenic differentiation of periosteal stem cells.

[0074] Figure 5 shows the effect of the product of this invention on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro under hypoxic conditions. Figure 5(a) shows that the migration ability of HUVECs was significantly improved after using the product of this invention. The left image of Figure 5(a) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 5(a) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). Figure 5(b) shows the effect of the product of this invention on HUVEC cell tube formation. The left image of Figure 5(b) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 5(b) is the polydopamine@oxygen / pravastatin membrane (i.e., the product of this patent). According to the Transwell experiment above, the product of this invention significantly promotes HUVEC cell migration, and immunofluorescence staining shows that its tube formation, including total length and number of nodes, is significantly increased.

[0075] Figure 6 shows the experimental results of repairing skull defects in rats using the present invention. Figure 6(a) shows the surgical procedure of implanting the present invention to repair skull defects in rats. From left to right, the surgical procedure is as follows: incision, periosteum removal, skull defect repair, periosteum implantation, and suturing. 48 hours after implantation, the implantation was periodically performed at a wavelength of 808 nm and a power of 1.0 W / cm². 2The rats were treated with NIR (near-infrared light irradiation) twice daily for 5 minutes each time. Figure 6(b) shows the results of imaging examinations of bone repair in the defect area of ​​rats at 4 and 8 weeks after using the product of this invention. The left image of Figure 6(b) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 6(b) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product). Figure 6(c) shows the results of imaging examinations of vascular perfusion in the defect area of ​​rats after using the product of this invention. The left image of Figure 6(c) is the control group with an oxygen-free polycaprolactone / perfluorotributylamine membrane, and the right image of Figure 6(c) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product). Micro-CT scanning and reconstruction showed that at 4 and 8 weeks, the present invention significantly promoted bone regeneration and angiogenesis in the bone defect area.

[0076] Figure 7 The group presented the biosafety test results after using the product of the present invention. It can be seen that after using the product of the present invention, the HE histological staining of the heart, liver, spleen, lungs and kidneys showed no obvious abnormalities, indicating that the product of the present invention has no significant effect on the major organs of the body after implantation.

[0077] Example 2

[0078] The following steps were taken to prepare a spectrally responsive oxygen-carrying artificial bone membrane:

[0079] Step 1: Prepare the inner core solution and the outer shell solution.

[0080] Step 1.1: Add lecithin to the benchtop solution at a mass concentration of 180 mg / mL, and treat it twice with ultrasonic power of 250 W at 0℃ for 15 seconds each time. Then add perfluorotributylamine solvent at a mass concentration of 1.8 g / mL, and treat it 10 times with ultrasonic power of 250 W at 0℃ to obtain perfluorotributylamine emulsion.

[0081] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 10% w / v, to produce the inner core solution.

[0082] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.2 g / mL in a 1:4 mixture of methanol and chloroform to obtain a polycaprolactone solution;

[0083] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 90 μmol / mL;

[0084] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 12 hours to obtain the shell solution;

[0085] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 10cm. The voltage of the coaxial electrospinning needle is 9kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.25mL / h for the inner needle and 1mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0086] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 10 mmol / L with phosphate buffer, adjust the pH of the solution to 8.5, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0 mg / mL;

[0087] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at 80 rpm at room temperature for 24 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0088] Example 3

[0089] Step 1: Prepare the inner core solution and the outer shell solution.

[0090] Step 1.1 Add lecithin to the benchtop solution at a mass concentration of 200 mg / mL and treat it with ultrasound at 350 W power for 4 times at 4℃. Then add perfluorotributylamine solvent at a mass concentration of 2 g / mL and treat it with ultrasound at 350 W power for 12 times at 4℃, each time for 20 seconds to obtain perfluorotributylamine emulsion.

[0091] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 15% w / v, to produce the core solution.

[0092] Step 1.3: Dissolve polycaprolactone in a 1:5 mixture of methanol and chloroform at a mass concentration of 0.3 g / mL to obtain a polycaprolactone solution;

[0093] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 100 μmol / mL;

[0094] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 16 hours to obtain the shell solution;

[0095] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 12cm. The voltage of the coaxial electrospinning needle is 10kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.3mL / h for the inner needle and 1.2mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0096] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 12 mmol / L with phosphate buffer, adjust the pH of the solution to 8.8, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.5 mg / mL.

[0097] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at 100 rpm at room temperature for 48 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0098] Example 4

[0099] Step 1: Prepare the inner core solution and the outer shell solution.

[0100] Step 1.1: Add lecithin to the benchtop solution at a mass concentration of 185 mg / mL, and treat it with ultrasound at 275 W power once at 1℃ for 16 seconds each time. Then add perfluorotributylamine solvent at a mass concentration of 1.85 g / mL, and treat it with ultrasound at 275 W power 10 times at 1℃ for 16 seconds each time to obtain perfluorotributylamine emulsion.

[0101] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 11.25% w / v, to produce the inner core solution;

[0102] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.225 g / mL in a mixture of methanol and chloroform at a volume ratio of 1:4.25 to obtain a polycaprolactone solution;

[0103] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 92.5 μmol / mL;

[0104] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 13 hours to obtain the shell solution;

[0105] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 10.5cm. The voltage of the coaxial electrospinning needle is 9.25kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.26 mL / h for the inner needle and 1.05 mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0106] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 11.5 mmol / L with phosphate buffer, adjust the pH of the solution to 8.6, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.1 mg / mL;

[0107] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at 85 rpm at room temperature for 30 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0108] Example 5

[0109] Step 1: Prepare the inner core solution and the outer shell solution.

[0110] Step 1.1: Add lecithin to the benchtop solution at a mass concentration of 195 mg / mL, and treat it with ultrasound at 325 W power for 4 times at 3℃ for 18 seconds each time. Then add perfluorotributylamine solvent at a mass concentration of 1.95 g / mL, and treat it with ultrasound at 325 W power for 12 times at 3℃ for 18 seconds each time to obtain perfluorotributylamine emulsion.

[0111] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 13.25% w / v, to produce the core solution;

[0112] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.2275 g / mL in a mixture of methanol and chloroform at a volume ratio of 1:4.75 to obtain a polycaprolactone solution;

[0113] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 97.5 μmol / mL;

[0114] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 15 hours to obtain the shell solution;

[0115] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11.5cm. The voltage of the coaxial electrospinning needle is 9.75kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.29 mL / h for the inner needle and 1.15 mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0116] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 10.5 mmol / L with phosphate buffer, adjust the pH of the solution to 8.7, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.7 mg / mL;

[0117] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at 95 rpm at room temperature for 42 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

[0118] Example 6

[0119] Step 1: Prepare the inner core solution and the outer shell solution.

[0120] Step 1.1: Add lecithin to the benchtop solution at a mass concentration of 190 mg / mL, and treat it with ultrasound at 290W power for 3 times at 3.5℃ for 18 seconds each time. Then add perfluorotributylamine solvent at a mass concentration of 1.9 g / mL, and treat it with ultrasound at 290W power for 11 times at 3.5℃ for 18 seconds each time to obtain perfluorotributylamine emulsion.

[0121] Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 14% w / v, to produce the inner core solution.

[0122] Step 1.3: Dissolve polycaprolactone at a mass concentration of 0.28 g / mL in a mixture of methanol and chloroform at a volume ratio of 1:4.2 to obtain a polycaprolactone solution;

[0123] Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 93 μmol / mL;

[0124] Step 1.5: Stir the mixed solution obtained in Step 1.4 at room temperature for 13 hours to obtain the shell solution;

[0125] Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner needle 14G and outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11cm. The voltage of the coaxial electrospinning needle is 9.6kV. The injection pumps pump out the inner core solution and outer shell solution at a rate of 0.27 mL / h for the inner needle and 1.1 mL / h for the outer needle. Spinning yields core-shell structured fiber filaments. The fiber membrane obtained by further spinning is the inner oxygen-carrying module.

[0126] Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution to 11 mmol / L with phosphate buffer, adjust the pH of the solution to 8.7, and add dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.3 mg / mL;

[0127] Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and shake it on a shaker at a speed of 88 rpm at room temperature for 38 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

Claims

1. A multispectral response oxygen-carrying artificial bone membrane, characterized in that, It includes an inner oxygen-carrying module and an outer control module that responds to light. The inner oxygen-carrying module is a fiber membrane woven from core-shell structured fiber filaments. The core of the core-shell structured fiber filaments is perfluorotributylamine carrying oxygen, and the outer shell is polycaprolactone carrying pravastatin. The outer control module is polydopamine nanoparticles.

2. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 1, characterized in that, Follow these steps: Step 1: Preparation of inner core solution and outer shell solution Step 2: Fill the inner core solution and outer shell solution obtained in Step 1 into two injection pumps respectively, and connect them to the inner and outer needles of the coaxial electrospinning needle. The injection pumps pump out the liquid, and electrospinning yields core-shell structure fiber filaments. Further weaving the core-shell structure fiber filaments to obtain a fiber membrane is the inner oxygen-carrying module. Step 3: Dilute the tris(hydroxymethyl)aminomethane hydrochloride solution with phosphate buffer to adjust the pH of the solution to 8.5-8-8, and add dopamine powder to the solution to obtain a dopamine solution; Step 4: Immerse the inner oxygen-carrying module obtained in Step 2 in the dopamine solution obtained in Step 3, and continuously shake at room temperature for 24-48 hours to form an outer control module covering the outside of the inner oxygen-carrying module.

3. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 2, characterized in that, In step 1, the specific steps for preparing the core solution are as follows: Step 1.1: Add lecithin to the tabletop liquid, perform the first ultrasonic treatment, then add perfluorotributylamine, and after a second ultrasonic treatment, obtain perfluorotributylamine emulsion. Step 1.2: Add carboxylated chitosan to the perfluorotributylamine emulsion to form a gel, producing an inner core solution; The specific steps for preparing the shell solution are as follows: Step 1.3: Dissolve polycaprolactone in a mixture of methanol and chloroform to obtain a polycaprolactone solution; Step 1.4: Dissolve pravastatin in the polycaprolactone solution obtained in step 1.3; Step 1.5: Stir the mixed solution obtained in step 1.4 at room temperature for 12-16 hours to obtain the shell solution.

4. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 3, characterized in that, In step 1.1, the mass concentration of lecithin relative to the styrosol solution is 180-200 mg / mL; the specific parameters for the first ultrasonic oscillation treatment are: oscillation treatment at 250-350 W power for 2-4 times at 0-4℃, each time for 15-20 seconds; the mass concentration of the added perfluorotributylamine solvent is 1.8-2 g / mL; and the specific parameters for the second ultrasonic oscillation treatment are: oscillation treatment at 250-350 W power for 10-12 times at 0-4℃, each time for 15-20 seconds.

5. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 3, characterized in that, In step 1.2, after adding carboxylated chitosan to form a gel, the concentration of carboxylated chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v.

6. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 3, characterized in that, In step 1.3, polycaprolactone is dissolved in a mixture of methanol and chloroform at a mass concentration of 0.2-0.3 g / mL, wherein the volume ratio of the methanol and chloroform mixture is 1:4-5.

7. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 3, characterized in that, In step 1.4, pravastatin is dissolved in polycaprolactone solution at a molar concentration of 90-100 μmol / mL.

8. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 3, characterized in that, In step 2, the inner needle of the coaxial electrospinning needle is 14G, the outer needle is 18G, the distance from the needle tip to the metal plate collector is 10-12cm, the voltage of the coaxial electrospinning needle is 9-10kV, the pumping rate of the inner core solution is 0.25-0.3 mL / h, and the pumping rate of the outer shell solution is 1.0-1.2 mL / h.

9. The method for preparing a multispectral response oxygen-carrying artificial bone membrane according to claim 2, characterized in that, In step 3, the tris(hydroxymethyl)aminomethane hydrochloride solution is diluted with phosphate buffer to 10-12 mmol / L, and the concentration of the dopamine solution is 2.0-2.5 mg / mL.

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