Biological piezoelectric scaffold for bone defect repair and preparation method thereof

Abstract

The preparation method comprises the following steps: by taking an oriented polylactic acid nanofiber membrane as a raw material, carrying out homogenization treatment to obtain polylactic acid piezoelectric short fibers; gelMA is used as a skeleton matrix, polylactic acid short fibers, LAP and DRG cells are compounded, a 0 / 90-degree alternate grid structure scaffold is constructed through pneumatic extrusion type biological printing, and the dual-network piezoelectric biological scaffold is formed through blue light crosslinking curing. The piezoelectric effect of polylactic acid short fibers is activated through ultrasonic stimulation, mechanical energy is converted into local electric signals, loaded DRG cells are directly driven to secrete CGRP in situ, macrophages are dynamically regulated and controlled to be polarized to M2 anti-inflammatory phenotypes, nerve-immune microenvironment disorder of diabetic bone defects is corrected, vascularization and bone regeneration are promoted, and the diabetic bone defect treatment effect is improved. The problems of short delivery half-life period of traditional cell factors, difficulty in nerve function reconstruction and chronic inflammation caused by non-degradable piezoelectric materials are solved, and non-invasive and degradable precise bone repair is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, and particularly relates to a biological piezoelectric scaffold for repairing bone defects and a preparation method thereof. BACKGROUND

[0002] Efficient repair of bone defects is a major challenge in the field of clinical orthopedics. In the process of bone regeneration, the imbalance of inflammatory response caused by trauma or disease can lead to disorder of immune microenvironment, which significantly reduces the efficiency of bone healing. Studies have shown that this disorder is often manifested as abnormal or persistent activation of pro-inflammatory macrophages (M1 type), which disrupts the balance of bone homeostasis, inhibits osteogenic differentiation and angiogenesis, and may exacerbate osteoclast activity, ultimately hindering the repair process of bone defects. To regulate the local immune microenvironment, current bone tissue engineering strategies mainly focus on using biological materials to load anti-inflammatory cytokines (according to IL-4, BMP4) or through ion release (according to lithium and magnesium ions), material surface modification (according to elastic modulus regulation, charge modification), and external field stimulation (electric field, ultrasound) to induce macrophages to polarize to the pro-repair anti-inflammatory phenotype (M2 type). For example, injectable gelatin microspheres releasing IL-4 have been shown to effectively promote bone regeneration in various bone defect models. However, these methods have obvious limitations: first, the half-life of cytokines is short, and local delivery is difficult to maintain long-term therapeutic concentrations; second, existing strategies mainly focus on a single immune regulation pathway, and the core regulatory role of the nervous system in the immune microenvironment is not adequately addressed.

[0003] The importance of neuro-immune interaction in bone regeneration is increasingly evident. Calcitonin gene-related peptide (CGRP) secreted by sensory neurons has been shown to inhibit M1 macrophage polarization, promote M2 transformation, and regulate inflammatory factor expression through activation of PI3K / AKT, Yap1, and other signaling pathways. However, bone defect patients have a severe deficiency in CGRP secretion due to neuropathy, and the biological half-life of CGRP is extremely short (about 18 minutes), making direct exogenous supplementation inefficient. Existing technologies attempt to induce dorsal root ganglion (DRG) cells to secrete CGRP by implanting magnesium metal or construct 3D-printed scaffolds loaded with CGRP, but still have two major defects: 1) the release of magnesium ions is uncontrollable, which may cause damage to cells in the local alkaline environment; 2) CGRP release scaffolds cannot restore nerve function and are difficult to dynamically respond to changes in the microenvironment.

[0004] In the field of electroactive bone scaffolds, piezoelectric materials (based on barium titanate, polyvinylidene fluoride) can generate electrical signals through mechanical stress, simulating the piezoelectric effect of bone tissue to promote regeneration. However, inorganic piezoelectric materials (based on barium titanate) are not degradable, and long-term retention in the body can easily trigger chronic inflammation; while degradable polymers (based on poly-L-lactic acid, PLLA) have piezoelectric properties, but the dense fiber structure formed by traditional processing methods (based on electrospinning) is difficult to construct a biomimetic multi-level vascular / nerve network. Although bioprinting technology can precisely regulate the macro / micro structure of the scaffold and co-load living cells, there is an inherent conflict between the crystal orientation requirements of piezoelectric materials and the rheological properties of bio-ink (based on shear thinning, rapid solidification), resulting in the fact that bioprinted scaffolds with high-efficiency piezoelectric response, cell compatibility and biomimetic structure have not been reported to date. In addition, existing piezoelectric scaffolds lack the ability to actively regulate nerve cells, and cannot achieve the functional reconstruction of the nerve-immune axis in the diabetic microenvironment. SUMMARY

[0005] The present application provides a kind of for bone defect repair biological piezoelectric scaffold and its preparation method, the scaffold is driven by ultrasonic response piezoelectric signal back root ganglion (DRG) cell calcitonin gene related peptide (CGRP) secretion, dynamically regulates the nerve-immune microenvironment of diabetic bone defect, promotes vascularized bone regeneration.

[0006] A kind of for diabetic bone defect repair biological piezoelectric scaffold, with polylactic acid short fiber as piezoelectric response material, methacrylated gelatin as matrix skeleton, compound light initiator phenyl-2,4,6-trimethyl benzoyl lithium phosphite and back root ganglion cell, construct 0 / 90 ° alternate grid structure, obtain the biological scaffold with piezoelectric performance and cell activity by blue light crosslinking solidification.

[0007] Further, the molecular weight of the polylactic acid short fiber of the present application is 530-870 thousand; the short fiber is obtained by electrospinning, shearing, homogenization and annealing treatment steps, and has a length range of 30-150 μm and a crystal orientation structure to enhance piezoelectric response.

[0008] Further, the mass concentration of the methacrylated gelatin of the present application is 5-7 wt%, dissolved in PBS as solvent at 37℃ and formed into a bioprinting ink.

[0009] Further, the mass concentration of the light initiator phenyl-2,4,6-trimethyl benzoyl lithium phosphite of the present application is 0.25-0.35 wt%, and crosslinking reaction occurs under blue light irradiation at a wavelength of 405 nm and a power density of 15 mW / cm 2 .

[0010] Further, the back root ganglion cells of the present application are 2×10 6The cells are uniformly distributed in the bio-ink and maintain activity and nerve regulation function.

[0011] Further, the bio-scaffold of the present application adopts pneumatic extrusion type bio-printing, the nozzle diameter is 300 mu m, the air pressure is 100 kPa, the printing speed is 8 mm / s, the cross structure with layer height 200 mu m and hole distance 1.5 mm is formed.

[0012] Further, the bio-scaffold of the present application can activate the piezoelectric effect of the polylactic acid short fiber in response to 1MHz frequency, 1.0W / cm 2 The intensity of ultrasonic stimulation, inducing dorsal root ganglion cells to secrete calcitonin gene-related peptide in situ.

[0013] Further, the bio-scaffold of the present application realizes immune microenvironment regulation through macrophage M2 polarization mediated by calcitonin gene-related peptide, and synchronously promotes angiogenesis and bone tissue regeneration.

[0014] Further, the polylactic acid short fiber, methacrylated gelatin, photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate and dorsal root ganglion cells of the present application are all degradable biological materials.

[0015] A preparation method of a bio-piezoelectric scaffold for repairing bone defects, comprising the following steps:

[0016] Step (1) preparation of polylactic acid short fiber: an oriented polylactic acid fiber membrane is prepared by electrospinning, short fibers are obtained by shearing and multi-stage homogenization, and then two-stage annealing treatment is performed to form a crystal structure;

[0017] Step (2) configuration of bio-printing ink: methacrylated gelatin is dissolved in PBS, polylactic acid short fibers, photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate are added and stirred uniformly in the dark, and dorsal root ganglion cells are loaded for standby use;

[0018] Step (3) printing and curing of the scaffold: the ink is printed into a cross grid structure using a pneumatic extrusion type printing device, and a blue light is irradiated to cure and form a bio-scaffold.

[0019] The present application adopts the above technical scheme, and has the following advantages compared with the prior art:

[0020] 1. The present application dynamically reconstructs the nerve-immune regulation axis through the ultrasonic responsive piezoelectric scaffold, converts the external ultrasound into a local electric signal by the piezoelectric effect of the polylactic acid short fiber, directly stimulates the dorsal root ganglion (DRG) cells to secrete CGRP in situ, significantly corrects the immune microenvironment disorder of diabetic bone defects, and solves the problems of poor targeting and delayed response caused by ignoring the innervation of the existing immune regulation materials.

[0021] 2、The application adopts a non-invasive ultrasonic intervention mode to replace a traditional implantable electrode, avoids secondary surgical injury, and significantly improves patient treatment compliance; at the same time, the stent bionic porous grid structure synchronously supports vascular ingrowth and innervation, realizes multi-tissue collaborative regeneration, and overcomes the defect that the dense structure of traditional piezoelectric materials hinders tissue infiltration.

[0022] 3、The biologic stent prepared by the application is made of all-degradable biomaterials, can be completely degraded without residue, and eliminates the risk of chronic inflammation caused by non-degradable piezoelectric ceramics. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a schematic diagram of SEM images of polylactic acid fiber film and short fibers.

[0024] Figure 2 It is a schematic diagram of XRD spectrum of polylactic acid fiber.

[0025] Figure 3 It is a schematic diagram of piezoelectric performance of the piezoelectric stent.

[0026] Figure 4 It is a schematic diagram of open-circuit voltage output of the piezoelectric hydrogel.

[0027] Figure 5 It is a real object diagram of the piezoelectric stent.

[0028] Figure 6 It is a schematic diagram of fluorescence images of the biologic piezoelectric stent.

[0029] Figure 7 It is a schematic diagram of CGRP secretion amount of DRG cells.

[0030] Figure 8 It is a schematic diagram of immune regulation of the biologic piezoelectric stent.

[0031] Figure 9 It is a schematic diagram of bone regeneration of the biologic piezoelectric stent. DETAILED DESCRIPTION

[0032] The technical solutions of the application will be described in detail as follows:

[0033] The application provides a biologic piezoelectric stent for bone defect repair and a preparation method thereof, and is used for solving the following problems.

[0034] (1) A composite hydrogel ink with both bioprinting and piezoelectric properties is constructed to solve the compatibility problem of piezoelectric stent bionic structure and function.

[0035] (2) In the scaffold, the dorsal root ganglion (DRG) cells are effectively loaded, and the piezoelectric response of the scaffold itself is utilized to achieve CGRP secretion through ultrasonic stimulation under the condition of no external power supply, solving the problems of low CGRP delivery efficiency and difficult nerve function reconstruction.

[0036] (3) CGRP-mediated macrophage immune polarization is achieved to improve the local inflammatory environment of diabetic bone defects, ultimately promoting vascularized osteogenic regeneration, and solving the problem of imbalance of immune microenvironment.

[0037] A biological piezoelectric scaffold for bone regeneration, using polylactic acid nanofiber as piezoelectric material and gelatin methacryloyl (GelMA) as scaffold matrix, a composite piezoelectric hydrogel scaffold is constructed by bioprinting. The technical solution specifically includes the following core steps:

[0038] Step (1) Preparation of oriented polylactic acid fiber film: Prepare the corresponding raw materials, use hexafluoroisopropanol as the solvent, left-handed polylactic acid (PLLA) or right-handed polylactic acid (PDLLA) as the solute, and configure 8-12wt% spinning solution. Stir the spinning solution for 24 hours to ensure that the polylactic acid is fully dissolved in hexafluoroisopropanol. Fill the well-stirred spinning solution into a syringe and install a G21 needle. The electrospinning device uses aluminum foil as the receiving device and is connected to a 12-14kv high-voltage power supply. At the same time, set the advancing speed to 1.5-1.7ml / h, the distance between the needle and the aluminum foil to 15-18cm, and the rotating speed of the receiving device to 1000-3000rpm, and ensure that the humidity is controlled at about 25-45%. Start electrospinning and gradually form an oriented polylactic acid fiber film on the aluminum foil.

[0039] Step (2) Preparation of polylactic acid short fibers: Use scissors to cut the oriented polylactic acid spinning film into pieces. Put these piece-shaped short fibers into a container filled with 0.2wt% PVA solution, start the high-speed homogenizer, set the rotating speed to 8000-10000rpm, and initially break the nanofiber film fragments, each time for 3-5 minutes, and repeat 3 times. Then transfer the short fiber sample to a container filled with tert-butyl alcohol solution, start the high-speed homogenizer again, adjust the rotating speed to 12000-15000rpm, and further break the sample, homogenization time for 10 minutes, also repeat 3 times. Wash the short fiber sample with water and ethanol in turn, after washing, use a nylon filter and Buchner funnel to filter the washed sample, and put it in an oven to dry overnight to obtain PLLA short fibers. Place the polylactic acid short fiber sample treated as above in a muffle furnace, raise the temperature to 105℃, and maintain this temperature for 10 hours. After annealing, let the sample cool slowly to room temperature in the annealing device. Raise the temperature to 160℃ again for 10 hours to obtain the final polylactic acid short fiber sample.

[0040] Step (3) Preparation of bio-piezoelectric ink: Prepare the corresponding raw materials and perform sterile processing. Prepare a 5-7wt% solution of gelatin methacrylate (GelMA) as the solute in PBS as the solvent, and stir in a 37°C constant temperature water bath for 30 minutes until completely dissolved. Then add polylactic acid short fibers at 1-10 mg / mL and 0.25-0.35wt% of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) in sequence, and transfer to a 4°C environment for 20 minutes of stirring in the dark to form a homogeneous composite solution; finally, suspend the SD rat dorsal root ganglion (DRG) cells at a density of 2×10 6 cells / mL in the above solution by gentle blowing, and quickly transfer to the bio-printer cartridge for standby.

[0041] Step (4) Preparation of bio-piezoelectric scaffold: Use a pneumatic extrusion type bio-printer, set the nozzle diameter to 300μm, the air pressure to 100kPa, and the travel speed to 8mm / s, and extrude the composite ink loaded with DRG cells at room temperature (25°C) to construct a 0 / 90° alternating grid structure (layer height 200μm, hole spacing 1.5mm); immediately after printing, irradiate with 405nm blue light (intensity 15mW / cm 2 ) vertically for 20 seconds to trigger photocrosslinking, forming a piezoelectric bio-scaffold with a double network structure.

[0042] Ultrasound stimulation and bone repair application: In vitro, place the scaffold in the culture medium and use a therapeutic grade ultrasound probe (frequency 1MHz, intensity 1.0W / cm 2 ) to stimulate the scaffold, and collect the culture medium at 1, 3, and 7 days after stimulation to quantify the amount of CGRP secretion; in vivo, implant the scaffold into the critical size defect of the femur of SD rats, and perform daily ultrasound intervention with the same parameters, and after 4-8 weeks, take the material for Micro-CT scanning to confirm that it promotes vascularized bone regeneration through neural immune regulation.

[0043] Example 1:

[0044] Preparation of oriented polylactic acid fiber membrane: Prepare the corresponding raw materials, and weigh 0.8g of L-polylactic acid and D-polylactic acid (molecular weight 530,000) respectively, then add 10ml of hexafluoroisopropanol to each. Stir the mixed solution for 24 hours to ensure that the polylactic acid is fully dissolved in the hexafluoroisopropanol. Pour the well-stirred spinning solution into a 10ml syringe and install a G21 needle. The electrospinning device uses aluminum foil as the receiving device and is connected to a 14kv high voltage power supply. The push speed is set to 1.7ml / h, the distance between the needle and the aluminum foil is 15cm, the rotating speed of the receiving device is set to 3000rpm, and the humidity is controlled at about 30%. Start electrospinning and gradually form an oriented polylactic acid fiber membrane on the aluminum foil.

[0045] Preparation of PLA short fibers: Using scissors to cut the oriented PLA filament membrane into 0.5 cm 2 Size of the sheet. Put these sheet short fibers into a container containing 100 ml of 0.2wt% PVA solution, open the high-speed homogenizer, set the speed to 10000 rpm, and the nanofiber membrane fragments are initially broken, each time homogenization time is 3 minutes, and needs to be repeated 3 times. Then transfer the short fiber sample to a container containing 100 ml of tert-butyl alcohol solution, open the high-speed homogenizer again, adjust the speed to 15000 rpm, and further break the sample, homogenization time is 10 minutes, also repeated 3 times. The short fiber sample is washed with water and ethanol in turn, and after washing, the sample is filtered with a nylon filter and a Buchner funnel, and the obtained PLLA short fibers are dried in an oven overnight. The above-processed poly lactic acid short fiber sample is placed in a muffle furnace, the temperature is raised to 105℃, and annealed for 10 hours. After annealing, the sample is slowly cooled to room temperature in the annealing device. The temperature is raised to 160℃ again and annealed for 10 hours to obtain the final poly lactic acid short fiber sample.

[0046] Figure 1 The upper half of Figure 6 is a high-magnification and low-magnification SEM image of the fiber membrane, and both magnifications can see the continuous oriented fiber structure, indicating that the oriented spinning fiber membrane in the application is successfully prepared. Figure 1 The lower half of Figure 6 is a high-magnification and low-magnification SEM image of the short fiber after homogenization, and both magnifications can see that the uniform short fiber is dispersed on the bottom plate, indicating that the short fiber in the application is successfully prepared. Figure 2 Figure 7 is an XRD pattern of poly lactic acid fiber, wherein the abscissa is the diffraction angle and the ordinate is the diffraction intensity. The PLLA sample has a clear crystallization peak, indicating that the PLLA in the application has crystallinity, and the PDLLA sample shows a gentle peak, indicating that the PDLLA sample does not have crystallinity, and the comparison of the three groups of PLLA samples indicates that the preparation process of the application improves the crystallinity.

[0047] Example 2:

[0048] Preparation of piezoelectric scaffold: 0.7 g of methacrylated gelatin (GelMA) was dissolved in 10 mL of phosphate buffer (PBS) in a 37°C constant temperature water bath for 30 minutes to completely dissolve; then 0, 10, 50, 100 mg of PLLA short fibers and 0.025 g of light initiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) were added in sequence, and the homogeneous composite solution was formed by stirring in the dark at 4°C for 20 minutes. A pneumatic extrusion bioprinter was used to set the nozzle diameter to 300 μm, the air pressure to 100 kPa, and the travel speed to 8 mm / s, and the composite ink was extruded at room temperature (25°C) to construct a 0 / 90° alternating grid structure (layer height 200 μm, hole spacing 1.5 mm). After printing, the piezoelectric scaffold was irradiated vertically with 405 nm blue light (intensity 15 mW / cm 2 ) for 20 seconds to trigger photocrosslinking, and piezoelectric scaffolds with different short fiber addition amounts were formed.

[0049] Figure 3 Fig. 4 is a schematic diagram of the piezoelectric performance of the piezoelectric scaffold, wherein the abscissa represents different samples, and the ordinate represents the D 33 value. The addition of piezoelectric PLLA shows obvious piezoelectric performance, indicating that the piezoelectric scaffold has certain piezoelectric performance, and the comparison of the four groups of materials shows that the piezoelectric performance also increases with the increase of the addition amount of piezoelectric PLLA. Figure 4 Fig. 5 is a schematic diagram of the open circuit voltage output of the piezoelectric hydrogel, wherein the abscissa represents time, and the ordinate represents the open circuit voltage. The left graph shows the open circuit voltage output of different piezoelectric PLLA addition amounts, and the piezoelectric output gradually increases with the increase of the addition amount. The right graph shows the open circuit voltage under different ultrasonic intensities, and the piezoelectric output gradually increases with the increase of the ultrasonic intensity. The above all shows that the piezoelectric scaffold has ultrasonic responsive piezoelectric output. Figure 5 Fig. 6 is a photograph of the piezoelectric scaffold, the left graph is during printing, and the right graph is the finished product, both of which show clear scaffold structure, indicating that the piezoelectric scaffold is successfully prepared.

[0050] Example 3:

[0051] Preparation of biological piezoelectric scaffold and biological non-piezoelectric scaffold: 0.7 g of sterile methacrylated gelatin (GelMA) was dissolved in 10 mL of phosphate buffer (PBS) in a 37°C constant temperature water bath for 30 minutes to completely dissolve; then 50 mg of PLLA short fibers and 50 mg of PDLLA short fibers were added, respectively, and finally 0.025 g of light initiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) was added, and the homogeneous composite solution was formed by stirring in the dark at 4°C for 20 minutes. Finally, the dorsal root ganglion (DRG) cells of SD rats were seeded at a density of 2×10 6cells / mL density gently resuspended in the above solution, and quickly moved into the bio-printer cartridge for standby. Using a pneumatic extrusion type bio-printer, set the nozzle diameter to 300 μm, air pressure to 100 kPa, and travel speed to 8 mm / s, the composite ink loaded with DRG cells was extruded at room temperature (25°C) to construct a 0 / 90° alternating grid structure (layer height 200 μm, hole spacing 1.5 mm); immediately after printing, 405 nm blue light (intensity 15 mW / cm 2 ) was vertically irradiated for 20 seconds to trigger photo-crosslinking, forming a bio-piezoelectric scaffold with a double network structure.

[0052] Figure 6 The fluorescence image of the bio-piezoelectric scaffold is shown in the figure, the left figure is the fluorescence image of the DRG cells, and the cells show green fluorescence, indicating that the cells have good activity. The structures of the scaffold and the uniform dispersion of the DRG cells can be clearly seen in the other two figures, indicating that the bio-piezoelectric scaffold in the application is successfully prepared.

[0053] Example 4:

[0054] Preparation of bio-piezoelectric scaffold and bio-non-piezoelectric scaffold: 0.7 g of sterile methyl methacrylate gelatin (GelMA) was dissolved in 10 mL of phosphate buffer (PBS) at 37°C in a constant temperature water bath for 30 minutes to completely dissolve; then 0 mg of PLLA short fibers, 50 mg of PLLA short fibers and 50 mg of PDLLA short fibers were added respectively, and finally 0.025 g of photo initiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) was added, and the homogeneous composite solution was transferred to a 4°C environment and stirred for 20 minutes in the dark. Finally, the dorsal root ganglion (DRG) cells of SD rats were resuspended in the above solution at a density of 2×10 6 cells / mL density gently resuspended in the above solution, and quickly moved into the bio-printer cartridge for standby. Using a pneumatic extrusion type bio-printer, set the nozzle diameter to 300 μm, air pressure to 100 kPa, and travel speed to 8 mm / s, the composite ink loaded with DRG cells was extruded at room temperature (25°C) to construct a 0 / 90° alternating grid structure (layer height 200 μm, hole spacing 1.5 mm); immediately after printing, 405 nm blue light (intensity 15 mW / cm 2 ) was vertically irradiated for 20 seconds to trigger photo-crosslinking, forming a bio-piezoelectric scaffold with a double network structure.

[0055] In vitro ultrasound stimulation and immune regulation: four groups of scaffolds were placed in the culture medium, and a therapeutic grade ultrasound probe (frequency 1 MHz, intensity 1.0 W / cm 2, 30min / day) to two groups of scaffolds, and the CGRP concentration in the conditioned medium was detected by ELISA after stimulation to quantify the CGRP secretion. The CGRP conditioned medium was co-cultured with macrophages, and then immunofluorescence staining was performed to characterize the immune regulation effect.

[0056] Figure 7 Figure 4 is a diagram of CGRP secretion of DRG cells, wherein the abscissa is the sample group, and the ordinate is the CGRP secretion. The left graph is the CGRP quantification detected by qPCR, and the right graph is the CGRP quantification detected by Elisa. Only the group with both PLLA and ultrasound has a significant increase in CGRP secretion, indicating that the bio-piezoelectric scaffold in the application can secrete CGRP on demand. Figure 8 Figure 5 is a diagram of immune regulation of bio-piezoelectric scaffolds, wherein the abscissa is the sample group, and the ordinate is the CD206 intensity. The left graph is an immunofluorescence staining graph. The CD206 index of the group containing piezoelectric has a significant increase, indicating that the bio-piezoelectric scaffold in the application can regulate the immune environment.

[0057] Example 5:

[0058] Preparation of bio-piezoelectric scaffolds and bio-non-piezoelectric scaffolds: 0.7 g of sterile methyl methacrylate gelatin (GelMA) was dissolved in 10 mL of phosphate buffer (PBS) in a 37°C constant temperature water bath for 30 minutes until completely dissolved; then 0 mg of PLLA short fibers, 50 mg of PLLA short fibers and 50 mg of PDLLA short fibers were added respectively, and finally 0.025 g of light initiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) was added. The homogeneous composite solution was formed by stirring in the dark at 4°C for 20 minutes. Finally, the dorsal root ganglion (DRG) cells of SD rats were gently resuspended in the above solution at a density of 2×10 6 cells / mL, and quickly transferred to the bioprinter cartridge for standby. A pneumatic extrusion type bioprinter was used, with a nozzle diameter of 300 μm, air pressure of 100 kPa and travel speed of 8 mm / s. The composite ink loaded with DRG cells was extruded to construct a 0 / 90° alternating grid structure (layer height 200 μm, pore spacing 1.5 mm) at room temperature (25°C). After printing, 405 nm blue light (intensity 15 mW / cm 2 ) was vertically irradiated for 20 seconds to trigger light crosslinking, forming a bio-piezoelectric scaffold with a double network structure.

[0059] In vivo ultrasound stimulation and bone defect repair and regeneration: In vivo experiments were performed by implanting scaffolds into the critical size defects of the femur of SD rats. After the operation, a therapeutic ultrasound probe (frequency 1 MHz, intensity 1.0 W / cm 2) to stimulate treatment, 4-8 weeks after taking material Micro-CT scanning, verify its through nerve immune regulation to promote bone regeneration.

[0060] Figure 9 The schematic diagram of bone regeneration of the biopiezoelectric scaffold, the group added with PLLA can promote the repair of bone defects to the maximum degree only under the stimulation of ultrasound, indicating that the biopiezoelectric scaffold in the application can promote bone regeneration.

[0061] The application discloses a biopiezoelectric scaffold for repairing diabetic bone defects and a preparation method thereof.

[0062] Those skilled in the art can understand that the above-mentioned embodiments are specific embodiments for realizing the application, and in actual application, various changes can be made in form and details without departing from the spirit and scope of the application. Any person skilled in the art can make respective changes and modifications without departing from the spirit and scope of the application, and therefore the protection scope of the application should be limited by the scope defined in the claims.

Claims

1. A bio-piezoelectric scaffold for bone defect repair, characterized in that: A polylactic acid short fiber is used as a piezoelectric response material, methacrylated gelatin is used as a matrix framework, a photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite is compounded with a dorsal root ganglion cell, a 0 / 90° alternating grid structure is constructed, and a biological scaffold with piezoelectric properties and cell activity is obtained through blue light cross-linking and curing.

2. The bio-piezoelectric scaffold for bone defect repair according to claim 1, wherein: The molecular weight of the polylactic acid short fiber is 530-870 thousand; the short fiber is obtained through the steps of electrospinning, shearing, homogenization and annealing treatment, has a length range of 30-150 μm, and has a crystal orientation structure to enhance piezoelectric response.

3. The bio-piezoelectric scaffold for bone defect repair according to claim 1, wherein: The mass concentration of the methacrylated gelatin is 5-7 wt%, which is dissolved in PBS as a solvent at 37°C and forms a biological printing ink.

4. The biopiezoelectric scaffold for bone defect repair according to claim 1, wherein: The photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite has a mass concentration of 0.25 - 0.35 wt%, and a crosslinking reaction occurs under irradiation of blue light with a wavelength of 405 nm, 15 mW / cm 2 2.

5. The biopiezoelectric scaffold for bone defect repair according to claim 1, wherein: The dorsal root ganglion cells are uniformly distributed in the bio-ink at a density of 2 x 10 6 cells / mL and maintain viability and neuroregulatory function.

6. The biopiezoelectric scaffold for bone defect repair according to claim 1, wherein: The biological scaffold is printed by a pneumatic extrusion type biological printing, the nozzle diameter is 300 μm, the air pressure is 100 kPa, the printing speed is 8 mm / s, the cross structure with a layer height of 200 μm and a hole distance of 1.5 mm is formed.

7. The biopiezoelectric scaffold for bone defect repair according to claim 1, wherein: The biological scaffold can activate the piezoelectric effect of the polylactic acid short fibers in response to an ultrasonic stimulus of 1 MHz frequency, 1.0 W / cm 2 The intensity of the ultrasonic stimulus induces the dorsal root ganglion cells to secrete calcitonin gene-related peptide in situ.

8. The bio-piezoelectric scaffold for bone defect repair according to claim 7, wherein: The biological scaffold realizes immune microenvironment regulation through calcitonin gene-related peptide-mediated macrophage M2 polarization, and simultaneously promotes angiogenesis and bone tissue regeneration.

9. The bio-piezoelectric scaffold for bone defect repair according to claim 1, wherein: The polylactic acid short fiber, the methacrylated gelatin, the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite and the dorsal root ganglion cell are all biodegradable biomaterials.

10. A method of preparing a bio-piezoelectric scaffold for bone defect repair according to any one of claims 1-9, characterized in that The method comprises the following steps: Step (1) preparation of polylactic acid short fiber: an oriented polylactic acid fiber film is prepared by electrospinning, short fibers are obtained through shearing and multi-stage homogenization, and then two-stage annealing treatment is performed to form a crystal structure; Step (2) configuration of biological printing ink: methacrylated gelatin is dissolved in PBS, polylactic acid short fibers, photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite are added, and the mixture is stirred and homogenized in the dark, and then dorsal root ganglion cells are loaded for standby; Step (3) printing and curing of the scaffold: the ink is printed into a cross grid structure using a pneumatic extrusion type printing device, and a biological scaffold is formed through blue light irradiation and curing.

Images