A PRP combined with carboxymethyl chitosan scaffold and its preparation method and application

Abstract

The present invention discloses a PRP combined with carboxymethyl chitosan scaffold and its preparation method and application, which belongs to the field of biomaterial technology. The preparation method comprises the following steps: mixing carboxymethyl chitosan powder with hydroxyapatite powder, and then adding genipin powder to mix evenly to obtain a mixture; thawing frozen PRP and mixing it with ddH2O to obtain a mixed solution; adding the mixture to the mixed solution and stirring evenly, and eliminating bubbles to obtain ink; printing the ink using a 3D printer, and then placing it in a CaCl2 solution for deformation treatment to obtain a PRP combined with carboxymethyl chitosan scaffold. The present invention uses 4D printing technology to turn PPR into part of the printing ink by stirring, and utilizes the microporous structure of the scaffold and the slow degradation of the scaffold to achieve the slow release of growth factors in the PRP.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a PRP combined with carboxymethyl chitosan scaffold and a preparation method and application thereof. Background Art

[0002] 3D printing technology enables the fabrication of three-dimensional scaffolds with high surface area, high porosity, and high mechanical strength using digitally controllable printing equipment, creating high-precision material models that perfectly match the defect site. Consequently, it has been widely used in fields such as healthcare, smart homes, and aerospace. In recent years, with the advancement of printing technology, 4D printing has gradually gained popularity, incorporating time as a fourth dimension into three-dimensional space and incorporating time-dependent programming into the printing design process. 4D printing specifically combines 3D printing with shape-memory materials. The initial shape is designed through computer programming, then printed using a 3D printer. The material can then undergo controlled secondary deformation under subsequent stimulation by heat, light, electricity, magnetism, or even water. Currently, 4D printing technology has been widely used in industries such as aerospace, smart homes, smart textiles, and automated equipment. However, due to the complex interactions between various chemical bonds involved in the programming, the complex preparation process has hindered its further expansion.

[0003] 4D printing technology has been widely used in the field of bone tissue engineering. Ideal biocompatibility, mechanical strength, matching degree with the bone defect area, and the ability of the scaffold to promote bone defect repair have become key indicators for evaluating 4D printed bone tissue engineering biomaterials. Carboxymethyl chitosan, as a derivative of water-soluble chitosan, greatly improves its water solubility by introducing the hydrophilic group -CH2COOH into the chitosan polymer chain. Due to the presence of amino groups (-NH 3+), carboxyl (-CH2COO-) and other active groups, and have a structure similar to that of natural extracellular matrix molecules, so they have good biocompatibility and in vivo degradation and absorption. At the same time, carboxymethyl chitosan has multiple functional activities, and has the characteristics of promoting cell growth and promoting the repair of damaged tissues, and is widely used in the development of absorbable medical devices and pharmaceuticals and health products. However, the disadvantage is that the mechanical properties of carboxymethyl chitosan are weak, and it cannot meet the requirements of bone tissue engineering for biomaterials. In addition, with the increasing number of patients with bone defects caused by trauma, infection, tumor treatment and radiotherapy, the bone defect areas of most patients are not regular and orderly, but present an irregular shape with uneven shapes. Due to the limitations of viscosity and fluidity of ordinary 3D printed carboxymethyl chitosan ink, its extruded 3D printed scaffold needs to be placed on a temperature-controlled flat platform. Therefore, 3D printed carboxymethyl chitosan scaffolds are almost all two-dimensional planar structures, which cannot match the shape of the clinical bone defect area. Therefore, it is crucial to develop a carboxymethyl chitosan scaffold with high adaptability to the affected area and strong bone repair ability.

[0004] In 1984, Assoian RK et al. first successfully prepared platelet-rich plasma (PRP) from human blood via centrifugation. PRP is a platelet concentrate obtained by centrifuging autologous whole blood using a specific centrifugation method. It is rich in growth factors, including platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, fibroblast growth factor, epidermal growth factor, and insulin-like growth factor. These growth factors play an important role in the anti-inflammatory response and granulation tissue formation during bone healing. Furthermore, because PRP is an autologous blood product derivative, it avoids immune rejection, providing a safer treatment option for bone healing. Numerous studies have demonstrated that PRP can promote bone defect repair by releasing a rich supply of growth factors. Currently, local injections of PRP or direct placement of PRP in the affected area are commonly used in clinical practice. These approaches often suffer from the drawbacks of a burst of growth factor release and rapid clearance. The efficient utilization of autologous blood derivatives to better serve the medical industry has become one of the factors limiting the development of blood product products. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a PRP combined with carboxymethyl chitosan scaffold to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing a PRP combined with carboxymethyl chitosan scaffold comprises the following steps:

[0008] After mixing carboxymethyl chitosan powder and hydroxyapatite powder, genipin powder is added and mixed evenly to obtain a mixture;

[0009] The frozen PRP was thawed and mixed with ddH2O to obtain a mixed solution;

[0010] The mixture is added to the mixed liquid, stirred evenly, and bubbles are eliminated to obtain ink;

[0011] The ink is printed using a 3D printer and then placed in a CaCl2 solution for deformation treatment to obtain the PRP combined with carboxymethyl chitosan scaffold.

[0012] Preferably, the mass ratio of the carboxymethyl chitosan powder to the hydroxyapatite powder is 5:(0.5-1.5).

[0013] Preferably, in the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8).

[0014] Preferably, the mass volume ratio of the genipin powder to the mixed solution is (0.1-0.3) g:100 mL.

[0015] Preferably, the mass volume ratio of the carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

[0016] Preferably, the step of printing the ink using a 3D printer and then placing the ink in a CaCl2 solution for deformation treatment specifically includes:

[0017] The ink is printed at different initial angles to control the deformation direction of the bracket. The printed bracket is then quickly dried and immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket is cross-linked and cured under the slow action of genipin.

[0018] Preferably, the concentration of the CaCl2 solution is 0.05-0.15 mol / L.

[0019] Another object of the embodiment of the present invention is to provide a PRP combined with carboxymethyl chitosan scaffold prepared by the above preparation method.

[0020] Another object of an embodiment of the present invention is to provide an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of a shape memory material with photothermal conversion properties.

[0021] Another object of the embodiment of the present invention is to provide a use of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of bone repair materials.

[0022] The method for preparing a PRP-based combined carboxymethyl chitosan scaffold provided in an embodiment of the present invention utilizes the swelling characteristics of carboxymethyl chitosan hydrogel and its ability to shrink under the stimulation of divalent calcium ions, resulting in a printed material that is easily processable, has adjustable shape responsiveness, is highly structurally designable, and is inexpensive. Furthermore, the embodiment of the present invention utilizes 4D printing technology to transform PPR into a portion of the printing ink through stirring, utilizing the scaffold's microporous structure and slow degradation to achieve the slow release of growth factors within the PRP. Furthermore, the addition of hydroxyapatite not only improves the scaffold's mechanical properties but also produces a three-dimensional scaffold that conforms to the topography of the affected area. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process for preparing a PRP combined with carboxymethyl chitosan scaffold provided in an embodiment of the present invention.

[0024] Figure 2 Graph showing the shear-viscosity characterization analysis results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0025] Figure 3 Graph showing the storage / loss modulus-strain characterization analysis results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0026] Figure 4 This is a graph showing the storage / loss modulus-angular frequency characterization analysis results of the brackets prepared in Examples 1-3 of the present invention and Comparative Examples 1-2.

[0027] Figure 5 Graph showing the viscosity-time characterization analysis results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0028] Figure 6 Graph showing the fidelity verification results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0029] Figure 7 The figure shows the swelling characterization results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0030] Figure 8 Graph showing the elastic modulus and compressive strength characterization results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0031] Figure 9 Graph showing degradation test results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0032] Figure 10 The diagram shows the basic printing deformation results of the brackets prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0033] Figure 11 This is a diagram showing the personalized printed deformation results of the bracket prepared in Example 1 of the present invention.

[0034] Figure 12 Graphs showing the qualitative and quantitative analysis results of the photothermal performance of the stents prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0035] Figure 13 This is a qualitative and quantitative analysis result diagram of the photothermal performance of the bracket prepared in Example 2 of the present invention under the stimulation of near-infrared light of different powers.

[0036] Figure 14 This is a graph showing the water contact angle characterization analysis results of the stents prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0037] Figure 15 Graph showing the results of CCK-8 cytotoxicity assays of the scaffolds prepared in Examples 1-3 and Comparative Example 2 of the present invention.

[0038] Figure 16 These are the alkaline phosphatase expression staining images of the blank control group, CH group, CHP40 group, and CHP40+NIR group after 7 and 14 days of bone induction.

[0039] Figure 17 These are Alizarin red staining images of the blank control group, CH group, CHP40 group, and CHP40+NIR group after 28 days.

[0040] Figure 18 Micro-CT reconstructed images of the skull defect area of ​​rats in the blank control group, CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery.

[0041] Figure 19 These are the He and Masson staining results of the skull defect area in the blank control group, CH group, CHP40 group, and CHP40+NIR group of rats 4 and 8 weeks after surgery. DETAILED DESCRIPTION

[0042] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] The present invention provides a PRP combined with carboxymethyl chitosan scaffold and its preparation method and application, which aims to solve the following two technical problems:

[0044] 1. 4D printing technology, due to its unique shape memory properties, has gradually emerged in the field of minimally invasive medicine. 4D printing specifically refers to the combination of 3D printing technology and shape memory materials. Initial shapes are designed through computer programming, then printed using a 3D printer. These materials can then undergo controlled secondary deformation under stimulation by heat, light, electricity, magnetism, or even water. While typical 3D printers currently available can only print regular, planar scaffolds, the shape memory properties of 4D printing materials enable these flat printed scaffolds to undergo secondary deformation according to pre-programmed designs, creating a variety of three-dimensional scaffolds. These scaffolds will also meet a wider range of clinical treatment needs, particularly for patients with severe alveolar ridge resorption encountered during oral medical treatment. However, 4D printing programming typically involves complex interactions between various chemical bonds, resulting in a cumbersome preparation process and other issues that have hindered further promotion. To address these issues, the present invention leverages the swelling and cross-linking properties of carboxymethyl chitosan and PRP in divalent calcium ion solutions, as well as the slow cross-linking properties of genipin in the mixed ink, to provide a simple and easy-to-use 4D printing programming method. By regulating the direction of printing, the direction of curling of the stent during secondary deformation is controlled, thereby forming various curved stents. The slow cross-linking of genipin and carboxymethyl chitosan in the ink is used to achieve the shaping and mechanical strength enhancement of the stent after deformation. At the same time, by regulating the original shape of the printed stent, the printing of various types of three-dimensional stents can be achieved, such as the "chrysanthemum" stent printed in the embodiment of the present invention. This programming method is suitable for 4D printing of most hydrogel-type stents. One of the purposes of the embodiment of the present invention is to provide a simple and easy-to-understand 4D printing programming design, so that the printing material has the advantages of easy design and easy printing, so that it can better meet the current clinical treatment requirements, and thus it is expected to be used in clinical minimally invasive implant treatments in the future.

[0045] Platelet-Rich Plasma (PRP) is a platelet concentrate rich in growth factors obtained by centrifuging whole blood using a specific centrifugation method. PRP contains growth factors such as platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, fibroblast growth factor, epidermal growth factor, and insulin-like growth factor. These growth factors are essential players in bone healing. Furthermore, because PRP is derived from autologous blood products, it avoids immune rejection, providing a safer treatment option for bone healing. Numerous studies have demonstrated that PRP can promote bone defect repair by releasing a rich supply of growth factors. Consequently, PRP has gained increasing favor among clinicians across various disciplines. However, clinical practice often involves local injection of PRP or direct application of PRP to the affected area. These methods often involve a burst of growth factor release, which is then rapidly cleared. Therefore, PRP is only effective in the early stages of bone repair and is not suitable for the complex and long-term process of bone regeneration. How to efficiently utilize PRP to achieve a slow release of blood-derived growth factors has become a key challenge in clinical treatment. This embodiment of the present invention uses 4D printing technology to prepare a carboxymethyl chitosan / hydroxyapatite / PRP (CMCS / HA / PRP) scaffold. By stirring the PRP into a portion of the printing ink, the scaffold's microporous structure and slow degradation enable the slow release of growth factors within the PRP. Furthermore, this embodiment of the present invention utilizes light stimulation in combination with 808nm near-infrared light to achieve controlled release of the PRP.

[0046] Specifically, in one embodiment of the present invention, Figure 1 As shown, a method for preparing a PRP combined with carboxymethyl chitosan scaffold is provided, which comprises the following steps:

[0047] S1, mixing carboxymethyl chitosan powder and hydroxyapatite powder, and then adding genipin powder and mixing evenly to obtain a mixture;

[0048] S2, thawing the frozen PRP and mixing it with ddH2O to obtain a mixed solution;

[0049] S3, adding the mixture to the mixed liquid, stirring evenly, and eliminating bubbles to obtain ink;

[0050] S4. After printing the ink using a 3D printer, the ink is placed in a CaCl2 solution for deformation treatment to obtain a PRP combined with carboxymethyl chitosan scaffold.

[0051] It should be noted that the extraction method of PRP is as follows:

[0052] Rat-derived PRP: Blood was collected from rat hearts and centrifuged at 3000 r / min for the first time for 15 min. The supernatant and the portion near the dividing line were collected. The supernatant obtained from the first centrifugation was centrifuged at 3500 r / min for a second time for 15 min. The upper 1 / 2 of the depleted PRP was discarded and the lower 1 / 2 was retained. The blood was resuspended by pipetting and stored in a -80°C freezer.

[0053] Human PRP: Collect blood from human veins, centrifuge it at 240g for 15 minutes, take the supernatant and the part close to the dividing line; centrifuge the supernatant obtained from the first centrifugation at 600g for 15 minutes, discard the upper 1 / 2 of the depleted PRP, retain the lower 1 / 2, resuspend it by pipetting and set aside, and freeze it in a -80℃ refrigerator.

[0054] In a preferred embodiment of the present invention, the mass ratio of carboxymethyl chitosan powder to hydroxyapatite powder is 5:(0.5-1.5); in the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8); the mass volume ratio of genipin powder to the mixed solution is (0.1-0.3) g:100 mL; and the mass volume ratio of carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

[0055] In a preferred embodiment of the present invention, the step of printing the ink using a 3D printer and then placing the ink in a CaCl2 solution for deformation treatment specifically includes:

[0056] S41. The ink is printed at different initial angles to control the deformation direction of the bracket. The printed bracket is then quickly dried and immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket is cross-linked and cured under the slow action of genipin.

[0057] In practical applications, the structure of the printed bracket can be designed as two parts. The first part (bottom layer) is a thin film structure without printing intervals. The printing path of the second part (upper layer) is set to a 2mm interval, and basic printing is performed at initial angles of 0°, 45°, and 90° to control the deformation direction of the bracket.

[0058] It should be noted that 4D scaffold printing can be performed using a 3D printer (Regenovo, China). The specific printing parameters were: printing temperature of 25-30°C, air pressure of 0.3-0.5 MPa, printing speed of 8 mm / s, layer height of 0.4 mm, nozzle inner diameter of 0.41 mm (22G), and scaffold size of 20 × 10 mm.

[0059] In a preferred embodiment of the present invention, the concentration of the CaCl2 solution is 0.05-0.15 mol / L.

[0060] In another embodiment of the present invention, a PRP combined with carboxymethyl chitosan scaffold prepared by the above preparation method is also provided.

[0061] In another embodiment of the present invention, there is provided an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in preparing a shape memory material with photothermal conversion properties.

[0062] Another object of an embodiment of the present invention is to provide an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of bone repair materials; specifically, the embodiment of the present invention can achieve controlled release of PRP by light stimulation in combination with 808nm near-infrared light.

[0063] The following embodiments are some specific implementation cases and experimental cases in practical applications of the present invention, but are not limited thereto.

[0064] Example 1: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0065] S1. After mixing 1 g of carboxymethyl chitosan powder (CMCS) and 0.2 g of hydroxyapatite powder (HA), 0.012 g of genipin powder was added and mixed uniformly to obtain a mixture;

[0066] S2. Take the frozen PRP out of the -80°C freezer and thaw it in a 37°C water bath. Mix 1.2 mL of the thawed PRP with 4.8 mL of ddH2O to obtain a mixed solution.

[0067] S3. The mixture obtained above was added to the mixed solution above, and stirred at 200 rpm / min for 15 minutes using a magnetic stirrer to obtain ink. The ink was then centrifuged at 3000 rpm for 5 minutes to eliminate bubbles. After the ink was completely uniform and free of bubbles, it was placed in a printing cartridge for later use. The above ink was recorded as CMCS / HA / PRP ink.

[0068] S4. The above inks were used to print 4D scaffolds using a 3D printer (Regenovo, China). Specific printing parameters were set as follows: printing at 30°C, air pressure set to 0.3-0.5 MPa, printing speed set to 8 mm / s, layer height set to 0.4 mm, a printhead with an inner diameter of 0.41 mm (22G), and scaffold dimensions set to 20 × 10 mm. Furthermore, a pre-programmed printing path was designed. Specifically, the printed scaffold structure was designed to be two-part: the first part (bottom layer) was a thin film structure with no printing gaps; the second part (top layer) had printing paths set with 2 mm gaps, and basic printing was performed at initial angles of 0°, 45°, and 90° to control the scaffold's deformation direction. After the printed hydrogel scaffolds were quickly dried at room temperature, they were immersed in a 0.1 mol / L CaCl2 solution to achieve deformation in different directions. The scaffold's deformation direction (angle) remained consistent with the angle of the top printing path (0°, 45°, 90°) during printing. Then, based on this fundamental deformation rule (printing angle determines deformation direction), different complex structures were designed through permutations and combinations of three basic printing angles and deformed in a 0.1 mol / L CaCl₂ solution. Finally, the deformed scaffolds were slowly crosslinked and cured under the action of genipin within the ink, causing the scaffold to change color. This resulted in a PRP-coated carboxymethyl chitosan scaffold with photothermal properties, designated as a CMCS / HA / 20% PRP scaffold, or CHP20 scaffold for short.

[0069] Example 2: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold. The only difference between this example and Example 1 is that the two examples use different inks.

[0070] Specifically, the preparation method of the ink used in Example 2 is as follows:

[0071] After mixing 1g of carboxymethyl chitosan powder with 0.2g of hydroxyapatite powder, 0.012g of genipin powder was added and mixed evenly to obtain a mixture; the frozen PRP was taken out of the -80°C refrigerator and placed in a 37°C water bath to thaw, and 2.4mL of the thawed PRP was mixed with 3.6mL of ddH2O to obtain a mixed solution; the above-obtained mixture was added to the above-mentioned mixed solution and stirred at 200rpm / min for 15min by a magnetic stirrer to obtain ink; then, to eliminate bubbles, the ink was centrifuged at 3000rpm for 5 minutes; after the ink was completely uniform and bubble-free, it was placed in the printing cartridge for use. The PRP combined with carboxymethyl chitosan scaffold prepared in Example 2 was recorded as CMCS / HA / 40% PRP scaffold, referred to as CHP40 scaffold.

[0072] Example 3: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold. The only difference between this example and Example 1 is that the two examples use different inks.

[0073] Specifically, the preparation method of the ink used in Example 3 is as follows:

[0074] 1g of carboxymethyl chitosan powder was mixed with 0.2g of hydroxyapatite powder, and then 0.012g of genipin powder was added and mixed evenly to obtain a mixture; the frozen PRP was taken out of the -80°C refrigerator and placed in a 37°C water bath to thaw, and 3.6mL of the thawed PRP was mixed with 2.4mL of ddH2O to obtain a mixed solution; the obtained mixture was added to the mixed solution and stirred at 200rpm / min for 15min by a magnetic stirrer to obtain ink; then, to eliminate bubbles, the ink was centrifuged at 3000rpm for 5 minutes; after the ink was completely uniform and bubble-free, it was placed in the printing cartridge for use. The PRP combined with carboxymethyl chitosan scaffold prepared in Example 3 was recorded as CMCS / HA / 60% PRP scaffold, referred to as CHP60 scaffold.

[0075] Example 4: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0076] S1. After mixing 0.8 g of carboxymethyl chitosan powder (CMCS) and 0.08 g of hydroxyapatite powder (HA), 0.006 g of genipin powder was added and mixed uniformly to obtain a mixture;

[0077] S2. Take the frozen PRP out of the -80°C freezer and thaw it in a 37°C water bath. Mix 1.5 mL of the thawed PRP with 4.5 mL of ddH2O to obtain a mixed solution.

[0078] S3. The mixture obtained above was added to the mixed solution above, and stirred at 200 rpm / min for 15 minutes using a magnetic stirrer to obtain ink. The ink was then centrifuged at 3000 rpm for 5 minutes to eliminate bubbles. After the ink was completely uniform and free of bubbles, it was placed in a printing cartridge for later use. The above ink was recorded as CMCS / HA / PRP ink.

[0079] S4. The above ink was used to print 4D scaffolds using a 3D printer (Regenovo, China); the specific printing parameters were set as follows: printing at 30°C, air pressure set to 0.3-0.5MPa, printing speed set to 8mm / s, layer height set to 0.4mm, print nozzle selected with an inner diameter of 0.41mm (22G), and scaffold size set to 20×10mm. The printed scaffold was placed in a 0.05mol / L CaCl2 solution for deformation. Finally, the deformed scaffold was cross-linked and cured under the slow action of genipin inside the ink, causing the scaffold to change color, thereby obtaining a PRP combined with carboxymethyl chitosan scaffold with photothermal properties.

[0080] Example 5: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0081] S1. After mixing 1.2 g of carboxymethyl chitosan powder (CMCS) and 0.36 g of hydroxyapatite powder (HA), 0.018 g of genipin powder was added and mixed uniformly to obtain a mixture;

[0082] S2. Take the frozen PRP out of the -80°C freezer and thaw it in a 37°C water bath. Mix 3 mL of the thawed PRP with 3 mL of ddH2O to obtain a mixed solution.

[0083] S3. The mixture obtained above was added to the mixed solution above, and stirred at 200 rpm / min for 15 minutes using a magnetic stirrer to obtain ink. The ink was then centrifuged at 3000 rpm for 5 minutes to eliminate bubbles. After the ink was completely uniform and free of bubbles, it was placed in a printing cartridge for later use. The above ink was recorded as CMCS / HA / PRP ink.

[0084] S4. The above ink was used to print 4D scaffolds using a 3D printer (Regenovo, China); the specific printing parameters were set as follows: printing at 30°C, air pressure set to 0.3-0.5MPa, printing speed set to 8mm / s, layer height set to 0.4mm, print nozzle selected with an inner diameter of 0.41mm (22G), and scaffold size set to 20×10mm. The printed scaffold was placed in a 0.15mol / L CaCl2 solution for deformation. Finally, the deformed scaffold was cross-linked and cured under the slow action of genipin inside the ink, causing the scaffold to change color, thereby obtaining a PRP-combined carboxymethyl chitosan scaffold with photothermal properties.

[0085] Comparative Example 1: This comparative example provides a method for preparing a printed stent. The only difference between this comparative example and Example 1 is that the two methods use different inks.

[0086] Specifically, the preparation method of the ink used in Comparative Example 1 is as follows:

[0087] 1.2 g of carboxymethyl chitosan powder and 0.012 g of genipin powder were mixed to obtain a mixture. This mixture was added to 6 mL of ddH2O and stirred at 200 rpm / min using a magnetic stirrer for 15 minutes to obtain ink. The ink was then centrifuged at 3000 rpm for 5 minutes to eliminate bubbles. Once the ink was completely uniform and bubble-free, it was placed in a printing cartridge for later use. The stent produced in Comparative Example 1 is designated as a CMCS stent.

[0088] Comparative Example 2: This comparative example provides a method for preparing a printed stent. The only difference between this comparative example and Example 1 is that the two methods use different inks.

[0089] Specifically, the preparation method of the ink used in Comparative Example 2 is as follows:

[0090] 1g of carboxymethyl chitosan powder was mixed with 0.2g of hydroxyapatite powder, followed by 0.012g of genipin powder. The mixture was then added to 6mL of ddH2O and stirred at 200rpm / min using a magnetic stirrer for 15 minutes to obtain ink. The ink was then centrifuged at 3000rpm for 5 minutes to eliminate bubbles. Once the ink was completely uniform and bubble-free, it was placed in a printing cartridge for later use. The stent produced in Comparative Example 2 is designated as a CMCS / HA stent, or CH stent for short.

[0091] Performance and effect test: 1. Shear-viscosity characterization analysis was performed on the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2. The results are as follows: Figure 2 As shown in the figure, it can be seen that all the inks in Examples 1-3 and Comparative Examples 1-2 have good shear thinning properties, ensuring smooth extrusion of the ink under high shear force. Among them, the addition of HA increases the viscosity of the printing ink, but as the PRP content increases, its viscosity continues to decrease.

[0092] 2. The CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2 were subjected to storage / loss modulus-strain characterization analysis. The results are as follows: Figure 3 As shown; it can be seen from the figure that the inks of Examples 1-3 and Comparative Examples 1-2 remain stable at a strain of 0.5% and can be used for angular frequency scanning analysis.

[0093] 3. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to storage / loss modulus-angular frequency characterization analysis. The results are as follows: Figure 4 As shown; it can be seen from the figure that the storage modulus of the ink of Examples 1-3 and Comparative Examples 1-2 is higher than the loss modulus under a strain of 0.5%, proving that the printed ink is in a good gel state.

[0094] 4. The CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2 were subjected to viscosity-time characterization analysis. The results are as follows: Figure 5 As shown in the figure, it can be seen that the addition of HA and PRP increases the thixotropy of the material, which not only ensures the smooth extrusion of the ink but also increases the stability of the hydrogel scaffold in the static state.

[0095] 5. The fidelity of the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2 was verified. Figure 6 shown; from Figure 6 As shown in Figures A and B, a 20×20×0.4mm single-layer array was designed using 3D Max. The dimensions of each microbead pillar within the array were (1.5×0.8×0.4mm). After printing, photos were taken. Ten points on the array were randomly selected, and the length and width of the micropillars were calculated using ImageJ software. The results show that the addition of HA and PRP increases the shape fidelity of the ink, allowing the extrusion-printed scaffold to more closely match the specified length and width (1.5×0.8mm).

[0096] VI. The CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2 were subjected to swelling characterization analysis. The results are as follows: Figure 7 As shown in the figure, it can be seen that the addition of HA reduces the swelling ratio of the CMC S scaffold, which in turn improves the shape fidelity of the scaffold.

[0097] VII. The elastic modulus and compressive strength of the CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were analyzed. Figure 8As shown in the figure, compared with the scaffold before cross-linking, Ca 2+ The scaffold cross-linked with genipin has excellent elastic modulus and compressive strength.

[0098] 8. Degradation tests were performed on the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, the CMCS scaffold prepared in Comparative Example 1, and the CH scaffold prepared in Comparative Example 2. The results are as follows: Figure 9 As shown; it can be seen from the figure that all the scaffolds of Examples 1-3 and Comparative Examples 1-2 slowly degraded within 8 weeks, and the addition of HA slightly reduced the degradation rate of the scaffolds, but the degradation rate of all the scaffolds matched the rate of new bone formation.

[0099] IX. The CHP20 bracket prepared in Example 1, the CHP40 bracket prepared in Example 2, the CHP60 bracket prepared in Example 3, and the CH bracket prepared in Comparative Example 2 were subjected to basic printing deformation display. The results are as follows: Figure 10 As shown; it can be seen from the figure that the deformation direction of all the brackets in Examples 1-3 and Comparative Example 2 in 0.1 mol / L calcium chloride solution is consistent with the pre-programmed printing direction of the bracket.

[0100] 10. The CHP20 stent prepared in Example 1 was subjected to personalized printing deformation display, and the results were as follows: Figure 11 As shown in the figure, it can be seen that through the combination of basic printing rules, personalized complex structures can be achieved by controlling the strain angle.

[0101] XI. The photothermal performance of the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were qualitatively and quantitatively analyzed. The qualitative analysis results are as follows: Figure 12 As shown in the left figure, the quantitative analysis results are as follows Figure 12 As shown in the right figure; it can be seen from the figure that the scaffold after cross-linking with genipin exhibits excellent photothermal conversion efficiency, at 0.75W / cm 2 The photothermal photography and thermal curves under different power levels showed that the addition of HA and PRP had no significant effect on the photothermal conversion efficiency of the scaffold.

[0102] 12. The photothermal performance of the CHP40 scaffold prepared in Example 2 was qualitatively and quantitatively analyzed under near-infrared light (NIR) stimulation of different powers. The qualitative analysis results are as follows: Figure 13 As shown in the left figure, the quantitative analysis results are as follows Figure 13 As shown in the right figure; it can be seen from the figure that 0.25W / cm 2 , 0.5W / cm 2 , 0.75W / cm 2and 1W / cm 2 Under high-power NIR irradiation, the temperatures of the black CHP40 scaffold after cross-linking with genipin can reach 28.1℃, 34.2℃, 39.1℃ and 43.3℃ respectively.

[0103] Thirteen, the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were subjected to water contact angle characterization analysis. The results are as follows: Figure 14 As shown; it can be seen from the figure that the stents prepared in Examples 1-3 and Comparative Example 2 all have excellent hydrophilicity.

[0104] 14. The CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were subjected to CCK-8 cytotoxicity test and compared with the blank control group (Blank). The results are as follows: Figure 15 As shown in the figure, it can be seen that from the results on days 1, 3, and 7, PRP significantly promoted cell proliferation. On the seventh day, the CHP60 scaffold with the highest PRP content showed the strongest cell proliferation ability.

[0105] 15. Under the same experimental conditions, the CH scaffold prepared in Comparative Example 2 was subjected to osteogenic induction experiment, recorded as CH group; the CHP40 scaffold prepared in Example 2 was subjected to osteogenic induction experiment, recorded as CHP40 group; the CHP40 scaffold prepared in Example 2 was subjected to osteogenic induction experiment under near-infrared light (NIR) stimulation, recorded as CHP40+NIR group, and the blank control group (Blank) was used as comparison; among them, the alkaline phosphatase (ALP) expression staining of the blank control group (Blank), CH group, CHP40 group, and CHP40+NIR group after 7 and 14 days of osteoinduction is shown as follows Figure 16 As shown in the figure, it can be seen that the CHP40 scaffold showed better ALP expression ability than the blank control group and CH scaffold, and near-infrared can effectively promote the expression of ALP, and the ALP expression of the CHP40+NIR group was the strongest.

[0106] The results of Alizarin Red (AR) staining of Blank control group, CH group, CHP40 group and CHP40+NIR group on day 28 were as follows Figure 17 As shown in the figure, it can be seen that the CHP40 scaffold showed better AR expression ability than the blank control group and CH scaffold, and near-infrared can effectively promote AR expression, and the AR expression in the CHP40+NIR group was the strongest.

[0107] Micro-CT reconstructed images of the skull defect area of ​​rats in the blank control group (Blank), CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery are shown in Figure 2. Figure 18 As shown in the figure, it can be seen that the recovery of bone defects in rats stimulated by NIR is better than that in rats without stimulation, and the bone regeneration quality of CHP40 group and CHP40+NIR group is better than that of blank control group and CH group.

[0108] The results of He and Masson staining of the skull defect area of ​​rats in the blank control group, CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery are shown in Figure 2 Figure 19 As shown in the figure, compared with the blank control group, the new bone formation in the CHP40+NIR group was significantly increased at 4 weeks and 8 weeks; in the Masson staining experiment, the CHP40 group and the CHP40+NIR group showed more collagen formation than the blank control group and the CH group, and near-infrared heating stimulation produced more microvessels.

[0109] Based on the above-mentioned ideal embodiment of the present invention, and through the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a PRP combined with carboxymethyl chitosan scaffold, characterized in that: The following steps are involved: After mixing carboxymethyl chitosan powder and hydroxyapatite powder, genipin powder is added and mixed evenly to obtain a mixture; The frozen PRP was thawed and mixed with ddH2O to obtain a mixed solution; Adding the mixture into the mixed liquid, stirring evenly, and eliminating bubbles to obtain ink; The ink is printed using a 3D printer and then placed in a CaCl2 solution for deformation treatment to obtain the PRP combined with carboxymethyl chitosan scaffold.

2. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: The mass ratio of the carboxymethyl chitosan powder to the hydroxyapatite powder is 5:(0.5-1.5).

3. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: In the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8).

4. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: The mass volume ratio of the genipin powder to the mixed solution is (0.1-0.3) g:100 mL.

5. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1 or 2, characterized in that: The mass volume ratio of the carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

6. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: After the ink is printed using a 3D printer, the steps of placing the ink in a CaCl2 solution for deformation treatment specifically include: The ink is printed at different initial angles to control the deformation direction of the bracket. The printed bracket is then quickly dried and immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket is cross-linked and cured under the slow action of genipin.

7. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1 or 6, characterized in that: The concentration of the CaCl2 solution is 0.05-0.15 mol / L.

8. A PRP combined with carboxymethyl chitosan scaffold prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the PRP combined with carboxymethyl chitosan scaffold as claimed in claim 8 in the preparation of a shape memory material with photothermal conversion properties.

10. Use of the PRP combined with carboxymethyl chitosan scaffold according to claim 8 in preparing bone repair materials.

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