Multispectral response oxygen-carrying artificial periosteum and preparation method thereof

Abstract

The invention discloses a multispectral response oxygen-carrying artificial periosteum which comprises an inner layer oxygen-carrying module and an outer layer control module controlled by outer layer response light. The preparation method of the artificial periosteum comprises the following steps: 1, preparing an inner core solution and a shell solution; 2, injecting the inner core solution and the shell solution into coaxial electrostatic spinning equipment to prepare the inner layer oxygen carrying module; 3, preparing a dopamine solution; and 4, soaking the inner layer oxygen carrying module in the dopamine solution to prepare the artificial periosteum. The problem that in the prior art, a tissue engineering periosteum cannot supply oxygen locally is solved; the aim of remotely adjusting the oxygen release speed through non-invasive intervention is achieved, oxygen is supplied according to the hypoxia tolerance characteristics of the stem cells, and the survival rate of the early stem cells is increased; a'sequential oxygen supply 'strategy is provided, that is, endogenous oxygen is provided for the damaged tissue microenvironment after exogenous oxygen is exhausted through early vascularization, so that the oxygen level of the microenvironment is maintained. Finally, the stem cell fate is regulated and controlled, and tissue regeneration and functional recovery are accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and relates to a multi-spectral responsive oxygen-carrying artificial periosteum. The invention also relates to a preparation method of the multi-spectral responsive oxygen-carrying artificial periosteum. Background Art

[0002] Oxygen-releasing biomaterials have been considered as an innovative and unique technology to regulate local oxygen supply during bone regeneration. This technology significantly improves the survival rate of transplanted cells and host cells by overcoming cytotoxic hypoxia. Perfluorotributylamine (PFTBA), a fluorocarbon, has attracted widespread attention in the industry due to its excellent oxygen solubility, stable chemical properties, biocompatibility and easy sterilization properties, and is regarded as a potential substitute for 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 results have shown that by encapsulating PFTBA in a reservoir structure, the release time of oxygen can be prolonged at specific injury sites, promoting oxygen supply to cell transplants and accelerating the regeneration of peripheral nerves and intervertebral disc tissues. However, existing oxygen-carrying strategies fail to fully consider the time dependence of specific stem cells' response to hypoxia, which may limit the effect of tissue regeneration, and existing artificial periosteum tissue engineering also has difficulty in regulating oxygen. Summary of the invention

[0004] The purpose of the present invention is to provide a multi-spectral responsive oxygen-carrying artificial periosteum, which solves the problem that the tissue engineering periosteum in the prior art cannot supply oxygen locally; by responding to multi-spectral stimulation, the goal of remotely regulating the oxygen release rate through non-invasive intervention is achieved, and personalized intervention is performed according to the characteristics of different stem cells' tolerance to hypoxia to improve the survival rate of early stem cells; a "sequential oxygen supply" strategy is proposed, that is, through early vascularization, endogenous oxygen is provided to the damaged tissue microenvironment after exogenous oxygen is exhausted to maintain the oxygen level of the tissue microenvironment, promote the long-term functional integration of stem cells, regulate the fate of stem cells, and accelerate tissue repair and regeneration.

[0005] Another object of the present invention is to provide a method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum.

[0006] The technical solution adopted by the present invention is to include an inner oxygen-carrying module and an outer control module that responds to light and is controlled. The inner oxygen-carrying module is a core-shell structure fiber membrane, the inner core of the core-shell structure fiber membrane is oxygen-carrying perfluorotributylamine, and the outer shell is polycaprolactone carrying pravastatin; the outer control module is polydopamine nanoparticles.

[0007] Another technical solution adopted by the present invention is to prepare a multi-spectral responsive oxygen-carrying artificial periosteum by following the steps below: Step 1, preparing an inner core solution and an outer shell solution; Step 1.1, adding lecithin to the benchtop solution, performing the first ultrasonic treatment, adding perfluorotributylamine, and performing the second ultrasonic treatment to obtain a perfluorotributylamine emulsion; Among them, the proportion of lecithin added to the benchtop solution is 180-200 mg / mL; the specific parameters of the first ultrasonic oscillation treatment are: 2-4 times of oscillation treatment at 250-350 W power at 0-4°C, each time for 15-20 seconds; the proportion of perfluorotributylamine solvent is 1.8-2 g / mL, and the specific parameters of the second ultrasonic oscillation treatment are: 10-12 times of oscillation treatment at 250-350 W power at 0-4°C, each time for 15-20 seconds; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel to produce an inner core solution; wherein, after adding carboxyl chitosan to form gel, the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v; The specific steps for preparing the shell solution are: Step 1.3, dissolving polycaprolactone in a mixture of methanol and chloroform to obtain a polycaprolactone solution; Wherein, polycaprolactone is dissolved in a mixture of methanol and chloroform at a ratio of 0.2-0.3 g / mL, and the ratio of the mixture of methanol and chloroform is 1:4-5; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3; Among them, pravastatin was dissolved in polycaprolactone solution at a ratio of 90-100 μmol / mL; Step 1.5: Stir the mixed solution obtained in step 1.4 at room temperature for 12-16 hours to obtain a shell solution.

[0008] Step 2, filling the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connecting them to the inner needle and the outer needle of the coaxial electrospinning needle, and the syringe pumps pump out the liquid to obtain an inner oxygen-carrying module of the core-shell structure; 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 speed of the inner core solution is 0.25-0.3 mL / h, and the pumping speed of the outer shell solution is 1.0-1.2 mL / h; Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution with a phosphate buffer solution, adjusting the pH of the solution to 8.5-8-8, and adding dopamine powder to the solution to obtain a dopamine solution; Wherein, the tris(hydroxymethyl)aminomethane hydrochloride) solution is diluted with phosphate buffer to 10-12 mmol / L, and the concentration of dopamine solution is 2.0-2.5 mg / mL; Step 4: Soak 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 outer side of the inner oxygen-carrying module. The artificial periosteum of the present invention non-invasively regulates local oxygen release to match the time-dependent effect of periosteal stem cells on hypoxia tolerance, thereby promoting the long-term survival of transplanted periosteal stem cells in the bone defect area. Pravastatin simultaneously promotes rapid vascularization to connect exogenous oxygen, provide long-term stable endogenous oxygen supply (sequential oxygen supply) for tissue repair, promote osteogenic differentiation and functional integration of periosteal stem cells, and accelerate bone regeneration and repair. At the same time, the oxygen release of this product can be controlled in response to near-infrared light, ultraviolet light and visible light spectra. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 (a) is a physical diagram of the present invention; FIG1( b ) is a scanning electron microscope image and a partial enlarged image of the present invention; FIG1( c ) is a scanning electron microscope image and a partial magnified image of the coaxial electrospinning structure of the present invention; FIG2 (a) is a temperature change curve diagram of the present invention under near-infrared continuous stimulation; FIG2( b ) is a graph showing the oxygen release curve of the present invention under continuous near-infrared stimulation; FIG2 (c) is a temperature variation curve of the present invention under periodic near-infrared stimulation; FIG2( d ) is a graph showing the oxygen release curve of the present invention under periodic near-infrared stimulation; FIG3 (a) is a detection curve diagram of the polydopamine coating promoting the sustained release of pravastatin in the present invention; FIG3( b ) is a detection curve diagram of pravastatin release in the present invention under the dual influence of pH / NIR; Figure 4 (a) is an immunofluorescence image of the effect of hypoxia on periosteal stem cells in a hypoxic environment; Figure 4 (b) is an immunofluorescence image showing the effect of hypoxia on the proliferation of periosteal stem cells (Ki67+ cells); Figure 4 (c) is a real picture of the early osteogenic differentiation (alkaline phosphatase staining) of periosteal stem cells under hypoxia; Figure 4 (d) is a real picture of late calcium deposition (Alizarin red staining) of periosteal stem cells under hypoxia; Figure 5 (a) is a general view and light microscopic image of HUVEC cell migration ability; Figure 5 (b) is an immunofluorescence image of HUVEC cell tube formation; FIG6 (a) is a photo of the implantation operation process when the present invention is used to repair a rat skull defect; FIG6 (b) is a three-dimensional reconstruction of the bone repair imaging of the rat skull defect area after implantation of the product of the present invention and related treatment; FIG6 (c) is a three-dimensional reconstruction of the vascular perfusion imaging examination of the skull defect area of ​​a rat after implantation of the product of the present invention and related treatment; Figure 7 These are actual pictures of HE histological staining of the heart, liver, spleen, lung and kidney after implantation of the product of the present invention. DETAILED DESCRIPTION

[0010] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0011] The multi-spectral responsive oxygen-carrying artificial periosteum of the present invention is composed of an inner oxygen-carrying module and an outer control module that responds to light and is controlled. The inner oxygen-carrying module is a fiber membrane woven from core-shell structure fiber silk, the inner core of the core-shell structure fiber silk is oxygen-carrying perfluorotributylamine, and the outer shell is polycaprolactone carrying pravastatin; the outer control module is polydopamine nanoparticles, and the polydopamine nanoparticles are linked to the polycaprolactone of the outer shell through hydrogen bonds.

[0012] The preparation method of the multi-spectral responsive oxygen-carrying artificial periosteum of the present invention is specifically as follows: Step 1: Prepare the inner core solution and the outer shell solution.

[0013] Step 1.1, adding lecithin to a benchtop solution at a mass concentration of 180-200 mg / mL, ultrasonically treating the solution at a power of 250-350 W for 2-4 times at a temperature of 0-4°C for 15-20 seconds each time, then adding a perfluorotributylamine solvent at a mass concentration of 1.8-2 g / mL, ultrasonically treating the solution at a power of 250-350 W for 10-12 times at a temperature of 0-4°C for 15-20 seconds each time, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.2-0.3 g / mL in a mixture of methanol and chloroform in a volume ratio of 1:4-5 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 90-100 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 12-16 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the 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, and the syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.25-0.3 mL / h for the inner needle and 1.0-1.2 mL / h for the outer needle. Spinning obtains core-shell structure fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module; Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution with phosphate buffer to 10-12 mmol / L, adjusting the pH of the solution to 8.5-8.8, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0-2.5 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake it on a shaker at 80-100 rpm for 24-48 hours at room temperature to form an outer control module covering the outer side of the inner oxygen-carrying module.

[0014] Among them, steps 1.1-1.5 are for constructing oxygen-carrying and drug-carrying membrane materials. The advantage of using perfluorotributylamine is that the product has excellent oxygen solubility, which is about 20 times that of water. It was first used in blood substitutes and has good biosafety. The advantage of using pravastatin is that it can achieve a transition from hydrophilic to hydrophobic properties at different pH values, and can achieve different degrees of binding with hydrophobic polycaprolactone, thereby achieving different drug release rates at different pH values, which is beneficial to accelerate the release rate of pravastatin according to the characteristic of gradually increasing pH after tissue damage.

[0015] Step 3-4 is to prepare an oxygen supply membrane system. The advantages of using polydopamine are: first, self-assembly with the surface of the membrane material can be achieved. The principle is that polydopamine can form hydrogen bonds with the shell component polycaprolactone. Second, the use of polydopamine has the advantage of being able to control the oxygen release rate. The principle is to increase the porosity by photothermal conversion under NIR, by affecting the conformational transition of the shell component polycaprolactone semi-crystal to fiber. At the same time, temperature changes will affect the solubility of oxygen in the perfluorotributylamine solution and accelerate the release rate of oxygen. Third, the use of polydopamine also has the advantage of slowing down the release rate of pravastatin. The principle is that pravastatin can form hydrogen bonds and π-π bonds with polydopamine. This non-covalent binding will reduce the sudden release of drugs. Fourth, polydopamine also has excellent hydrophilicity and intracellular ROS scavenging ability. Decorating on the surface of the material can significantly increase the biocompatibility and hydrophilicity of the material.

[0016] At the same time, polydopamine colloid particles of different sizes have light absorption in the ultraviolet, visible and near-infrared regions, and the absorbance gradually decays with the increase of light wavelength, which is completely consistent with the spectral characteristics of sunlight. In the entire ultraviolet, visible and near-infrared bands, polydopamine particles are high-performance light-absorbing materials, and the interference from light scattering is almost negligible. Therefore, polydopamine is an efficient broadband photothermal conversion material, and photothermal reactions can occur in the above three bands, so that polycaprolactone undergoes a semi-crystalline to fibrous state under heat, and the oxygen in the internal oxygen-carrying module can change its release rate synchronously after the temperature and conformational changes of polycaprolactone occur, thereby controlling the oxygen release rate of the artificial periosteum of the present invention.

[0017] Example 1 Prepare the spectrally responsive oxygen-carrying artificial periosteum as follows: Step 1, adding lecithin to a benchtop solution at a mass concentration of 190 mg / ml, ultrasonically treating the solution twice at 300 W power at 4°C for 17.5 seconds each time, then adding perfluorotributylamine at a mass concentration of 1.883 g / mL, ultrasonically treating the solution 11 times at 300 W power at 4°C for 17.5 seconds each time, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 12.5% ​​w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.25 g / mL in a mixture of methanol and chloroform in a ratio of 1:4 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 3 at a molar concentration of 100 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 14 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11 cm. The voltage of the coaxial electrospinning needle is 9.5 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.3 mL / h for the inner needle and 1.2 mL / h for the outer needle. Spinning obtains core-shell structure fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 10 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.65, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake it continuously at room temperature for 36 hours at a speed of 90 rpm on a shaker to form an outer control module covering the outer side of the inner oxygen-carrying module.

[0018] The multi-spectral responsive oxygen-carrying artificial periosteum manufactured according to Example 1 was tested.

[0019] As shown in Figure 1, Figure 1 (a) is a physical picture of the present invention; Figure 1 (b) is a scanning electron microscope picture and a local enlarged picture of the present invention, and the yellow arrow marks the deposition of polydopamine nanoparticles; Figure 1 (c) is a scanning electron microscope picture and a local enlarged picture of the coaxial electrospinning structure of the present invention.

[0020] As shown in Figure 2, Figure 2 shows the photothermal conversion performance and oxygen release performance of the product of the present invention, wherein the horizontal axis of Figure 2 (a) is time and the vertical axis is temperature. At time 0, the wavelength is 808nm and the power is 1.0W / cm 2 Temperature change curve of polydopamine@oxygen / pravastatin membrane (i.e., the patented product) after NIR (near infrared light irradiation) treatment; Figure 2 (b) shows the time on the abscissa and the dissolved oxygen content in water on the ordinate. The treatment was started at time 0 with a wavelength of 808 nm and a power of 1.0 W / cm 2 After NIR (near infrared light irradiation) treatment, the dissolved oxygen content change curve of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) in the 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 is time, the vertical axis is temperature, and the red area is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) subjected to 808nm wavelength and 1.0W / cm 2 The blue part is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) that has not been exposed to NIR (near infrared light) at a wavelength of 808 nm and a power of 1.0 W / cm 2 The temperature change curve of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) formed during the NIR (near infrared light irradiation) treatment period; Figure 2 (d) shows the time on the horizontal axis and the dissolved oxygen content in the water on the vertical axis. The red area is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) irradiated with 808nm wavelength and 1.0W / cm 2 The blue part is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) that has not been exposed to NIR (near infrared light) at a wavelength of 808 nm and a power of 1.0 W / cm 2The change curve of oxygen release of the finally formed polydopamine@oxygen / pravastatin film (i.e., the patented product) during the period of NIR (near-infrared light irradiation) treatment; Based on the above test results, it can be seen that the product of the present invention can rapidly undergo temperature changes under the influence of near-infrared light, and then the temperature and oxygen release rate change with periodic near-infrared light stimulation.

[0021] As shown in FIG3 , FIG3 shows the drug release of the product of the present invention. FIG3 (a) shows the time on the horizontal axis and the cumulative pravastatin release percentage on the vertical axis. The cumulative pravastatin release percentage change curve of the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) is marked with a yellow square. As a control, the cumulative pravastatin release percentage change curve of the oxygen-containing material without polydopamine deposition is marked with a black circle. FIG3 (b) shows the time on the horizontal axis and the cumulative pravastatin release percentage on the vertical axis. The red area is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) subjected to 808 nm wavelength and 1.0 W / cm 2 The blue part is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product) that has not been exposed to NIR (near infrared light) at a wavelength of 808 nm and a power of 1.0 W / cm 2 During the period of NIR (near-infrared light irradiation) treatment, the cumulative pravastatin release percentage change curve of the polydopamine@oxygen / pravastatin film (i.e., the patented product) in a pH 7.4 environment is marked with a yellow square. As a control, the cumulative pravastatin release percentage change curve of the polydopamine@oxygen / pravastatin film (i.e., the patented product) in a pH 6.0 environment 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. The release rate of pravastatin is accelerated in a higher pH environment, and it can respond to periodic near-infrared light stimulation.

[0022] As shown in Figure 4, Figure 4 shows the effect of the product of the present invention on the fate of periosteal stem cells under hypoxic conditions in vitro. The specific test method is: periosteal stem cells are cultured on the product of the present invention and placed in an oxygen-free environment. After 48 hours, 808nm NIR (1.0W / cm 2) stimulation. Figure 4 (a) shows the effect of the product of the present invention on the hypoxia of periosteal stem cells in a hypoxic environment, where green is the fluorescence display of the hypoxia probe. The left picture of Figure 4 (a) is the control group of the anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 4 (a) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Figure 4 (b) shows the effect of the product of the present invention on the proliferation of periosteal stem cells (Ki67+ cells) in a hypoxic environment. The left picture of Figure 4 (b) is the control group of the anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 4 (b) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Figure 4 (c) The figure shows the early osteogenic differentiation (alkaline phosphatase staining) of periosteal stem cells under hypoxia after using the product of the present invention. The left picture of Figure 4 (c) is the control group of the anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 4 (c) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Figure 4 (d) shows the late calcium deposition (alizarin red staining) of periosteal stem cells under hypoxia after using the product of the present invention. The left picture of Figure 4 (d) is the control group of the anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 4 (d) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Based on the above actual shots, it can be seen through immunofluorescence staining that the product of the present invention can significantly promote the improvement of periosteal stem cell hypoxia and proliferation rate, and the actual photos of alkaline phosphatase and alizarin red staining show that the present invention significantly promotes the early and late osteogenic differentiation of periosteal stem cells.

[0023] Figure 5 shows the effect of the product of the present invention on the angiogenesis of human umbilical vein endothelial cells (HUVECs) cultured in vitro under hypoxic conditions. Figure 5 (a) shows that after using the product of the present invention, the migration ability of HUVEC cells is significantly improved. The left picture of Figure 5 (a) is the control group of anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 5 (a) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); Figure 5 (b) shows the situation of the product of the present invention promoting HUVEC cell tube formation. The left picture of Figure 5 (b) is the control control group of anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of Figure 5 (b) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); According to the above Transwell experiment, it can be seen that the product of the present invention significantly promotes the migration of HUVEC cells, and immunofluorescence staining shows that its tube formation, including total length and number of nodes, has significantly increased.

[0024] Figure 6 shows the experimental results of the present invention in repairing skull defects in rats. Figure 6 (a) shows the surgical process of using the present invention to repair skull defects in rats. The surgical process from left to right is incision, periosteum removal, skull defect, periosteum implantation, and suturing. 48 hours after implantation, the laser was periodically injected with a wavelength of 808nm and a power of 1.0W / cm 2The treatment was carried out by NIR (near infrared light irradiation) twice a day for 5 minutes each time; FIG6 (b) shows the results of imaging examination of bone repair in the defect area of ​​rats 4 weeks and 8 weeks after using the product of the present invention. The left picture of FIG6 (b) is the control group of anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of FIG6 (b) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); FIG6 (c) shows the results of imaging examination of vascular perfusion in the defect area of ​​rats after using the product of the present invention. The left picture of FIG6 (c) is the control group of anaerobic polycaprolactone / perfluorotributylamine membrane, and the right picture of FIG6 (c) is the polydopamine@oxygen / pravastatin membrane (i.e., the patented product); micro-CT scanning and reconstruction show that at 4 weeks and 8 weeks, the present invention significantly promotes bone regeneration and angiogenesis in the bone defect area.

[0025] Figure 7 The group shows the results of biosafety testing after using the product of the present invention. It can be seen that there are no obvious abnormalities in HE histological staining of the heart, liver, spleen, lung and kidney after using the product of the present invention. It can be seen that the product of the present invention has no significant effect on the main organs of the whole body after implantation.

[0026] Example 2 Prepare the spectrally responsive oxygen-carrying artificial periosteum as follows: Step 1: Prepare the inner core solution and the outer shell solution.

[0027] Step 1.1, adding lecithin to the benchtop solution at a mass concentration of 180 mg / mL, ultrasonically treating it twice at a power of 250 W at 0°C, each time for 15 seconds, then adding perfluorotributylamine solvent at a mass concentration of 1.8 g / mL, ultrasonically treating it 10 times at a power of 250 W at 0°C, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 10% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.2 g / mL in a mixture of methanol and chloroform in a ratio of 1:4 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 90 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 12 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 10 cm. The voltage of the coaxial electrospinning needle is 9 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.25 mL / h for the inner needle and 1 mL / h for the outer needle. Spinning obtains core-shell structured fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 10 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.5, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.0 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake on a shaker at 80 rpm for 24 hours at room temperature to form an outer control module covering the outer side of the inner oxygen-carrying module.

[0028] Example 3 Step 1: Prepare the inner core solution and the outer shell solution.

[0029] Step 1.1: adding lecithin to a benchtop solution at a mass concentration of 200 mg / mL, ultrasonically treating the solution at 350 W power for 4 times at 4°C, and then adding a perfluorotributylamine solvent at a mass concentration of 2 g / mL, ultrasonically treating the solution at 350 W power for 12 times at 4°C, and obtaining a perfluorotributylamine emulsion after each ultrasonic treatment for 20 seconds; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 15% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.3 g / mL in a mixture of methanol and chloroform in a ratio of 1:5 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 100 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 16 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 12 cm. The voltage of the coaxial electrospinning needle is 10 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.3 mL / h for the inner needle and 1.2 mL / h for the outer needle. Spinning obtains core-shell structure fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 12 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.8, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.5 mg / mL; Step 4: Soak 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 outer side of the inner oxygen-carrying module.

[0030] Example 4 Step 1: Prepare the inner core solution and the outer shell solution.

[0031] Step 1.1, adding lecithin to the benchtop solution at a mass concentration of 185 mg / mL, ultrasonically treating once at 275 W power at 1°C for 16 seconds each time, then adding perfluorotributylamine solvent at a mass concentration of 1.85 g / mL, ultrasonically treating 10 times at 275 W power at 1°C for 16 seconds each time, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 11.25% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.225 g / mL in a mixture of methanol and chloroform in a volume ratio of 1:4.25 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 92.5 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 13 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 10.5 cm. The voltage of the coaxial electrospinning needle is 9.25 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.26 mL / h for the inner needle and 1.05 mL / h for the outer needle. Spinning obtains core-shell structure fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 11.5 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.6, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.1 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake on a shaker at 85 rpm for 30 hours at room temperature to form an outer control module covering the outer side of the inner oxygen-carrying module.

[0032] Example 5 Step 1: Prepare the inner core solution and the outer shell solution.

[0033] Step 1.1, adding lecithin to the benchtop solution at a mass concentration of 195 mg / mL, ultrasonically treating it at 325 W power for 4 times at 3°C, each time for 18 seconds, and then adding perfluorotributylamine solvent at a mass concentration of 1.95 g / mL, ultrasonically treating it at 325 W power for 12 times at 3°C, each time for 18 seconds, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 13.25% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.2275 g / mL in a mixture of methanol and chloroform in a volume ratio of 1:4.75 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 97.5 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 15 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11.5 cm. The voltage of the coaxial electrospinning needle is 9.75 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.29 mL / h for the inner needle and 1.15 mL / h for the outer needle. Spinning obtains core-shell structured fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 10.5 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.7, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.7 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake on a shaker at 95 rpm for 42 hours at room temperature to form an outer control module covering the outer side of the inner oxygen-carrying module.

[0034] Example 6 Step 1: Prepare the inner core solution and the outer shell solution.

[0035] Step 1.1, adding lecithin to the benchtop solution at a mass concentration of 190 mg / mL, ultrasonically treating it at a power of 290 W for 3 times at 3.5°C for 18 seconds each time, then adding a perfluorotributylamine solvent at a mass concentration of 1.9 g / mL, ultrasonically treating it at a power of 290 W for 11 times at 3.5°C for 18 seconds each time, to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel, so that the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 14% w / v, to produce an inner core solution; Step 1.3, dissolving polycaprolactone at a mass concentration of 0.28 g / mL in a mixture of methanol and chloroform in a volume ratio of 1:4.2 to obtain a polycaprolactone solution; Step 1.4, dissolving pravastatin in the polycaprolactone solution obtained in step 1.3 at a molar concentration of 93 μmol / mL; Step 1.5, stirring the mixed solution obtained in step 1.4 at room temperature for 13 hours to obtain a shell solution; Step 2: Fill the inner core solution and the outer shell solution obtained in step 1 into two syringe pumps respectively, and connect them to the inner needle 14G and the outer needle 18G of the coaxial electrospinning needle. The distance from the needle tip to the metal plate collector is 11 cm. The voltage of the coaxial electrospinning needle is 9.6 kV. The syringe pump pumps out the inner core solution and the outer shell solution at a speed of 0.27 mL / h for the inner needle and 1.1 mL / h for the outer needle. Spinning obtains core-shell structured fiber filaments, and the fiber membrane further woven is the inner oxygen-carrying module. Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution to 11 mmol / L with phosphate buffer, adjusting the pH of the solution to 8.7, and adding dopamine powder to the solution to obtain a dopamine solution with a mass concentration of 2.3 mg / mL; Step 4: Soak the inner oxygen-carrying module obtained in step 2 in the dopamine solution obtained in step 3, and shake on a shaker at 88 rpm for 38 hours at room temperature to form an outer control module covering the outer side of the inner oxygen-carrying module.

Claims

1. Multi-spectral responsive oxygen-carrying artificial periosteum, characterized in that: It includes an inner oxygen-carrying module and an outer control module that responds to light control.

2. The multi-spectral responsive oxygen-carrying artificial periosteum according to claim 1, characterized in that: The inner oxygen-carrying module is a fiber membrane woven from core-shell structure fibers, the inner core of the core-shell structure fibers is oxygen-carrying perfluorotributylamine, the outer shell is polycaprolactone carrying pravastatin, and the outer control module is polydopamine nanoparticles.

3. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 1, characterized in that: Follow these steps to implement: Step 1: Prepare the inner core solution and the outer shell solution Step 2, the inner core solution and the outer shell solution obtained in step 1 are respectively filled into two syringe pumps, and connected to the inner needle and the outer needle of the coaxial electrospinning needle, the syringe pumps pump out the liquid, and electrospin to obtain core-shell structure fiber filaments, and the fiber membrane obtained by further spinning the core-shell structure fiber filaments is the inner oxygen carrying module; Step 3, diluting the tris(hydroxymethyl)aminomethane hydrochloride) solution with a phosphate buffer solution, adjusting the pH of the solution to 8.5-8-8, and adding dopamine powder to the solution to obtain a dopamine solution; Step 4: Soak 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 outer side of the inner oxygen-carrying module.

4. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 3, characterized in that: In step 1, the specific steps of preparing the inner core solution are: Step 1.1, adding lecithin to the benchtop solution, performing the first ultrasonic treatment, adding perfluorotributylamine, and performing the second ultrasonic treatment to obtain a perfluorotributylamine emulsion; Step 1.2, adding carboxyl chitosan to the perfluorotributylamine emulsion to form a gel to produce an inner core solution; The specific steps for preparing the shell solution are: Step 1.3, dissolving polycaprolactone in a mixture of methanol and chloroform to obtain a polycaprolactone solution; Step 1.4, dissolving 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 a shell solution.

5. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 4, characterized in that: In the step 1.1, the mass concentration of lecithin relative to the desktop liquid is 180-200 mg / ml; the specific parameters of the first ultrasonic oscillation treatment are 2-4 times of oscillation treatment at 250-350 W power at 0-4°C, each time for 15-20 seconds; the mass concentration of the added perfluorotributylamine solvent is 1.8-2 g / mL, and the specific parameters of the second ultrasonic oscillation treatment are 10-12 times of oscillation treatment at 250-350 W power at 0-4°C, each time for 15-20 seconds.

6. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 4, characterized in that: In the step 1.2, after adding carboxyl chitosan to form gel, the concentration of carboxyl chitosan in the perfluorotributylamine emulsion reaches 10-15% w / v.

7. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 4, characterized in that: In the step 1.3, polycaprolactone is dissolved in a mixture of methanol and chloroform at a mass concentration of 0.2-0.3 g / ml, and the volume ratio of the mixture of methanol and chloroform is 1:4-5.

8. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 4, characterized in that: In the step 1.4, pravastatin is dissolved in the polycaprolactone solution at a molar concentration of 90-100 μmol / ml.

9. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 3, characterized in that: In the 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 speed of the inner core solution is 0.25-0.3 mL / h, and the pumping speed of the outer shell solution is 1.0-1.2 mL / h.

10. The method for preparing the multi-spectral responsive oxygen-carrying artificial periosteum according to claim 3, characterized in that: In the 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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