Cell-loaded composite platelet hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of composite platelet hydrogel, in particular to cell-loaded composite platelet hydrogel which comprises a hydrogel mixture, and the hydrogel mixture comprises methacrylated gelatin, platelet-rich lysate, endothelial cell growth factors, a photoinitiator, a cross-linking agent and a solvent. A blood vessel network is constructed through the preparation steps of preparation of methacrylated gelatin, preparation of a platelet-rich lysate, mixing preparation, PRP redissolution, 3D printing and the like, and at present, most of biological ink printing strategies are based on sacrificial microgel, firstly, sacrificial material ink is deposited and embedded into a hydrogel matrix, and the sacrificial material ink is deposited and embedded into the hydrogel matrix; the sacrificial bio-ink is then removed to obtain a hollow cardiovascular structure, these channels can be filled with endothelial cells to further obtain a biological vascular function, the mechanical properties of the platelet rich plasma gel are improved, the endothelial cells can form a microvascular network structure, and healing of chronic wounds is promoted to a greater extent.

Description

Technical Field

[0001] The invention relates to the technical field of composite platelet hydrogels, and in particular to a cell-loaded composite platelet hydrogel and a preparation method thereof. Background Art

[0002] Chronic wounds are defined as tissues that have impaired healing abilities due to conditions such as diabetes, obesity, chemotherapy, stress, etc. Chronic wounds do not go through the normal healing phase and cannot be repaired in an orderly and timely manner. In addition, some wounds may be complicated by infection and tissue defects. Platelets play a key role in the wound healing process.

[0003] When blood vessels are damaged, platelets will quickly aggregate at the damaged area and release growth factors after activation, including platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, etc. The growth factors released by platelets will stimulate various cells in the surrounding tissues, including fibroblasts and epithelial cells, which helps cell adhesion and migration. Cell adhesion and migration promote new tissue formation and angiogenesis. In addition, platelets also participate in the formation of matrix scaffolds, providing cells with a platform for movement and differentiation, which helps the formation of new tissues.

[0004] The mechanism of platelet-rich plasma gel formation involves platelet activation and the action of coagulation factors. Platelet activation can be achieved in various ways, such as the addition of calcium ions, the action of thrombin, etc. After activation, platelets release a series of growth factors and other bioactive substances they are rich in, including fibrinogen, prothrombin, fibrin adhesion protein, etc. The bioactive substances and coagulation factors released by platelets participate in the coagulation cascade reaction together with the activated platelets. This series of reactions includes platelet aggregation, fibrinogen conversion to fibrin, and finally the formation of platelet gel. Platelet-rich plasma is prepared as a biological scaffold by exposing it to thrombin or calcium chloride to stimulate fibrin formation. Platelet gel can be used as a dressing to help restore homeostasis and tissue repair. However, compared with fibrin gel, platelet gel has poor mechanical strength, which further reduces the healing efficiency of chronic wounds. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a cell-loaded composite platelet hydrogel and a preparation method thereof, which can effectively solve the technical problems raised in the background technology.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The invention provides a cell-loaded composite platelet hydrogel, comprising a hydrogel mixture, wherein the hydrogel mixture comprises methacrylylated gelatin, platelet-rich lysate, endothelial cell growth factor, a photoinitiator, a crosslinking agent and a solvent.

[0008] Furthermore, the methacryloylated gelatin is a hydrogel prepared by methacryloylating gelatin, and the platelet concentration in the platelet-rich lysate reaches 1X109 / ml.

[0009] Furthermore, the photoinitiator is one of i2959 and LAP, the cross-linking agent is one of a solution containing calcium ions and a solution of thrombin, and the solvent is one of PBS and culture medium.

[0010] Furthermore, the platelet-rich lysate reacts with a cross-linking agent containing calcium ions or thrombin to produce a cross-linked network. At the same time, under the action of a photoinitiator, the methacrylylated gelatin undergoes a cross-linking reaction under irradiation with visible light of 405 nm. The platelet-rich lysate in the hydrogel will further dissolve to obtain a hydrogel with a porous structure. Cells, such as human umbilical vein endothelial cells, can be loaded in the hydrogel precursor solution. Under the dual action of endothelial cell growth factor and platelet-rich lysate, the loaded human umbilical vein endothelial cells will gradually form a microvascular network system.

[0011] A method for preparing a cell-loaded composite platelet hydrogel comprises the following preparation processes:

[0012] S1. Preparation of methacrylic acid-acylated gelatin. The methacrylic acid-acylated gelatin usually selects one of water and PBS buffer as a solvent. An appropriate amount of gelatin is added to the solvent to obtain a gelatin solution of appropriate concentration. The gelatin is heated and stirred during the dissolution process. Methacrylic anhydride is added dropwise to the gelatin solution at a certain speed. The solution is continuously stirred during the dropping process. The mixed solution is placed under appropriate reaction conditions to carry out a methacrylic acid-acylation reaction. After the reaction is completed, the product is neutralized. After neutralization, the product can be further purified by dialysis, centrifugation and the like. The purified product is washed. Finally, the washed product is dried to obtain a final Ge l MA product.

[0013] S2, preparation of platelet-rich lysate, wherein the platelet-rich lysate is prepared by collecting fresh whole blood from a donor, and immediately adding an anticoagulant to prevent blood coagulation, injecting the fresh anticoagulated whole blood into a centrifuge tube, and adding a density gradient medium as needed, placing the centrifuge tube in a centrifuge, and centrifuging at an appropriate speed and time. After the centrifugation, carefully aspirate the blood components of different layers using a pipette or a pipette, and collect them in different containers respectively. According to the experimental needs, further process and purify these components, freeze the obtained components with liquid nitrogen for a certain period of time, and then melt them at 35-40 degrees, and repeat 2-3 times as required to obtain platelet lysate;

[0014] S3, mixing preparation, mixing a certain volume fraction of PRP solution with a specific concentration of Gel MA. After mixing, it is activated by Ca2+ and thrombin and solidified into a hydrogel by ultraviolet light irradiation. It is necessary to ensure that all operations are carried out under sterile conditions to avoid contamination and infection. In practical applications, the mixing ratio needs to be optimized and adjusted according to specific needs.

[0015] S4. PRP redissolves. During clinical application, the PRP in the hydrogel will further dissolve. On the one hand, it can provide nutrition for the cells in the gel. On the other hand, as the PRP dissolves, a hydrogel with a porous structure is obtained.

[0016] S5. 3D printing. Due to the temperature-sensitive property of methacrylated gelatin, it can be 3D printed into a fixed shape to form a 3D printed model.

[0017] Compared with the known prior art, the technical solution provided by the present invention has the following beneficial effects:

[0018] Vascular networks are constructed through 3D printing technology. Currently, most of them are based on the bio-ink printing strategy of sacrificial microgels. First, the sacrificial material ink is deposited and embedded in the hydrogel matrix, and then the sacrificial bio-ink is removed to obtain a hollow blood vessel structure. These channels can be filled with endothelial cells to further obtain biological vascular functions, such as gelatin, agarose, etc. as sacrificial materials. For example, using caramel as a sacrificial biomaterial, Lei et al. constructed a biodegradable, perfusable and permeable hierarchical microchannel network and found that the material can not only maintain high-density cell growth in vitro, but also promote angiogenesis in vivo. It not only improves the mechanical properties of platelet-rich plasma gel, but also enables endothelial cells to form a microvascular network structure, thereby promoting the healing of chronic wounds to a greater extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 An ear model printed with Ge l MA having a concentration of 2.5% (w / v) according to the present invention;

[0021] Figure 2 Observation of HUVECs cell culture in the Gel MA hydrogel of the present invention (day 3): (a) HUVECs cell morphology in the Gel MA hydrogel with a concentration of 2.5% (w / v); (b) HUVECs cell morphology in the Gel MA hydrogel with a concentration of 5% (w / v);

[0022] Figure 3 The microscopic images of the fibroblast scratch test of the present invention at 0, 5, 10, and 15 hours (ad) The experimental group is 2 ml of culture medium containing 2.5% (w / v) Gel MA, 50% (w / v) PRP, 20 (ng / ml) VEGF, and 0.4% (w / v) LAP; (eh) The control group is 2 ml of MEM culture medium;

[0023] Figure 4 Figure 1 shows the live / dead staining results of HUVECs cells cultured in a 2.5% (w / v) Gel MA hydrogel for a period of time under a fluorescence microscope of the present invention (2nd, 5th, and 7th days): (a) the morphology of HUVECs cells in the Gel MA hydrogel on the 2nd day; (b) the morphology of HUVECs cells in the Gel MA hydrogel on the 5th day; (c) the morphology of HUVECs cells in the Gel MA hydrogel on the 7th day;

[0024] Figure 5 The experimental results of the time it takes for PRP to form gel under the action of different cross-linking agents of the present invention are shown in Figure A. Group A is 5 ml PRP; Group B is 5 ml PRP and 0.5 ml 10% calcium chloride solution; Group C is 5 ml PRP, 0.5 ml 10% calcium chloride solution, and 100 U thrombin. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0026] The present invention will be further described below in conjunction with the embodiments.

[0027] Embodiment 1:

[0028] A cell-loaded composite platelet hydrogel comprises a hydrogel mixture, wherein the hydrogel mixture comprises methacrylylated gelatin (Gel MA), platelet-rich lysate (PRP), endothelial cell growth factor (VEGF), a photoinitiator, a crosslinker and a solvent, wherein the methacrylylated gelatin is a hydrogel prepared by methacrylylating gelatin, the platelet concentration in the platelet-rich lysate reaches 1×109 / ml, the photoinitiator is one of i 2959 and LAP, the crosslinker is one of a solution containing calcium ions and a solution containing thrombin, and the solvent is one of PBS and a culture medium;

[0029] The hydrogel is a double cross-linked network system, in which PRP reacts with a cross-linking agent containing calcium ions or thrombin to produce a cross-linked network. At the same time, under the action of a photoinitiator, Gel MA undergoes a cross-linking reaction under 405nm visible light irradiation, and the PRP in the hydrogel will further dissolve to obtain a hydrogel with a porous structure, thereby improving cell adhesion, promoting cell growth and migration, and especially the formation of microvessels. Cells can be loaded in the hydrogel precursor solution, such as human umbilical vein endothelial cells (HUVEC). Under the dual action of VEGF and PRP, HUVEC will gradually form a microvascular network system. The hydrogel can be used as a biological ink for extrusion 3D printing, for example: when the concentration of Gel MA is 2.5% and the concentration of PRP is 50%, the temperature of the print head is 4°C, the air pressure is 170kPa, and the printing speed is 7mm / s; when the concentration of Gel MA is 3% and the concentration of PRP is 50%, the temperature of the print head is 6°C, the air pressure is 130kPa, and the printing speed is 7mm / s.

[0030] Embodiment 2:

[0031] This embodiment is different from the first embodiment in that: a method for preparing a cell-loaded composite platelet hydrogel includes the following preparation processes:

[0032] S1. Preparation of methacrylic acid-acylated gelatin. The methacrylic acid-acylated gelatin usually uses water or PBS buffer as a solvent. An appropriate amount of gelatin is added to the solvent to obtain a gelatin solution of appropriate concentration. The dissolution process of the gelatin may require heating and stirring to ensure its complete dissolution. Methacrylic anhydride is added dropwise to the gelatin solution at a certain speed (such as 0.5 mL / min). During the addition process, the solution is continuously stirred to ensure that the methacrylic anhydride and the gelatin are fully mixed. The mixed solution is placed under appropriate reaction conditions (such as temperature, pH value, etc.) to carry out a methacrylic acid-acylation reaction. The reaction time may take several hours to ensure that the reaction is complete. The progress of the reaction can be monitored by detecting the residual amount of methacrylic anhydride in the reaction solution or observing the change in the properties of the solution. After the reaction is completed, the product needs to be neutralized to remove unreacted methacrylic anhydride and other by-products. An alkaline solution (such as NaOH) is usually used for neutralization. After neutralization, the product can be further purified by dialysis, centrifugation, etc. to remove impurities and unreacted reagents. The purified product is washed to remove residual solvents and impurities. Finally, the washed product is dried to obtain the final Gel MA product. The drying method can be freeze-drying or natural drying.

[0033] S2, preparation of platelet-rich lysate, wherein the platelet-rich lysate is prepared by collecting fresh whole blood from a donor and immediately adding an anticoagulant to prevent blood coagulation. The selection and amount of the anticoagulant should be determined according to specific experimental requirements. A suitable centrifuge and centrifuge tube are selected to ensure that the centrifuge tube can accommodate enough blood samples and can withstand the pressure generated by high-speed centrifugation. Fresh anticoagulated whole blood is injected into the centrifuge tube, and a density gradient medium (such as Fico ll, Perco ll, etc.) is added as needed. The centrifuge tube is placed in a centrifuge and centrifuged at an appropriate speed and time. During the centrifugation process, blood components of different densities will be arranged in layers under the action of the gradient medium. After the centrifugation is completed, the centrifuge tube is carefully removed to avoid disturbing the layers. The blood components of different layers are carefully aspirated using a pipette or a pipette and collected in different containers. These components can be further processed or purified according to experimental requirements. The components are frozen with liquid nitrogen for a certain period of time and then melted at 35-40 degrees. The process is repeated 2-3 times as required to obtain platelet lysate. The specific steps are as follows:

[0034] 1. Collect 30ml of peripheral blood;

[0035] 2. Transfer to a centrifuge tube and centrifuge at 2000 rpm for 10 minutes to remove the red blood cells and white blood cells in the lower layer, and take the upper serum layer into a new 15 ml centrifuge tube;

[0036] 3. Centrifuge the serum at 3000 rpm for 15 minutes, and platelet precipitation can be seen;

[0037] 4. Adjust the platelets to the appropriate density and remove the remaining supernatant;

[0038] 5. Use the gun tip to blow the platelet precipitate evenly;

[0039] 6. Freeze in liquid nitrogen for 10 minutes and thaw at 37 degrees. Repeat 2-3 times as required to obtain platelet lysate.

[0040] S3. Mixing preparation: PRP solution with a certain volume fraction is mixed with Gel MA with a specific concentration. The commonly used mixing ratio can be adjusted according to the specific application scenario. For example, in the culture of mouse insulinoma cells MIN6, PRP solution with a volume fraction of 10%, 30%, and 50% can be mixed with 50g / L Gel MA. After mixing, it is activated by Ca2+ and thrombin and solidified into a hydrogel by ultraviolet light irradiation. It is necessary to ensure that all operations are carried out under sterile conditions to avoid contamination and infection. Different application scenarios may require different mixing ratios. Therefore, in actual applications, the mixing ratio needs to be optimized and adjusted according to specific needs.

[0041] S4. PRP redissolves. During clinical application, the PRP in the hydrogel will further dissolve. On the one hand, it can provide nutrition for the cells in the gel. On the other hand, as the PRP dissolves, a hydrogel with a porous structure is obtained, which improves cell adhesion, promotes cell growth and migration, especially the formation of microvessels, and provides blood supply for repairing diabetic foot.

[0042] S5. 3D printing. Due to the temperature-sensitive properties of methacrylated gelatin, 3D printing can be used to form fixed shapes and form 3D printed models, such as ear models, etc.

[0043] The remaining structures are the same as those of Example 1.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell-loaded composite platelet hydrogel, characterized in that: The hydrogel mixture comprises methacrylated gelatin, platelet-rich lysate, endothelial cell growth factor, photoinitiator, cross-linking agent and solvent.

2. The cell-loaded composite platelet hydrogel according to claim 1, characterized in that: The methacryloyl gelatin is a hydrogel prepared by methacryloylating gelatin, and the platelet concentration in the platelet-rich lysate reaches 1×10 9 / ml.

3. The cell-loaded composite platelet hydrogel according to claim 1, characterized in that: The photoinitiator is one of i2959 and LAP, the cross-linking agent is one of a solution containing calcium ions and a solution of thrombin, and the solvent is one of PBS and a culture medium.

4. The cell-loaded composite platelet hydrogel according to claim 1, characterized in that: The platelet-rich lysate reacts with a cross-linking agent containing calcium ions or thrombin to produce a cross-linked network. The platelet-rich lysate in the hydrogel will further dissolve to obtain a hydrogel with a porous structure. Human umbilical cord vein endothelial cells are loaded in the hydrogel precursor solution. Under the dual effects of endothelial cell growth factor and platelet-rich lysate, the loaded human umbilical cord vein endothelial cells will gradually form a microvascular network system.

5. A method for preparing a cell-loaded composite platelet hydrogel as claimed in claims 1 to 4, characterized in that: The preparation process includes the following: S1. Preparation of methacrylated gelatin. The methacrylated gelatin usually uses water or PBS buffer as a solvent. An appropriate amount of gelatin is added to the solvent to obtain a gelatin solution of appropriate concentration. The gelatin is heated and stirred during dissolution. Methacrylic anhydride is added dropwise to the gelatin solution at a certain speed. The solution is continuously stirred during the addition. The mixed solution is placed under appropriate reaction conditions to perform a methacrylation reaction. After the reaction is completed, the product is neutralized. After neutralization, the product is further purified by dialysis and centrifugation methods. The purified product is washed. Finally, the washed product is dried to obtain a final methacrylated gelatin product. S2, preparation of platelet-rich lysate, wherein the platelet-rich lysate is prepared by collecting fresh whole blood from a donor, and immediately adding an anticoagulant to prevent blood coagulation, injecting the fresh anticoagulated whole blood into a centrifuge tube, and adding a density gradient medium as needed, placing the centrifuge tube in a centrifuge, and centrifuging at an appropriate speed and time. After the centrifugation, carefully aspirate the blood components of different layers using a pipette or a pipette, and collect them in different containers respectively. According to the experimental needs, further process and purify these components, freeze the obtained components with liquid nitrogen for a certain period of time, and then melt them at 35-40 degrees, and repeat 2-3 times as required to obtain platelet lysate; S3, mixing preparation, mixing a certain volume fraction of platelet-rich lysate solution with a specific concentration of methacryloyl gelatin. After mixing, it is solidified into a hydrogel through Ca2+ and thrombin activation and ultraviolet light irradiation. It is necessary to ensure that all operations are carried out under sterile conditions to avoid contamination and infection. In actual applications, the mixing ratio needs to be optimized and adjusted according to specific needs. S4. The platelet-rich lysate is re-dissolved. During clinical application, the platelet-rich lysate in the hydrogel will further dissolve, providing nutrition to the cells in the gel while obtaining a hydrogel with a porous structure. S5. 3D printing. Due to the temperature-sensitive property of methacrylated gelatin, it can be 3D printed into a fixed shape to form a 3D printed model.

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