A bio-ink and a preparation method and application thereof

Abstract

The application provides a bio-ink and a preparation method and application thereof, and the bio-ink comprises the following components in the following concentrations: 50-150 g / L of methacrylated gelatin, 2.5-10 g / L of a photoinitiator and 5-10 g / L of bioceramic nanofibers, wherein the dispersing agent is a culture medium or a phosphate buffer solution. The bio-ink is obtained by modifying GelMA with bioceramic nanofibers, the bio-ink has good bone induction capacity, can support the in-vitro and in-vivo growth and osteogenic differentiation of seed cells, and thus promotes the bone regeneration of a bone defect area; the bio-ink has a shear thinning performance, can protect cells and quickly form during a micro-extrusion printing process, and has a simple process and is easy to popularize.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical devices, in particular to a bio-ink and a preparation method and application thereof. BACKGROUND

[0002] Bone, as the support structure of the organism, has great significance for maintaining the appearance and normal function. Bone defects caused by tumors, trauma, infection, etc. bring psychological and physiological distress to patients, and are also a major clinical problem faced by surgeons. At present, the commonly used bone defect repair method in clinical practice is to use autologous bone, allogeneic bone, xenogeneic bone and artificial bone repair materials for transplantation repair. Autologous bone is considered the "gold standard" for bone repair due to its non-immunogenicity and good osteoinductive properties. However, the source of autologous bone is limited, and it will cause secondary trauma to the patient. Although allogeneic bone and xenogeneic bone have a wide source, they have problems such as immune rejection and infection, which limit their wide application. Although artificial bone repair materials have good biological safety, bone conduction and biodegradability, they are limited to the migration and differentiation of endogenous cells when repairing large bone defects. Therefore, bone tissue engineering as a new bone repair method has attracted great attention.

[0003] Conventional bone tissue engineering constructs scaffolds in vitro, and then seeds cells are inoculated onto the scaffolds, and after a certain period of in vitro culture, the scaffolds are implanted into the bone defect area in vivo. However, this method is limited by uncontrollable distribution of seed cells, limited number of cells, etc., and cannot meet the needs of bone tissue regeneration. At the same time, in clinical cases of bone defects, in order to preserve the patient's original bone tissue as much as possible, the bone defect area is usually irregular. Therefore, in order to match the bone graft with the bone defect area, 3D bioprinting of a repair body by bio-ink encapsulated seed cells has become a new development direction for bone defect repair. GelMA hydrogel is considered to be able to better simulate the extracellular matrix and provide a biomimetic microenvironment for bone tissue engineering due to its good biological safety and biocompatibility. However, pure GelMA hydrogel also has certain limitations, such as low mechanical strength and poor osteoinductive ability. Therefore, developing a bio-ink with biological activity by modifying GelMA hydrogel and applying it to bone regeneration is one of the key paths to solve this clinical problem. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a bio-ink and a preparation method and application thereof, which solve the problems of poor osteoinductive ability of pure methacrylated gelatin and low mechanical strength of the repair body obtained by bone regeneration in the prior art.

[0005] To achieve the above-mentioned objects and other related objects, the present application is obtained by including the following technical solutions.

[0006] The present application provides a kind of biological ink, the biological ink includes the following concentrations of components based on the total volume of dispersant: methyl methacrylate gelatin (GelMA) 50-150 g / L, photoinitiator 2.5-10 g / L, bioceramic nanofiber 5-10 g / L, and the dispersant is culture medium or phosphate buffer.

[0007] Preferably, the diameter of the bioceramic nanofiber is 10-30 nm, and the length is 0.1-2.0 μm.

[0008] Preferably, the bioceramic nanofiber is xonotlite or strontium-doped xonotlite.

[0009] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0010] The second object of the present application is to provide a method for preparing a biological ink, wherein methyl methacrylate gelatin, a photoinitiator and bioceramic nanofiber are uniformly dispersed in a dispersant to obtain the biological ink.

[0011] Preferably, the methyl methacrylate gelatin and the photoinitiator are first uniformly dispersed in the dispersant, and then the bioceramic nanofiber is added.

[0012] Preferably, the temperature of the system during the dispersion process is 35-40 °C.

[0013] The third object of the present application is to provide a use of the biological ink in the preparation of a drug or a medical device for repairing bone defects.

[0014] The fourth object of the present application is to provide a method for 3D bioprinting a bone regeneration repair body, wherein a three-dimensional model of the bone regeneration repair body is constructed, the biological ink is mixed with seed cells to serve as a printing raw material for 3D bioprinting, and a bone regeneration repair body is formed after photocuring.

[0015] Preferably, blue light with a wavelength of 365-405 nm is used for photocuring. More preferably, the photocuring time is 50-60 s.

[0016] Preferably, the seed cells are adult cells or stem cells.

[0017] Preferably, the amount of the seed cells added to the biological ink is 10 6 ~10 7 ml.

[0018] Preferably, the air pressure for 3D bioprinting is 0.1-0.2 Mpa.

[0019] Preferably, the speed for 3D bioprinting is 6-12 mm / s.

[0020] The fifth object of the present application is to provide a bone regeneration repair body obtained by the above method.

[0021] As described above, the bio-ink and the preparation method and application thereof have the following beneficial effects: the bio-ink is obtained by modifying GelMA with bioceramic nanofibers, has good bone induction capacity, can support the in-vitro and in-vivo growth and osteogenic differentiation of seed cells, and thus promotes the bone regeneration of the bone defect area; the bio-ink has shear thinning performance, can protect cells and quickly form during the micro-extrusion printing process, and is simple and easy to implement and promote. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A shear thinning performance diagram of the bio-ink prepared in Example 1 is shown.

[0023] Figure 2 A live and dead cell staining diagram of the bone regeneration repair body prepared in Example 1 is shown.

[0024] Figure 3 An alkaline phosphatase staining diagram of the bone marrow mesenchymal stem cells of the bone regeneration repair body prepared in Example 1 is shown.

[0025] Figure 4 Micro-CT diagrams of the blank group, the control group and the experimental group in the in-vivo osteogenesis experiment of the bone regeneration repair body prepared in Example 1 are shown.

[0026] Figure 5 A column diagram of the data obtained after quantitative analysis of the micro-CT diagrams of Figure 4 is shown. DETAILED DESCRIPTION

[0027] The embodiments of the present application are described below by way of specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the present specification. The present application can also be implemented or applied by different specific embodiments, and the details in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application.

[0028] It should be understood that the process equipment or device not specifically mentioned in the following examples all uses the conventional equipment or device in the art.

[0029] Furthermore, it should be understood that any steps of the methods mentioned in the present application do not exclude that other steps can be present before and / or after the mentioned steps or that other steps can be inserted between the mentioned steps, unless otherwise specified; it should also be understood that any combination of one or more devices / apparatuses mentioned in the present application does not exclude that other devices / apparatuses can be present before and / or after the mentioned devices / apparatuses or that other devices / apparatuses can be inserted between the mentioned two devices / apparatuses, unless otherwise specified. Moreover, the numbering of the steps of the methods is only a convenient tool to identify the steps of the methods and does not limit the arrangement order of the steps of the methods or define the scope of the present application, and the change or adjustment of the relative relationship thereof is also deemed as the scope of the present application, without substantial change of the technical content.

[0030] The embodiment of the present application provides a specific bio-ink, which comprises the following components in the following concentrations based on the total volume of the dispersant: 50-150 g / L of methacrylated gelatin (GelMA), 2.5-10 g / L of a photoinitiator, and 5-10 g / L of bioceramic nanofibers, wherein the dispersant is a culture medium or a phosphate buffer solution (PBS).

[0031] In a specific embodiment, each liter of sterile culture medium comprises the following components: 90-110 ml of fetal bovine serum, 10-20 ml of a penicillin-streptomycin solution, and 850-900 ml of minimum essential medium.

[0032] In a more specific embodiment, each liter of sterile culture medium comprises the following components: 100 ml of fetal bovine serum, 10 ml of a penicillin-streptomycin solution, and 890 ml of minimum essential medium (MEM). The minimum essential medium (MEM) is purchased from Gibco or Hyclone.

[0033] In a specific embodiment, the concentrations of the components in the phosphate buffer solution (PBS) are 30-40 g / L of sodium dihydrogen phosphate and 5.0-5.5 g / L of disodium hydrogen phosphate, and the solvent of the phosphate buffer solution is water.

[0034] In a more specific embodiment, the concentrations of the components in the phosphate buffer solution (PBS) are 38 g / L of sodium dihydrogen phosphate and 5.04 g / L of disodium hydrogen phosphate, and the solvent of the phosphate buffer solution is water.

[0035] In a specific embodiment, the bioceramic nanofibers have a diameter of 10-30 nm and a length of 0.1-2.0 μm.

[0036] In a specific embodiment, the bioceramic nanofibers are calcium silicate hydrate (CSH) or strontium-doped calcium silicate hydrate (Sr-CSH).

[0037] In one specific embodiment, the xonotlite is synthesized by hydrothermal method using calcium nitrate and sodium silicate as raw materials.

[0038] In a more specific embodiment, the molar ratio of the calcium nitrate and sodium silicate is (0.9-1.2):1, such as specifically 1:1.

[0039] In one specific embodiment, the strontium-doped xonotlite is synthesized by hydrothermal method using strontium nitrate, calcium nitrate and sodium silicate as raw materials.

[0040] In a more specific embodiment, the molar ratio of the strontium nitrate, calcium nitrate and sodium silicate is 1:(3-4):(4-5), such as specifically 1:4:5.

[0041] In one specific embodiment, the hydrothermal temperature is 180-200℃, and the hydrothermal treatment time is 24-48h.

[0042] In a more specific embodiment, the hydrothermal temperature is 200℃, and the hydrothermal treatment time is 24h.

[0043] In one specific embodiment, after the hydrothermal reaction, a post-treatment is further included, which comprises washing and drying.

[0044] In one specific embodiment, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). LAP is a blue light initiator, which is applied in bio-ink and can be photo-cured by blue light, and is safer than ultraviolet light curing, without damaging cells.

[0045] The embodiment of the present application further provides a specific preparation method of bio-ink, which uniformly disperses methacrylated gelatin, photoinitiator and bioceramic nanofiber in a dispersant to prepare the bio-ink.

[0046] In one specific embodiment, the methacrylated gelatin and photoinitiator are first uniformly dispersed in a dispersant, and then the bioceramic nanofiber is added.

[0047] In one specific embodiment, the system temperature during the dispersion process is 35-40℃, such as specifically 35℃, 37℃ or 40℃.

[0048] The embodiment of the present application further provides an application of the bio-ink in preparing a drug or medical device for repairing bone defects.

[0049] The embodiment of the present application also provides a method for 3D bioprinting bone regeneration repair body, a three-dimensional model of the bone regeneration repair body is constructed, and the biological ink is mixed with seed cells to be used as printing raw materials for 3D bioprinting, and the bone regeneration repair body is formed after photocuring. The obtained bone regeneration repair body is filled in a bone defect, and the bone membrane and skin are sutured layer by layer, so as to promote the regeneration of the bone defect. When the seed cells in the bone repair body differentiate into osteogenic cells under the stimulation of the biological ink, the bone matrix is secreted, so as to promote the regeneration of the bone defect.

[0050] In a specific embodiment, the 3D bioprinting is performed by using a micro-extrusion type bioprinter.

[0051] In a specific embodiment, the blue light with a wavelength of 365-405 nm is used for photocuring. The photocuring is performed by using the blue light, which is safer than ultraviolet photocuring and does not damage cells.

[0052] In a specific embodiment, the photocuring time is 50-60 s.

[0053] In a specific embodiment, the seed cells are adult cells or stem cells.

[0054] In a specific embodiment, the seed cells are added in the biological ink in an amount of 10 6 ~10 7 individuals / ml.

[0055] In a specific embodiment, the air pressure for 3D bioprinting is 0.1-0.2 Mpa.

[0056] In a specific embodiment, the speed for 3D bioprinting is 6-12 mm / s.

[0057] The embodiment of the present application also provides a bone regeneration repair body obtained by using the above method.

[0058] The following specific embodiments illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the present specification.

[0059] In each of the following embodiments of the present application, each liter of sterile culture medium comprises the following components: 100 ml fetal bovine serum, 10 ml penicillin-streptomycin solution and 890 ml minimum essential medium (MEM, purchased from Gibco Company).

[0060] The concentration of each component in the phosphate buffer (PBS) is: 38 g / L sodium dihydrogen phosphate and 5.04 g / L disodium hydrogen phosphate, and the solvent of the phosphate buffer is water.

[0061] In the following examples of the present application, the diameter of the xonotlite (CSH) is 10-30 nm and the length is 0.1-2 μm, which is prepared by the following method:

[0062] 1) 118.075 g of calcium nitrate tetrahydrate is dissolved in 1 L of deionized water to prepare a 0.5 M calcium nitrate solution;

[0063] 2) 142.1 g of sodium silicate nonahydrate is dissolved in 1 L of deionized water to obtain a 0.5 M sodium silicate solution;

[0064] 3) The calcium nitrate solution is slowly added to the sodium silicate solution under stirring at room temperature, and after the addition is completed, it is transferred to a stainless steel hydrothermal kettle lined with polytetrafluoroethylene, and is hydrothermally treated at 200 °C for 24 hours; the white precipitate is filtered and washed with deionized water and anhydrous ethanol three times, respectively. After drying in a vacuum drying oven at 180 °C for 24 hours, the white powder obtained is xonotlite (CSH).

[0065] In the following examples of the present application, the diameter of the strontium-doped xonotlite (Sr-CSH) is 10-30 nm and the length is 0.1-2 μm, which is prepared by the following method:

[0066] 1) 10.5815 g of strontium nitrate and 106.2675 g of calcium nitrate tetrahydrate are dissolved in 1 L of deionized water to obtain a 0.5 M mixed solution;

[0067] 2) 142.1 g of sodium silicate nonahydrate is dissolved in 1 L of deionized water to obtain a 0.5 M sodium silicate solution;

[0068] 3) The mixed solution is slowly added to the sodium silicate solution under stirring at room temperature, and after the addition is completed, it is transferred to a stainless steel hydrothermal kettle lined with polytetrafluoroethylene, and is hydrothermally treated at 200 °C for 24 hours; the white precipitate is filtered and washed with deionized water and anhydrous ethanol three times, respectively. After drying in a vacuum drying oven at 180 °C for 24 hours, the white powder obtained is strontium-doped xonotlite (Sr-CSH).

[0069] Example 1

[0070] In this example, a bio-ink is provided, which comprises the following components at the following concentrations, based on the total volume of the dispersant: 100 g / L of methacrylated gelatin, 5 g / L of a photoinitiator, and 5 g / L of bioceramic nanofibers, the dispersant being a sterile culture medium, the photoinitiator being lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and the bioceramic nanofibers being Sr-CSH.

[0071] In this example, a method for preparing a bio-ink is provided, which comprises the following steps:

[0072] 1) 1 g of GelMA and 0.05 g of photo initiator (LAP) were weighed into 10 ml of sterile medium and stirred magnetically at 37°C for 2 hours;

[0073] 2) 0.05 g of Sr-CSH was weighed into the above mixed solution and stirred magnetically at 37°C for 2 hours, and a biological ink was prepared after mixing uniformly.

[0074] A method for 3D bioprinting of bone regeneration repair body was also provided in the embodiment. A three-dimensional model of the bone regeneration repair body was constructed, and the above biological ink mixed with bone marrow mesenchymal stem cells (2 x 10 6 cells / mL) was used as a printing raw material for 3D bioprinting. The printing air pressure was 0.18 MPa, the printing speed was 8 mm / s, and the bone regeneration repair body was obtained after curing for 60 s under 405 nm blue light.

[0075] The biological ink prepared in the embodiment was placed in a cone-plate fixture by using a rheometer (Mars40, Thermo Fisher, USA), and experiments were performed in a shear rate range of 0.001-100 s -1 . The viscosity of the biological ink under different shear rates was detected. The results are shown in Figure 1 . The viscosity of the biological ink in the embodiment decreased with the increase of the shear rate, and had the performance of shear thinning, which could meet the requirements of protecting cells and rapid forming in the micro-extrusion printing process.

[0076] The activity of bone marrow mesenchymal stem cells in the bone regeneration repair body obtained in the embodiment was detected. Specifically, live and dead cell staining kits were used to stain the living cells and dead cells, and the observation was performed under a fluorescence microscope. The results are shown in Figure 2 . The BMSCs in the 3D bioprinted bone regeneration repair body in the embodiment showed good cell survival rate, which embodied the biological safety of the biological ink.

[0077] Pure GelMA hydrogel was used as a biological ink as a control group, and an alkaline phosphatase staining kit was used to stain the alkaline phosphatase of the cells in the bone regeneration repair body of the embodiment, and the observation was performed under a stereoscope. The results are shown in Figure 3 . The bone marrow mesenchymal stem cells in the bone regeneration repair body of the embodiment showed high alkaline phosphatase activity, which proved that they had osteogenic differentiation ability.

[0078] The bone regeneration repair body obtained in the embodiment was used for in vivo osteogenesis experiment, and pure GelMA hydrogel was used as a biological ink as a control group, and the blank group was not treated.

[0079] The model used in the in vivo osteogenesis experiment was as follows: adult SD rats, weighing 200-250 grams, male.

[0080] The rats are randomly divided into 3 groups, each group of 3, corresponding to a blank group, a control group and an experimental group

[0081] Two symmetrical defects with a diameter of 5 mm are prepared on the top of the rat skull, and the bone regeneration repair body is implanted into the bone defect. Pure GelMA hydrogel is used as a biological ink as a control group, and the blank group is not treated. Layered suture muscle layer and skin.

[0082] At 8 weeks, the new bone volume of each group is tested by micro-CT, and the experimental results are shown in Figure 4 and Figure 5 As shown, the experimental group using the biological ink of the present application to prepare the bone regeneration repair body has a higher new bone volume, while the new bone volume of the control group and the blank group is very limited, so the effect can be obviously seen. The bone regeneration repair body prepared by using the biological ink in the present application can significantly promote bone regeneration.

[0083] Example 2-5

[0084] Example 2-5 differs from Example 1 in that the formula of the biological ink is different, and for reference, see Table 1, and the rest of the process is exactly the same.

[0085] Table 1. Formula table of biological ink of Examples 1-5

[0086]

[0087] The performance of the biological ink and the bone regeneration repair body in Example 2-5 is comparable to that of Example 1, and will not be repeated here.

[0088] In summary, the present application obtains a biological ink by modifying GelMA with bioceramic nanofibers. The biological ink has good bone induction ability and can support the in vitro and in vivo growth and osteogenic differentiation of seed cells, thereby promoting bone regeneration in the bone defect area. The biological ink has shear thinning properties, can protect cells during micro-extrusion printing, and can be quickly formed. The process is simple and easy to implement and easy to promote. Therefore, the present application effectively overcomes the various shortcomings in the prior art and has a high industrial utilization value.

[0089] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A bio-ink, characterized in that, The bio-ink is composed of the following components at the following concentrations, based on the total volume of the dispersant: 50-150 g / L of methacrylated gelatin, 2.5-10 g / L of a photoinitiator, and 5-10 g / L of bioceramic nanofibers, the dispersant being a culture medium or a phosphate buffer; The bioceramic nanofibers have a diameter of 10-30 nm and a length of 0.1-2.0 μm; The bioceramic nanofibers are xonotlite or strontium-doped xonotlite; The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate; The strontium-doped xonotlite is synthesized by a hydrothermal method using strontium nitrate, calcium nitrate and sodium silicate as raw materials, and the molar ratio of strontium nitrate, calcium nitrate and sodium silicate is 1:(3-4):(4-5).

2. A method of preparing a bio-ink as claimed in claim 1, characterized in that: The methacrylated gelatin, the photoinitiator and the bioceramic nanofibers are uniformly dispersed in the dispersant to obtain the bio-ink.

3. The method of claim 2, wherein: The methacrylated gelatin and the photoinitiator are first uniformly dispersed in the dispersant, and then the bioceramic nanofibers are added. During the dispersion process, the temperature of the system is 35-40℃.

4. Use of the bio-ink of claim 1 in the preparation of a drug or a medical device for repairing bone defects.

5. A method of 3D bioprinting a bone regenerative implant, characterized by: The bio-ink of claim 1 is mixed with seed cells, and then 3D bioprinting is performed, and a bone regenerative repair body is formed after photocuring.

6. The method of claim 5, wherein: Blue light with a wavelength of 365-405 nm is used for photocuring. The seed cells are adult cells or stem cells. and / or the seed cells are added to the bio-ink in an amount of 10 6 ~ 10 7 cells / ml; The 3D bioprinting is performed at a gas pressure of 0.1-0.2 MPa. The 3D bioprinting is performed at a speed of 6-12 mm / s.

7. A bone regenerative repair body obtained by the method of claim 5 or 6.

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