An organizational engineering hydrogel scaffold, its preparation method and application

Abstract

The present invention belongs to the technical field of tissue engineering, and particularly relates to a tissue engineering hydrogel scaffold, a preparation method thereof, and an application. The tissue engineering hydrogel scaffold includes collagen microspheres containing bone marrow mesenchymal stem cells and endothelial cells and a hydrogel formed by a methacrylated gelatin cross-linked network and fibrin. The collagen microspheres are dispersed in the hydrogel. Among them, the mass ratio of methacrylated gelatin to fibrin in the methacrylated gelatin cross-linked network is 3-8:1. The tissue engineering hydrogel scaffold of the present invention has good mechanical properties and stable structure, is suitable for the field of 3D printing technology to prepare tissue function scaffolds with complex structures, can ensure a high survival rate of the loaded seed cells (BMSCs and HUVECs), good viability of the surviving cells, has good pre-vascularization network formation ability and osteogenic differentiation effect, excellent bone repair performance, and has good application prospects in clinical practice.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and particularly relates to a tissue engineering hydrogel scaffold, a preparation method thereof, and an application thereof. Background Art

[0002] Bone tissue plays a crucial role in the human body, such as protecting various organs, maintaining hematopoiesis, storing minerals, and providing mechanical support for people to move. Severe trauma, tumor resection, degenerative osteoarthritis, obesity, and aging-related diseases can all cause bone defects. In recent years, the incidence of bone defects has been increasing year by year. Due to the lack of blood vessels and nerves, once a bone injury occurs, its self-repair ability is very limited, which requires surgical bone grafting to restore its structural integrity and functionality. Clinically, surgical methods such as autologous bone grafting and allogeneic bone grafting are the main methods for treating severe bone defects. The most commonly used clinical methods include autologous bone grafting, allogeneic bone grafting, etc. However, after a large number of clinical verifications, the foregoing methods have many drawbacks, such as limited availability, morbidity and pain at the donor site, high failure rate, and risk of disease transmission, etc.

[0003] With the development of the discipline of tissue engineering, the bone tissue engineering transplantation technology constructed by tissue engineering strategies combining cells, biocompatible scaffolds, and growth factors has unique advantages. Its principle is to organically combine a biocompatible scaffold with osteoconductive ability and biodegradable in vivo with autologous therapeutic cells (such as bone marrow mesenchymal stem cells) and / or cytokines with osteoinductive activity to construct a living bone tissue engineering graft, which is implanted into the defect site. It has the dual characteristics of osteoconduction and induction, which is more conducive to repairing the defective bone tissue. Moreover, while rapidly forming bone, the implanted scaffold material gradually degrades and is finally metabolized and absorbed by the human body. Therefore, the treatment strategy based on bone tissue engineering transplantation technology provides a new idea and method for the repair of bone defects. The bone tissue engineering transplantation technology mainly studies scaffold materials, seed cells, and cytokines. Among them, the scaffold material, as the framework for bone tissue regeneration, its properties directly affect the biological properties of seed cells, affect the survival, migration, proliferation, and metabolic functions of cells, and is therefore one of the key elements in bone tissue engineering. The design and development of new scaffold materials have become a research hotspot for bone injury repair. The mechanical properties of commonly used bone injury repair materials such as metals and ceramics are very different from those of human bones. After implantation, it is easy to cause stress shielding, resulting in loosening of the surrounding bone tissue, and it is also easy to produce rejection reactions. Hydrogel is a polymer material with water as the dispersion medium, hydrophilic and insoluble in water, capable of absorbing a large amount of water, and having a cross-linked structure. It has good water permeability and biocompatibility, can reproduce the natural tissue microenvironment in vivo and in vitro, can maintain good cell viability, and promote cell proliferation, migration, and differentiation. As a graft, it can reduce adverse reactions, and thus has received increasing attention in biomedicine.

[0004] With the rapid development of 3D printing technology, constructing a precise three-dimensional hydrogel scaffold using 3D printing technology can better simulate the bone tissue structure of the human body, providing strong support for the application of hydrogels in bone tissue engineering grafts. Currently, the hydrogel scaffold materials developed for 3D printing have their own advantages and disadvantages. For example, natural collagen and fibrin hydrogels have good tissue compatibility but poor mechanical properties, and their compressive strength is generally lower than 1.6 MPa; biodegradable synthetic materials such as polylactic acid have controllable mechanical properties but poor tissue compatibility. In addition, the printing of bone tissue engineering transplantation scaffolds usually adopts the method of printing first and then crosslinking. On the one hand, poor mechanical properties will cause the scaffold structure to easily collapse before crosslinking, reducing the fidelity of the scaffold. On the other hand, the crosslinking process will affect the activity of the seeded cells loaded in the hydrogel scaffold, and due to the damage of the cells by the shear force during the printing process, the survival rate of the seeded cells loaded in the hydrogel scaffold after crosslinking is generally about 40%, greatly reducing the osteogenic effect and repair function. Therefore, it is of great significance to develop a hydrogel scaffold material with good biocompatibility, excellent mechanical properties, and high cell survival rate for bone injury repair. Summary of the Invention

[0005] In view of the above problems in the prior art, the present invention provides a tissue engineering hydrogel scaffold and its preparation method, and provides the application of the tissue engineering hydrogel scaffold in the field of tissue engineering scaffolds, especially bone tissue engineering transplantation scaffolds, to solve or at least alleviate some or all of the technical problems in the prior art.

[0006] To achieve the above object, the present invention is specifically realized through the following technical solutions:

[0007] In a first aspect of the present invention, a tissue engineering hydrogel scaffold is provided, which includes collagen microspheres containing bone marrow mesenchymal stem cells and endothelial cells and a hydrogel formed by a methacrylated gelatin crosslinked network and fibrin. The collagen microspheres are dispersed in the hydrogel, wherein the mass ratio of methacrylated gelatin to fibrin in the methacrylated gelatin crosslinked network is 3 - 8:1.

[0008] Further, the mass ratio of methacrylated gelatin to fibrin in the methacrylated gelatin crosslinked network is 5:1.

[0009] Further, each square millimeter of the cross-section of the tissue engineering hydrogel scaffold contains 3 - 12 of the collagen microspheres, and the diameter of the collagen microspheres is 150 - 350 μm.

[0010] Further, the collagen microspheres contain a cell suspension, and the cell concentration of the cell suspension is 4×10 5 -8×105 cells / mL.

[0011] Furthermore, the concentration ratio of the endothelial cells to the bone marrow mesenchymal stem cells in the cell suspension is 1:1 - 5. Preferably, it is 1:3.

[0012] Further, the collagen is type I collagen.

[0013] Further, the methacrylated gelatin cross - linked network is a methacrylated gelatin photocross - linked network.

[0014] Furthermore, the methacrylated gelatin photocross - linked network is formed by cross - linking and polymerization of methacrylated gelatin under the action of a photoinitiator.

[0015] The second aspect of the present invention provides a method for preparing the tissue - engineered hydrogel scaffold as described above, comprising the following steps:

[0016] S1. Add a cell suspension containing bone marrow mesenchymal stem cells and endothelial cells to a collagen solution to obtain a microsphere precursor solution. By using a microfluidic technique and adopting a water - in - oil structure, prepare collagen droplets, and after solidifying the collagen droplets, remove the oil phase to obtain collagen microspheres;

[0017] S2. Culture the collagen microspheres in a hybrid growth medium to obtain a collagen microsphere solution with good cell activity;

[0018] S3. Mix a methacryloyl gelatin solution and a fibrin solution, the mass ratio of the methacrylated gelatin to the fibrin is 3 - 8:1, and then add an initiator to obtain a hydrogel prepolymer solution;

[0019] S4. Mix the collagen microsphere solution and the hydrogel prepolymer solution to obtain a bioink, and place the bioink at a low temperature to change it from a liquid state to a gel state;

[0020] S5. Print and shape the bioink by 3D printing technology and solidify it to obtain a tissue - engineered hydrogel scaffold.

[0021] Further, in step S1, the cell concentration in the microsphere precursor solution is 4×10 5 - 8×10 5 cells / mL, and the concentration ratio of the endothelial cells to the bone marrow mesenchymal stem cells is 1:1 - 5, and the collagen concentration is 8 - 11 mg / mL.

[0022] Further, in step S1, when preparing the collagen droplets by means of microfluidics technology with a water-in-oil structure, the microsphere precursor solution is used as the dispersed phase and sterile mineral oil is used as the continuous phase. The pumping flow rate of the dispersed phase is 5 - 30 μL / min, and the pumping flow rate of the continuous phase is 50 - 300 μL / min. The diameter of the prepared collagen microspheres is 150 - 350 μm.

[0023] Further, in step S2, the hybrid growth medium comprises the following components: high-glucose DMEM medium, 10% fetal bovine serum, and 1% double antibody, and the double antibody includes penicillin and streptomycin.

[0024] Further, in step S4, the collagen microsphere solution and the hydrogel prepolymer solution are mixed at a volume ratio of 4:1 to obtain the bioink. The bioink is pre-cooled at 10 - 18°C for 15 - 20 min to change from a liquid state to a gel state.

[0025] Further, in step S5, through a direct writing printing process, at 15°C and an air pressure intensity of 0.5 - 0.7 MPa, the gel-state bioink is extruded at a printing speed of 2 - 5 mm / s and irradiated under an ultraviolet radiation lamp for 15 - 20 s with a photocuring power of 30% - 50% to obtain the tissue engineering hydrogel scaffold. Each square millimeter of the cross-section of the tissue engineering hydrogel scaffold contains 3 - 12 of the collagen microspheres.

[0026] The third aspect of the present invention provides the application of the tissue engineering hydrogel scaffold as described above or the tissue engineering hydrogel scaffold prepared by the preparation method of the tissue engineering hydrogel scaffold as described above in the preparation of tissue engineering scaffolds.

[0027] The advantages and positive effects of the present invention are as follows:

[0028] The tissue engineering hydrogel scaffold of the present invention has good mechanical properties and high compressive strength, and its compressive strength is up to more than 1.8 MPa. It has a stable structure, is suitable for the 3D printing technology field to prepare tissue function scaffolds with complex structures, and can ensure a high survival rate of the loaded seed cells (BMSCs and HUVECs), with a cell survival rate of up to more than 80%. Moreover, the surviving cells have good activity, have good pre-vascularization network formation ability and osteogenic differentiation effect, and have excellent bone repair performance, showing good application prospects in clinical practice. Description of the Drawings

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0030] Figure 1 It is a schematic diagram of the composition structure of the tissue engineering hydrogel scaffold according to the embodiment of the present invention;

[0031] Figure 2 It is a flowchart for the preparation of collagen microspheres according to the embodiment of the present invention;

[0032] Figure 3 It is a flowchart for the preparation of the tissue engineering hydrogel scaffold according to the embodiment of the present invention and the tissue engineering scaffold prepared therefrom. Detailed implementation manners

[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Based on the information included in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for schematically illustrating specific aspects of the present invention. In fact, various changes that those skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0035] To better understand the present invention rather than limit its scope, all numbers representing amounts, percentages, and other numerical values used in the present invention should be understood as being modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired properties. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by the conventional rounding method. In addition, the meanings of the terms "comprising", "including", "containing", "having" and other similar words are non-restrictive, that is, other steps and other components can be added without affecting the results.

[0036] To make the above objects, features and advantages of the present invention more obvious and understandable, the following details the specific embodiments of the present invention in combination with the drawings.

[0037] An embodiment of the present invention provides a tissue engineering hydrogel scaffold, as Figure 1 shown, comprising collagen microspheres containing bone marrow mesenchymal stem cells and endothelial cells and a hydrogel formed by a methacrylated gelatin crosslinking network and fibrin, wherein the collagen microspheres are dispersed in the hydrogel, and the mass ratio of methacrylated gelatin to fibrin in the methacrylated gelatin crosslinking network is 3-8:1.

[0038] In the present invention, bone marrow mesenchymal stem cells (BMSCs) and endothelial cells (HUVECs) are encapsulated by collagen. Collagen belongs to a thermosensitive crosslinking gel material, which is in a liquid state at 4°C and transforms into a gel state when the temperature rises to 37°C, thereby forming a multi-cellular collagen microsphere structure containing bone marrow mesenchymal stem cells and endothelial cells ( Figure 1 abbreviated as multi-cellular microspheres in the following). Then, the collagen microspheres are mixed with a methacrylated gelatin (GelMA) and fibrin (FIB) solution, and then a hydrogel composed of a methacrylated gelatin crosslinking network and fibrin is formed by photocuring or thermocuring. The collagen microspheres are dispersed therein to obtain the tissue engineering hydrogel scaffold of the present invention. The tissue engineering hydrogel scaffold of the present invention has the following characteristics:

[0039] 1. Good biocompatibility: Collagen and fibrin are two main components of the extracellular matrix microenvironment, and methacrylated gelatin contains a gelatin network produced by partial hydrolysis of collagen. There is excellent compatibility among the three, and they have good biocompatibility with human tissues, which can greatly reduce the immune rejection reaction occurring after implantation of the tissue engineering scaffold made therefrom. Moreover, it can be degraded after implantation, and the degradation products are low-toxic and have no side effects, and can be metabolized and absorbed by the human body.

[0040] 2. Excellent mechanical properties: Fibrin, as an amorphous fibrous elastic solid, is mixed with GelMA. After the GelMA crosslinking network is formed, many fibrin bind tightly to form fibrin polymers, which are dispersed in the GelMA crosslinking network to improve the mechanical properties and mechanical strength of the scaffold material. In addition, collagen microspheres with good compressive strength are dispersed in the hydrogel structure, which can further improve the mechanical properties and mechanical strength of the material. Therefore, through the cooperation of collagen microspheres, fibrin polymers and the GelMA crosslinking network, the compressive strength of the tissue engineering hydrogel scaffold of the present invention can reach 1.8 MPa or more.

[0041] 3. Good cell viability and high cell survival rate: In the present invention, collagen is used as the extracellular matrix, which can protect cells and mix BMSCs and HUVECs at a high density. Compared with the common use of photocrosslinkable material hydrogel as the microsphere matrix, using collagen as the microsphere extracellular matrix can be solidified into spheres directly by using a 37°C water bath, which can avoid the damage to cells caused by the ultraviolet lamp irradiation during the solidification of the cell droplets inside into spheres, and is beneficial to improving the activity and survival rate of the cells in the microspheres. Moreover, the collagen microspheres are dispersed in the hydrogel structure, and the hydrogel structure can buffer the extrusion of external forces on the microspheres and maintain the microsphere morphology, thereby reducing the damage to cells caused by external forces such as shear force during the printing process using 3D printing technology, further improving the cell activity and survival rate, and also being beneficial to increasing the cell concentration in the material. After detection, the survival rate of cells in the tissue engineering hydrogel scaffold prepared by the present invention reaches more than 80% after 3D printing and forming.

[0042] 4. Excellent bone repair ability: On the one hand, through the synergistic mechanism in the co-culture system of BMSCs and HUVECs, the generation of a pre-vascular network can be achieved; on the other hand, the hydrogel formed by the crosslinking network of fibrin and GelMA can provide a supporting role for the bone defect area in the initial stage of implantation, and provide a stable environment for the proliferation and differentiation of cells, thereby helping the BMSCs and HUVECs cells cultured in vitro to generate a self-assembled vascular network, efficiently induce angiogenesis and promote osteoblast differentiation, so as to effectively solve the problem of preventing central bone tissue necrosis in the pre-vascularized network of large bone tissue engineering scaffolds, and enable the newly formed bone tissue to repair the defect area as soon as possible.

[0043] In summary, the tissue engineering hydrogel scaffold of the present invention has good mechanical properties and stable structure, is suitable for the field of 3D printing technology to prepare tissue function scaffolds with complex structures, and can ensure that the seeded cells (BMSCs and HUVECs) have a high survival rate and good activity, have good pre-vascularized network formation ability and osteogenic differentiation effect, excellent bone repair performance, and have good application prospects in clinical practice.

[0044] The solidification and forming of the tissue engineering hydrogel scaffold mainly depends on the crosslinking network of methacrylated gelatin, and the fibrin polymer formed by the tight binding of fibrin is the main factor to improve the mechanical strength of the scaffold. The proportion range of the two can be 3 - 8:1. With the increase of the proportion of the methacrylated gelatin crosslinking network, the forming state of the scaffold is better, but due to the decrease of the proportion of the fibrin polymer in the scaffold, its compressive strength will gradually decrease. Therefore, considering the forming state and compressive performance of the hydrogel scaffold comprehensively, preferably, the mass ratio of methacrylated gelatin to fibrin in the methacrylated gelatin crosslinking network is 4 - 7:1, and more preferably 5:1.

[0045] Generally, after the tissue engineering hydrogel scaffold is printed and formed, it exists in the state of fiber filaments. The number of microspheres contained in the cross-section of the fiber filaments will affect its mechanical properties, and further affect the mechanical strength (the force received by the tissue engineering scaffold after implantation in the body is the pressure borne by a surface, that is, the compressive strength) and structural stability of the tissue engineering scaffold made of fiber filaments. Generally speaking, the more the number of collagen microspheres contained in the cross-section of the fiber filaments, the better the mechanical properties of the prepared scaffold. However, the influence of the size of the collagen microspheres on the survival rate of internal cells also needs to be comprehensively considered. When the size of the microspheres is appropriate, their morphology is regular spheres and they are evenly dispersed. In this state, the mechanical properties of the collagen microspheres themselves are better, and the survival rate of internal cells is also better. Optionally, the fiber filaments contain 3-12 of the above-mentioned collagen microspheres per square millimeter, preferably 4-10 collagen microspheres, and the diameter of the collagen microspheres is 150-350 μm. At this time, the synchronous improvement of cell survival rate and mechanical properties can be taken into account.

[0046] Optionally, the collagen microspheres contain a cell suspension, and the cell concentration in the cell suspension is 4×10 5 -8×10 5 cells / mL, that is, the total cell concentration of bone marrow mesenchymal stem cells (BMSCs) and endothelial cells (HUVECs) in the cell suspension is 4×10 5 -8×10 5 cells / mL.

[0047] The growth states of the two types of cells in the collagen microspheres will affect the cell survival rate in the hydrogel scaffold material to a certain extent. In order to improve cell activity, better induce osteogenic differentiation and form a pre-vascularized network, in a preferred embodiment, the concentration ratio of the endothelial cells (HUVECs) to the bone marrow mesenchymal stem cells (BMSCs) in the cell suspension is 1:1-5. Exemplarily, it can be 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5. Preferably, the concentration ratio of the endothelial cells to the bone marrow mesenchymal stem cells is 1:2-4, and more preferably 1:3. Under the foregoing ratio, the growth states of each cell are the best, and the osteogenic differentiation effect is good.

[0048] The collagen used to prepare the above-mentioned collagen microspheres is type I collagen.

[0049] Optionally, the methacrylated gelatin cross-linked network is a methacrylated gelatin photo-cross-linked network, which can be specifically formed by cross-linking and polymerization of methacrylated gelatin under the action of a photoinitiator. It can be understood that the photoinitiator is decomposed into free radicals under light excitation at the polymerization temperature, and the free radicals initiate the polymerization of methacrylated gelatin (GelMA) molecular chains with each other. The present invention does not make special limitations on the type of photoinitiator.

[0050] In some specific embodiments, the photoinitiator may specifically be lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). GelMA is a photosensitive biomaterial, which can be crosslinked and cured under the action of blue light or ultraviolet light when used in combination with a photoinitiator. Thus, the hydrogel material of the present invention can be printed into a three-dimensional scaffold by 3D printing technology and then cured by light.

[0051] Another embodiment of the present invention provides a method for preparing the tissue engineering hydrogel scaffold as described above. See Figures 2 - 3 , which includes the following steps:

[0052] S1. Prepare collagen microspheres: Add a cell suspension containing bone marrow mesenchymal stem cells and endothelial cells to a collagen solution to obtain a precursor solution of microspheres. By using a microfluidic technique and adopting a water-in-oil structure, prepare collagen droplets, and after curing the collagen droplets, remove the oil phase to obtain collagen microspheres;

[0053] S2. Culture the collagen microspheres: Culture the collagen microspheres in a hybrid growth medium to obtain a collagen microsphere solution with good cell activity;

[0054] S3. Prepare a hydrogel prepolymer solution: Mix a methacrylated gelatin solution and a fibrin solution, where the mass ratio of the methacrylated gelatin to the fibrin is 3-8:1, and then add an initiator to obtain a hydrogel prepolymer solution;

[0055] S4. Prepare bioink for printing: Mix the collagen microsphere solution and the hydrogel prepolymer solution to obtain bioink, and place the bioink at a low temperature to change it from a liquid state to a gel state for subsequent printing;

[0056] S5. Printing and forming: Print and form the bioink by 3D printing technology and cure it to obtain a tissue engineering hydrogel scaffold.

[0057] The method for preparing the tissue engineering hydrogel scaffold has the same advantages as the tissue engineering hydrogel scaffold described above over the prior art, and will not be elaborated here.

[0058] It should be noted that although the present invention has limited the order of steps S1-S3 in the above preparation process, those skilled in the art should know that this order limitation is only for descriptive purposes and for easy understanding, and does not mean that the present invention must be carried out in this order. For example, there is no sequential relationship between S1 and S3. S1 can be carried out first and then S3, or S3 can be carried out first and then S1, or the two steps can be carried out simultaneously.

[0059] Optionally, in step S1, the cell concentration in the microsphere precursor solution is 4×10 5 -8×10 5 cells / mL, and the concentration ratio of the endothelial cells to the bone marrow mesenchymal stem cells is 1:1-5, and the collagen concentration is 8-11 mg / mL.

[0060] Microfluidic technology is a commonly used technology for preparing microspheres. Its basic principle is that two immiscible phase solutions (such as aqueous phase and oil phase) meet in a microchannel and are split into micron-sized emulsion droplets under the action of external force or flow shear force, and finally the solvent is evaporated and solidified to obtain microspheres. Specifically, in the present invention, the microsphere precursor solution is used as the dispersed phase (aqueous phase), and sterile mineral oil is used as the continuous phase (oil phase). The dispersed phase is pumped into the primary inlet of the microfluidic device, and the continuous phase is pumped into the secondary inlet in an orthogonal direction to perform water-in-oil emulsification to generate collagen droplets containing two types of cells. Then, the formed collagen droplets are solidified in the microchannel with a 37°C water bath, and finally, the formed multi-cellular collagen microspheres with a diameter of 150-350 μm are collected at the outlet of the device. After that, the residual mineral oil is removed by washing multiple times.

[0061] The mineral oil used generally adopts sterile mineral oil (anhydrous, purity 99.9%) in the medical field, such as paraffin oil.

[0062] It should be noted that if the flow rates of the continuous phase and the dispersed phase are different, the shear force generated on the dispersed phase solution will be different, resulting in different sizes of the formed droplets. When different flow rate ratios are used, the distance between adjacent two droplets will also be different, and finally the morphology and dispersibility of the formed microspheres will be different. Irregular microsphere morphology and poor dispersion effect will cause certain damage to the cells contained inside, thereby reducing the cell survival rate. How to regulate the flow rates of the continuous phase and the dispersed phase to prepare microspheres with regular morphology and uniform dispersion is a conventional technical means in the art. Exemplarily, the pumping flow rate of the dispersed phase microsphere precursor solution is 5-30 μL / min, and the pumping flow rate of the continuous phase sterile mineral oil is 50-300 μL / min.

[0063] Optionally, in step S2, the hybrid growth medium includes the following components: DMEM high-glucose medium (purchased from Servicebio, product number: G4513-500ML), 10% fetal bovine serum (purchased from Biological Industries, product number: 04-001-1ACS), and 1% double antibody. The double antibody includes penicillin and streptomycin (purchased from Gibco brand penicillin-streptomycin, product number: 15140122).

[0064] Optionally, in step S3, the methacryloyl gelatin solution and the fibrin solution are stirred and mixed evenly under the condition of constant temperature at 37°C, then an initiator is added and stirred in the dark, and then centrifuged at 1000 r / min for 5 min to obtain a hydrogel prepolymer solution. The initiator is preferably a photoinitiator, specifically it can be LAP.

[0065] Optionally, in step S4, the collagen microsphere solution and the hydrogel prepolymer solution are mixed at a volume ratio of 4:1 to obtain the bioink, and the bioink is pre-cooled at 10-18°C for 15-20 min to change it from a liquid state to a gel state.

[0066] Optionally, in step S5, the 3D printing technology adopts direct writing printing technology. The gel-state bioink is injected into the feeding device of a bio-3D printer equipped with a dispensing needle. Using the direct writing printing process, it is printed into fiber filaments. Each square millimeter of the cross-section of the fiber filaments contains 3-12 of the collagen microspheres, and then it is cured under ultraviolet light, which is the tissue engineering hydrogel scaffold of the present invention. The inner diameter of the dispensing needle is determined according to the diameter of the fiber filaments to be printed. When fiber filaments with a diameter of 1000 μm are to be prepared, the inner diameter of the dispensing needle is 0.9-1.5 mm.

[0067] Another embodiment of the present invention provides the application of the tissue engineering hydrogel scaffold as described above or the tissue engineering hydrogel scaffold prepared by the preparation method of the tissue engineering hydrogel scaffold as described above in the preparation of tissue engineering scaffolds, especially bone tissue engineering scaffolds.

[0068] The application advantages of the tissue engineering hydrogel scaffold in the preparation of tissue engineering scaffolds are the same as those of the tissue engineering hydrogel scaffold as described above relative to the prior art, and will not be elaborated here.

[0069] The shape and structure of the tissue engineering scaffold are 3D printed and customized according to actual needs, such as according to the bone defect morphology of different areas to be repaired, or manufactured into a scaffold with a fixed shape by 3D printing. The present invention exemplarily provides a structure. See Figure 3 , first the gel-state bioink is printed into fiber filaments by the direct writing printing process, and then it is made by splicing the fiber filaments at different layers and angles. Specifically, the tissue engineering scaffold includes multiple layers, and the fiber filaments in the upper layer are arranged perpendicular to the fiber filaments in the lower layer to form a grid structure in the shape of a well.

[0070] Direct writing printing parameters: Under the conditions of 15°C and an air pressure intensity of 0.5 - 0.7 MPa, the bioink is extruded at a printing speed of 2 - 5 mm / s to print into fiber filaments. The tissue engineering scaffold is formed by discontinuously extruding and printing eight layers through a bio-3D printer. Each layer is irradiated with ultraviolet light for 15 - 20 s. The ultraviolet light source is a 365 nm ultraviolet radiation lamp, and the photocuring power is 30% - 50%.

[0071] The present invention will be further described below in conjunction with specific embodiments. The experimental methods without specific conditions noted in the following embodiments are generally carried out according to the conditions recommended by the manufacturer.

[0072] Example 1

[0073] A tissue engineering hydrogel scaffold includes collagen microspheres containing bone marrow mesenchymal stem cells and endothelial cells with a cell concentration ratio of 3:1 and a hydrogel formed by a methacrylated gelatin cross-linked network and fibrin. The collagen microspheres are dispersed in the hydrogel. Among them, the methacrylated gelatin cross-linked network is formed by cross-linking and polymerizing methacrylated gelatin under the action of a photoinitiator LAP. The mass ratio of methacrylated gelatin to fibrin is 5:1. Its preparation method includes the following steps:

[0074] S1. Preparation of collagen microspheres: Add 8 mL of cell suspension C containing bone marrow mesenchymal stem cells and endothelial cells to 2 mL of type I collagen solution with a concentration of 9 mg / mL, stir and mix evenly at 4°C to obtain a microsphere precursor solution. At 4°C, pump the microsphere precursor solution as the dispersed phase solution into the first inlet of the microfluidic device, and pump sterile mineral oil as the continuous phase into the second inlet in an orthogonal direction. The flow rate of the dispersed phase is 15 μL / min, and the flow rate of the continuous phase is 75 μL / min for oil-in-water emulsification to obtain collagen droplets containing BMSCs and HUVECs. Solidify in a 37°C water bath in the channel to obtain a collagen microsphere suspension with an average diameter of 350 μm. Gently shake the suspension and precipitate for 5 min to separate from the oil phase, and then wash 3 times in 1×PBS to remove residual mineral oil to obtain collagen microspheres;

[0075] The preparation method of the cell suspension C containing bone marrow mesenchymal stem cells and endothelial cells includes the following steps:

[0076] Preparation of BMSCs cell suspension: Harvest the cultured BMSCs with 0.25% trypsin, centrifuge at 1000 r / min for 5 min, and then add BMSCs medium to obtain suspension A containing BMSCs with a concentration of 6×10 5 cells / mL;

[0077] Preparation of HUVECs cell suspension: The cultured HUVECs were harvested with 0.25% trypsin, centrifuged at 1000 r / min for 5 min, and then HUVECs medium was added to obtain suspension B containing HUVECs with a concentration of 6×10 5 cells / mL;

[0078] Suspension A and suspension B were mixed in a ratio of 3:1 to obtain 8 mL of cell suspension C containing two types of cells, which was composed of a mixed cell population of 75% BMSCs and 25% HUVECs;

[0079] S2. Culturing collagen microspheres: The collagen microspheres were cultured in a hybrid growth medium for 1 - 2 weeks to obtain a collagen microsphere solution with good cell viability; The hybrid growth medium includes the following components: high-glucose DMEM medium (purchased from Servicebio, product number: G4513 - 500ML), 10% fetal bovine serum (purchased from Biological Industries, product number: 04 - 001 - 1ACS), and 1% double antibody, and the double antibody includes penicillin and streptomycin (purchased from Gibco brand penicillin - streptomycin, product number: 15140122);

[0080] S3. Preparation of hydrogel prepolymer solution: A 50 mg / mL methacrylated gelatin solution and a 20 mg / mL fibrin solution were mixed in a volume ratio of 2:1 to obtain 10 mL of a mixed solution, and then 0.025 g of photoinitiator LAP was added. The mixture was stirred evenly in the dark under the condition of constant temperature at 37℃ to obtain a hydrogel prepolymer solution;

[0081] S4. Preparation of bioink for printing: 40 mL of the collagen microsphere solution and 10 mL of the hydrogel prepolymer solution were mixed to obtain bioink. The bioink was injected into a polypropylene syringe equipped with a 0.9 mm threaded needle, and the syringe was installed in the temperature-controlled photocuring nozzle device of a bio 3D printer and pre-cooled at 15℃ for 15 min to obtain a gel-state bioink that was easy to print;

[0082] S5. Printing and forming: Using the direct writing printing extrusion forming process, with a bio 3D printer, at 15℃, the bioink was extruded into fiber filaments. The fiber filaments were cylindrical structures. The feeding pressure (air pressure intensity) for extrusion forming was 0.7 MPa, the movement speed of the temperature-controlled photocuring nozzle device was 5 mm / s, the temperature of the printing platform was controlled at 10℃, the photocuring power of the 365 nm ultraviolet radiation lamp was 50%, and the irradiation time of the ultraviolet lamp was 16 s to obtain the tissue engineering hydrogel scaffold of the present invention. The fiber filament diameter was 950 μm, and the cross-section of the collagen microspheres was 3, that is, there were 4 microspheres per square millimeter (mm 2 ) of the fiber filament cross-section.

[0083] Example 2

[0084] This example is basically the same as Example 1, except that: when preparing collagen microspheres in step S1, the flow rate of the dispersed phase is 15 μL / min, the flow rate of the continuous phase is 160 μL / min, and the average diameter of the obtained collagen microspheres is 250 μm. The number of collagen microspheres in the cross-section of the fiber filament is 6.

[0085] Example 3

[0086] This example is basically the same as Example 2, except that: in step S3, a 30 mg / mL methacryloyl gelatin solution and a 20 mg / mL fibrin solution are mixed at a volume ratio of 2:1 to obtain 10 mL of a mixed solution, and the mass ratio of methacryloyl gelatin to fibrin in the 10 mL of the mixed solution is 3:1.

[0087] Example 4

[0088] This example is basically the same as Example 2, except that: in step S3, a 70 mg / mL methacryloyl gelatin solution and a 20 mg / mL fibrin solution are mixed at a volume ratio of 2:1 to obtain 10 mL of a mixed solution, and the mass ratio of methacryloyl gelatin to fibrin in the 10 mL of the mixed solution is 7:1.

[0089] Comparative Example 1

[0090] This comparative example is basically the same as Example 2, except that: collagen microspheres containing BMSCs and collagen microspheres containing HUVECs are prepared separately, and then the two cell-loaded collagen microsphere solutions are mixed with the polymer precursor solution to obtain a bioink.

[0091] Preparation of BMSCs cell suspension: The cultured BMSCs are harvested with 0.25% trypsin, centrifuged at 1000 r / min for 5 min, and then BMSCs medium is added to obtain 6 mL of suspension A containing BMSCs with a concentration of 6×10 5 cells / mL;

[0092] Preparation of HUVECs cell suspension: The cultured HUVECs are harvested with 0.25% trypsin, centrifuged at 1000 r / min for 5 min, and then HUVECs medium is added to obtain 2 mL of suspension B containing HUVECs with a concentration of 6×10 5 cells / mL;

[0093] 1.5 mL of a type I collagen solution with a concentration of 9 mg / mL is added to the suspension A containing BMSCs, and the mixture is stirred and mixed evenly at 4°C to obtain 7.5 mL of a type I collagen solution C1 containing BMSCs;

[0094] Add 0.5 mL of type I collagen solution with a concentration of 9 mg / mL to suspension B containing HUVECs, and stir and mix evenly at 4 °C to obtain 2.5 mL of type I collagen solution C2 containing HUVECs;

[0095] Respectively use type I collagen solutions C1 and C2 as the dispersed phase, and prepare collagen microspheres loaded with BMSCs and collagen microspheres loaded with HUVECs according to the method of Example 2.

[0096] Comparative Example 2

[0097] This comparative example is basically the same as Example 2, except that: fibrin is not added in step S3.

[0098] Detect the cell viability, vascularization, osteogenic differentiation, morphology and dispersion effect of the prepared microspheres, the number of microspheres in the cross-section of the fiber filaments, and the mechanical properties of the fiber filaments prepared in Examples 1-4 and Comparative Examples 1-2 of the present invention. The results are shown in Table 1.

[0099] Cell viability and vitality analysis experiment: Use a live / dead cell double staining reagent (live / dead staining reagent, Shanghai Jiqi Biotechnology Co., Ltd.) to analyze cell vitality. First, wash the fiber filaments three times with PBS before staining. Next, add 100 μL of the prepared live / dead staining assay reagent (live / dead staining reagent formula: 1.5 ml 10×Assay Buffer + 3 μL PI Solution + 1 μL Calcein AM) to the fiber filaments for staining. Then incubate them in the dark for 40 min and wash three times with PBS to remove the residual reagent. Finally, under a confocal fluorescence microscope, observe and image by obtaining two images in each frame (red and green for live and dead cells respectively).

[0100] Vascularization analysis experiment: The immunohistochemistry method was used to determine the expression level of platelet endothelial cell adhesion molecule (PECAM-1, an endothelial marker) to reflect the vascularization situation in the scaffold. The first step was to take the fiber filament sections (4 μm thick) prepared by the present invention, place them on adhesive slides, and bake the slides in an incubator at 60 °C overnight; the second step was conventional clearing and hydration; the third step was to soak in 3% H2O2 at room temperature for 10 min, wash with distilled water for 2 min, and wash 3 times with 1×PBS; the fourth step was to repair antigens by water bath. Immerse the sections in the EDTA antigen repair solution, heat to boiling at maximum heat and then turn off the heat, let the sections cool naturally to room temperature, and wash 3 times with 1×PBS; the fifth step was to add 5% BSA blocking solution, let it stand at 37 °C for 30 min, and then discard the excess liquid; the sixth step was to add rabbit anti-mouse CD31 antibody (Wuhan Aiboteck Co., Ltd.) diluted 1:150, incubate overnight at 4 °C, and wash 3 times with 1×PBS; the seventh step was to add DAB chromogenic agent diluted 1:200, let it stand for 90 s, and then rinse with tap water; the eighth step was to counterstain with hematoxylin for 20 s, rinse with tap water, soak in 1% hydrochloric acid alcohol for 1 s, and then rinse with tap water; the eighth step was dehydration treatment, wash 3 times with 95% ethanol, wash 3 times with absolute ethanol, and wash 3 times with xylene; the ninth step was to randomly select 20 non-overlapping fields of view for each obtained section for observation, and use Image-J image analysis software for automatic measurement and analysis, calculate the optical density value of each field of view and calculate the average value.

[0101] Osteogenic differentiation analysis experiment: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression levels of osteogenic specific genes type I collagen (COL-1), alkaline phosphatase (ALP), and Runt-related transcription factor 2 (Runx 2) genes to evaluate the osteogenic differentiation situation of the bone scaffold. Total RNA was extracted according to the manufacturer's protocol using a cell / tissue total RNA extraction kit ( Cell / Tissue Total RNA Kit), and its content was measured at 260 nm using a micro ultraviolet spectrophotometer. The first strand of cDNA was prepared using EasyQuick RT MasterMix. Then, RT-qPCR was performed on a PCR instrument using SYBRGreen 2x qPCR MasterMix, forward and reverse primers. The expression levels of different samples were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0102] Observation of microsphere morphology, dispersion effect, and the number of microspheres in the cross-section of fiber filaments: Observed through an inverted microscope (NIKON EC-LIPSE TS100).

[0103] Mechanical property analysis experiment of the scaffold: Place the fiber filaments between the load cells (25 kgf) with circular metal plates, and the metal plates are equipped in a universal testing machine. Use a metal compression head to compress the sample until 70% strain occurs, and record the maximum stress that can be borne during the experiment. Then analyze the compressive strength of the fiber filaments through the stress-strain curve between 20% and 40%. The metal compression head moves at a speed of 1.0 mm / min throughout the process.

[0104] Table 1 Performance test of the tissue engineering hydrogel scaffolds prepared in Examples 1-4 and Comparative Examples 1-2

[0105]

[0106]

[0107] Evaluate the vascularization in the bone scaffold by comparing the mRNA expression levels of platelet endothelial cell adhesion molecule (PECAM-1, an endothelial marker), and evaluate the osteogenic differentiation in the bone by measuring the mRNA expression levels of osteoblast-specific type I collagen (COL-1, an early osteogenic differentiation marker), alkaline phosphatase (ALP, an early osteogenic differentiation marker), and Runt-related transcription factor 2 (RUNX2, a mid-osteogenic differentiation marker). In the experiments of measuring the expression of vascularization and osteogenic differentiation markers, the test data of Example 2 are used as the reference standard value.

[0108] The tissue engineering hydrogel materials prepared in Examples 1-4 of the present invention have good mechanical properties, with a compressive strength above 1.8 MPa, and the cell survival rate is also greatly improved, reaching more than 80%. Through the mRNA expression level of the endothelial marker PECAM-1, it can be seen that the vascularization effect of Examples 1-4 is significantly improved; through the mRNA expression levels of the osteogenic differentiation markers COL-1, ALP, and RUNX2, it can be seen that the osteogenic differentiation effect of Examples 1-4 is significantly improved. In summary, the hydrogel scaffold materials of the examples of the present invention have good biocompatibility, are beneficial to vascularization and osteogenic differentiation, can promote bone tissue regeneration, and contribute to the treatment of large bone defects. Moreover, the collagen microspheres and good mechanical properties can reduce the damage to cells caused by shear force during the direct writing printing extrusion molding process, and improve the cell survival rate in the material.

[0109] It can be found from Examples 1-2 that the mechanical properties of Example 2 are better than those of Example 1 because the more microspheres contained in the cross-section of the fiber filaments, the better the mechanical properties of a single fiber filament; and the cell survival rate of Example 2 is also better than that of Example 1 because the morphology and dispersion effect of the prepared collagen microspheres will affect the cells inside the microspheres. The more regular the microsphere morphology and the more uniform the dispersion, the higher the cell survival rate inside the microspheres. In addition, the better the mechanical properties of the fiber filaments, the better the buffering and protection effects on the internal microspheres, and the combined effect results in a higher final cell survival rate. Therefore, it is preferred that there are 4-10 collagen microspheres per square millimeter of the cross-section of the fiber filaments.

[0110] It can be found from Examples 2-4 that the ratio of methacrylated gelatin to fibrin will affect the curing and forming of the hydrogel scaffold and to a certain extent affect its mechanical properties, and after reaching relatively high mechanical properties, it has little or no effect on the activity of cells in the microspheres. Thus, the PECAM-1 mRNA expression level, COL-1 mRNA expression level, ALP mRNA expression level, and RUNX2 mRNA expression level are basically the same. When Example 3 adopts a ratio of 3:1, due to the decrease in the cross-linking network ratio of methacrylated gelatin, small-scale collapse phenomena (poor forming state) will occur in the whole or part of the formed scaffold. However, since the materials at the collapsed parts aggregate into clusters, the strength of the scaffold in this example is relatively high; when Example 4 adopts a ratio of 7:1, due to the decrease in the fibrin polymer ratio in the scaffold, the compressive strength of the scaffold will drop to nearly 2.0 MPa, and the cell activity also decreases slightly. Therefore, it is preferred that the mass ratio of methacrylated gelatin to fibrin is 5:1.

[0111] By comparing Example 2 and Comparative Example 1, it can be found that the compressive strength of Example 2 remains unchanged and the cell survival rate remains unchanged. At the same time, due to the existence of the multi-cell co-culture system of BMSCs and HUVECs in Example 2, the mRNA expression levels of the endothelial marker PECAM-1 and the osteogenic differentiation markers COL-1, ALP, and RUNX2 are significantly increased, further proving that the multi-cell co-culture system of BMSCs and HUVECs is beneficial to vascularization and osteogenic differentiation. In Comparative Example 1, BMSCs and HUVECs cannot be in direct contact and cannot form a multi-cell co-culture system, and the vascularization and osteogenic differentiation ability will decrease by 60%-70%.

[0112] Comparing Example 2 and Comparative Example 2, it can be found that the lack of fibrin significantly reduces the mechanical properties of the fiber filaments, resulting in a certain degree of decrease in cell viability. In the previous experiments, without adding GelMA and lacking the GelMA photocrosslinking network, a formed structure could not be formed. Therefore, the combination of GelMA, fibrin, and collagen microspheres in the present invention is an essential component for forming a tissue engineering material with excellent mechanical properties, high cell viability and survival rate, and excellent bone repair ability.

[0113] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A tissue engineering hydrogel scaffold, characterized in that, Comprising collagen microspheres containing bone marrow mesenchymal stem cells and endothelial cells and a hydrogel formed by a methacrylated gelatin crosslinked network and fibrin, wherein the collagen microspheres are dispersed in the hydrogel, and the mass ratio of methacrylated gelatin to the fibrin in the methacrylated gelatin crosslinked network is 5:1; A method for preparing a tissue engineering hydrogel scaffold, comprising the following steps: S1. Adding a cell suspension containing bone marrow mesenchymal stem cells and endothelial cells into a collagen solution to obtain a microsphere precursor solution, and preparing collagen droplets by using a microfluidic technique with a water-in-oil structure. After solidifying the collagen droplets, removing the oil phase to obtain collagen microspheres; S2. Culturing the collagen microspheres in a hybrid growth medium to obtain a collagen microsphere solution with good cell activity; S3. Mixing a methacryloyl gelatin solution and a fibrin solution, with the mass ratio of methacrylated gelatin to the fibrin being 5:1, and then adding an initiator to obtain a hydrogel prepolymer solution; S4. Mixing the collagen microsphere solution and the hydrogel prepolymer solution to obtain a bioink, and pre-cooling the bioink at 10-18 °C for 15-20 min to change it from a liquid state to a gel state; S5. Injecting the gel-state bioink into a feeding device of a bio-3D printer equipped with a dispensing needle, and using a direct writing printing process to print and form filaments. Each square millimeter of the cross-section of the filaments contains 3-12 collagen microspheres, and then curing under ultraviolet light to obtain a tissue engineering hydrogel scaffold; In step S1, the cell concentration in the microsphere precursor solution is 4×10 5 -8×10 5 cells / mL, and the concentration ratio of the endothelial cells to the bone marrow mesenchymal stem cells is 1:1-5, and the collagen concentration is 8-11 mg / mL; When preparing collagen droplets by using a microfluidic technique with a water-in-oil structure, using the microsphere precursor solution as the dispersed phase and sterile mineral oil as the continuous phase. The pumping flow rate of the dispersed phase is 5-30 μL / min, and the pumping flow rate of the continuous phase is 50-300 μL / min. The diameter of the prepared collagen microspheres is 150-350 μm.

2. The tissue engineering hydrogel scaffold according to claim 1, wherein The collagen is type I collagen; The methacrylated gelatin crosslinked network is a methacrylated gelatin photo-crosslinked network, which is formed by crosslinking and polymerization of methacrylated gelatin under the action of a photoinitiator.

3. The tissue engineering hydrogel scaffold according to claim 1, wherein In step S4, the collagen microsphere solution and the hydrogel prepolymer solution are mixed at a volume ratio of 4:1 to obtain the bioink.

4. Use of the tissue engineering hydrogel scaffold according to any one of claims 1-3 in the preparation of a tissue engineering scaffold.

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