3D printing degradable bone defect repair stent and preparation method thereof

Abstract

The invention provides a 3D printing degradable bone defect repair scaffold and a preparation method thereof, and belongs to the technical field of bone repair materials. The preparation method provided by the invention comprises the following steps: dissolving methylacryloylated gelatin (GelMA), sodium alginate, mesoporous bioactive glass and a photoinitiator in water to obtain 3D printing slurry; performing 3D printing on the slurry to obtain a bracket semi-finished product, and then performing cross-linking reaction on the bracket semi-finished product in visible light and a calcium chloride solution in sequence to obtain the 3D printing degradable bone defect repair bracket. The 3D printing degradable bone defect repair scaffold provided by the invention has good performance, can provide basic conditions for attachment, proliferation, differentiation and osteogenesis of cells, and realizes the process of bone regeneration and repair.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair materials, and in particular to a 3D-printed degradable bone defect repair scaffold and a preparation method thereof. Background Art

[0002] In recent years, the incidence of bone-related diseases has increased dramatically worldwide. According to the World Health Organization, approximately 2 million new bone defects are diagnosed worldwide each year. With the aging population and rising living standards, the demand for bone defect repair and treatment is increasing. Bone defect treatment is long and expensive. Long-term medical and surgical treatments impose significant physical and psychological strain on patients, while also placing a heavy burden on health systems worldwide. According to statistical analysis, bone transplantation is the second most common tissue transplantation procedure worldwide, second only to blood transfusions. Bone tissue possesses the ability to repair and regenerate itself. Small bone defects typically heal without additional treatment. However, when bone defects exceed the critical defect size (approximately >2 cm), are 2–3 times the diameter, or exceed 50% bone circumference loss, nonunion, malunion, or pathological fracture may result. Currently, the treatment of subcritical bone defects remains a challenge, with bone transplantation being the primary surgical option. Bone transplantation is widely used in clinical practice and can be categorized as autologous, allogeneic, and xenogeneic bone transplantation. Autologous bone transplantation has the dual effects of osteoinduction and osteoconduction and is the "gold standard" for bone repair. However, it is restricted in practical application due to limited donor tissue, chronic pain, secondary injury and infection. Allogeneic bone transplantation has no donor site complications, but is mainly limited by the risk of immune rejection and pathogen transmission. Xenogeneic bone transplantation is not well integrated and vascularized in the host area, and also has the potential risk of immune rejection and pathogen transmission. Although the occurrence of immune rejection can be reduced through treatment, studies have found that its mechanical strength and osteoinduction activity will be reduced accordingly. Therefore, it is of great significance to prepare a filling scaffold for the bone defect area through bone tissue engineering to achieve the process of bone regeneration and repair. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a 3D-printed degradable bone defect repair scaffold and a preparation method thereof. The preparation method provided by the present invention obtains a scaffold with a three-dimensional structure, which has good mechanical properties, biocompatibility, and osteogenesis properties, and is a more economical, safe and effective bone defect repair material.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] One of the technical solutions of the present invention is a method for preparing a 3D-printed degradable bone defect repair scaffold, comprising the following steps:

[0006] Methacryl-coated gelatin, sodium alginate, mesoporous bioactive glass and a photoinitiator are dissolved in water to obtain a 3D printing slurry;

[0007] The 3D printing slurry is 3D printed to obtain a scaffold semi-finished product, and then the scaffold semi-finished product is subjected to cross-linking reaction in visible light and calcium chloride solution in sequence to obtain the 3D printed degradable bone defect repair scaffold.

[0008] The second technical solution of the present invention is a 3D printed degradable bone defect repair scaffold prepared by the preparation method described in the above technical solution.

[0009] The present invention discloses the following technical effects:

[0010] The preparation method provided by the present invention can be carried out under mild conditions and does not require the use of a chemical cross-linking agent (such as glutaraldehyde), thereby avoiding the potential biosafety issues of the bone defect repair scaffold.

[0011] The 3D-printed degradable bone defect repair scaffold provided by the present invention has good performance and can provide basic conditions for cell attachment, proliferation, differentiation and osteogenesis, thereby realizing the process of bone regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 This is a photograph of the structure and dimensions of the bone defect repair scaffold obtained in Example 1;

[0014] Figure 2 This is a scanning electron microscope image of the bone defect repair scaffold obtained in Example 1;

[0015] Figure 3 The displacement-load curve of the bone defect repair scaffold obtained in Example 1;

[0016] Figure 4 The cck8 data of the bone defect repair scaffold at 1, 3, and 7 days in the effect verification example are shown;

[0017] Figure 5 This is the D3 live-dead staining image of the bone defect repair scaffold in the effect verification example;

[0018] Figure 6 This is the D7 live-dead staining image of the bone defect repair scaffold in the effect verification example;

[0019] Figure 7 The qRT-PCR results of D7 and D14 of the bone defect repair scaffold in the effect verification example, where (a) is D7 and (b) is D14;

[0020] Figure 8 This is the ALP staining image of D3 of the bone defect repair scaffold in the effect verification example;

[0021] Figure 9 This is the ALP staining image of the bone defect repair scaffold at D7 in the effect verification example;

[0022] Figure 10 This is the ALP quantitative result of the bone defect repair scaffold in the effect verification example. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] In scaffold-based bone tissue engineering, a scaffold with good performance is an important foundation for the success of bone tissue engineering, and it is required to provide basic conditions for cell attachment, proliferation, differentiation and osteogenesis. The bone tissue engineering scaffold materials currently studied mainly include metals, bioceramics, and polymers. Bioceramics such as bioactive glass are mechanically brittle when used alone and are usually used as substrates for polymer addition. In the preparation of bone tissue engineering scaffolds, natural polymers have attracted the attention of scholars because they have good biodegradability and the biofunctional molecules on the surface are conducive to cell integration and differentiation. However, they usually have poor mechanical properties and cannot provide sufficient mechanical support when used alone. Therefore, the present invention combines the composite scaffold formed by multiple materials to give full play to the advantages of the materials while overcoming the limitations of their application in certain aspects.

[0029] Specifically, the present invention provides a method for preparing a 3D-printed degradable bone defect repair scaffold, comprising the following steps:

[0030] Methacrylamide gelatin (GelMA), sodium alginate, mesoporous bioactive glass and photoinitiator were dissolved in water to obtain 3D printing slurry;

[0031] The 3D printing slurry is 3D printed to obtain a scaffold semi-finished product, and then the scaffold semi-finished product is subjected to cross-linking reaction in visible light and calcium chloride solution in sequence to obtain the 3D printed degradable bone defect repair scaffold.

[0032] In some embodiments of the present invention, the mass concentration of methacrylated gelatin in the 3D printing slurry is 10%; the mass ratio of methacrylated gelatin, sodium alginate, mesoporous bioactive glass, and photoinitiator is 10:6:10:0.25; and the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). The present invention has no particular requirements for the source of the methacrylated gelatin, which can be obtained from commercial sources or prepared using methods well known to those skilled in the art.

[0033] In some embodiments of the present invention, the pore size of the mesoporous bioactive glass is 2-50 nm.

[0034] The present invention introduces methacryloyl groups into gelatin, allowing the methacrylated gelatin to undergo photocrosslinking when exposed to a photoinitiator and ultraviolet or visible light. This polymerization can be performed under mild conditions, avoiding the potential biosafety concerns associated with crosslinking using chemical reagents. Furthermore, the gelatin retains its arginine-glycine-aspartic acid (RGD) sequence, which promotes cell adhesion, and its matrix metalloproteinase (MMP) sequence, which is suitable for cell remodeling. Furthermore, the present invention utilizes mesoporous bioactive glass with a pore size of 2-50 nm. Its high specific surface area allows it to form a hydroxyapatite layer upon contact with body fluids, enhancing its bone-forming capacity.

[0035] In the present invention, the content selection of methacrylated gelatin, sodium alginate, and mesoporous bioactive glass has a significant impact on the mechanical strength and osteogenic properties of the scaffold. Gelatin is a product obtained by moderate hydrolysis and thermal denaturation of collagen. It is a low-cost natural polymer biomaterial with good biocompatibility. However, due to its poor mechanical strength and rapid degradation rate, it is rarely used alone in its natural form in bioprinting. Its printability is usually improved by mixing it with other biomaterials. Methacrylated gelatin is obtained by combining methacrylate groups with amino groups of gelatin through a grafting reaction. It has the characteristics of both natural and synthetic biomaterials and has a three-dimensional structure suitable for cell growth and differentiation. Its increased content improves biocompatibility, but its degradation rate is rapid. Alginate is an anionic polysaccharide polymer that is widely used in bioprinting due to its good biocompatibility, low cost, and convenient cross-linking. It is also a bioinert material that cannot produce specific enzymes in mammals to degrade this polymer. Therefore, the addition of sodium alginate can prolong the degradation time of the scaffold. At the same time, the present invention has found that increasing sodium alginate can improve the printability of printing ink. Mesoporous bioactive glass is an inorganic bioactive component that can stably release calcium, phosphorus and other ions in the scaffold, activate osteogenesis-related signaling pathways, promote osteoblast differentiation, and enhance osteogenic induction. Experimental results show that only the scaffold obtained under the specific ratio of the specific raw materials of the present invention has the highest osteogenic activity. Therefore, the concentration selection of the components mentioned in the present invention is easier to mix evenly, is more suitable for 3D printing, and has better osteogenic performance.

[0036] In some embodiments of the present invention, the 3D printing parameters are set as follows: using a 0.42 mm aperture needle, printing at 0.30 MPa air pressure, 10-15° C., and a printing speed of 15 mm / s.

[0037] The printing parameters adjusted by the present invention match the 3D printing slurry prepared by the present invention to form a uniform porous network structure, thereby ensuring the structural strength of the bracket, thereby increasing its elastic modulus and providing good mechanical support.

[0038] In some embodiments of the present invention, the wavelength of the visible light is 405 nm; and the time for the cross-linking reaction under the visible light condition is 1 min.

[0039] In some embodiments of the present invention, the concentration of the calcium chloride solution is 9 wt% to 11 wt%, and the time for the cross-linking reaction in the calcium chloride solution is 8 to 15 minutes, preferably 10 minutes.

[0040] In the present invention, when the calcium ion concentration is too low, the exchange between sodium alginate and calcium ions is incomplete, and the formed gel is weak and has low strength. In order to ensure the strength of the gel, the concentration of the calcium chloride solution is preferably the above parameters.

[0041] In some embodiments of the present invention, after the cross-linking reaction is completed, the steps of washing and drying are further included; the drying method is preferably freeze-drying.

[0042] The present invention also provides a 3D printed degradable bone defect repair scaffold prepared by the above preparation method.

[0043] The scaffold prepared by the present invention is a multi-layer, mutually cross-linked porous mesh structure; the first layer includes multiple parallel lines, the second layer is multiple parallel lines perpendicularly connected to the upper surface of the first layer of lines, the third layer is multiple parallel lines perpendicularly connected to the upper surface of the second layer of lines, and so on; the diameter of each layer of lines in the 3D printed bone defect repair scaffold is 300-500μm, and the distance between adjacent lines is 700-1000μm.

[0044] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0045] Unless otherwise specified, the raw materials and reagents used in the examples can be obtained through commercial channels.

[0046] Example 1

[0047] Step 1. Dissolve 1 g of methacrylated gelatin, 0.6 g of sodium alginate, 1 g of mesoporous bioactive glass, and 0.025 g of photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) in 10 mL of water and stir to obtain a 3D printing slurry.

[0048] Step 2. The obtained slurry is 3D printed using a 0.42 mm aperture needle, and printing is performed at a printing speed of 15 mm / s under 0.30 MPa air pressure and 10°C to obtain a semi-finished scaffold. The semi-finished scaffold is then photocrosslinked under 405 nm visible light for 1 minute, and then placed in a 10 wt% calcium chloride solution for chemical crosslinking for 10 minutes. Finally, it is washed and freeze-dried to obtain the 3D printed degradable bone defect repair scaffold.

[0049] Effect verification example

[0050] The performance test of the bone defect repair scaffold obtained in Example 1 was performed as follows:

[0051] (1) Take photos and 3D print the structural dimensions of the bone defect repair scaffold. Figure 1 , it can be seen that the scaffold is a uniform multi-layer cross-linked porous network structure.

[0052] (2) Observation of the surface structure of the scaffold: The 3D printed bone defect repair scaffold was freeze-dried and then sprayed with gold, and the surface structure of the scaffold was observed under a scanning electron microscope. Figure 2 As shown, it can be seen that the surface of the scaffold is loose and porous, and the mesoporous bioactive glass is evenly distributed.

[0053] (3) Mechanical property test: Soak the 3D printed bone defect repair scaffold in PBS, take it out after 24 hours, dry it with filter paper, place it in a universal mechanical tester, load it with 10kN pressure, and draw the stress-displacement curve. Figure 3 As shown, it can be seen that the average Young's modulus of the 3D printed bone defect repair scaffold is 210-220 MPa.

[0054] (4) Cell proliferation performance on composite scaffolds: Bone marrow mesenchymal stem cells (BMSCs) were cultured at a rate of 1×10 5 Cells were seeded on the scaffold at a density of 100 cells / well and cultured in low-glucose DMEM containing 10% FBS, with the medium changed every two days. The cell count was determined using a cell counting kit on days 1, 3, and 7. Figure 4 The results showed that after co-culturing the scaffold with BMSCs, the cell viability of the scaffold group exceeded that of the control group on days 1, 3, and 7, confirming that the scaffold had good cell compatibility.

[0055] (5) Live and dead staining of cells on composite scaffolds: Bone marrow mesenchymal stem cells (BMSCs) were plated at 1×10 5 Cells were seeded on the scaffold at a density of 100 cells / well and cultured in low-glucose DMEM containing 10% FBS. The medium was changed every two days. The scaffold was removed on the 3rd and 7th days, rinsed three times with phosphate buffer, and then stained for live and dead cells. Observation was performed using an inverted fluorescence microscope. Figure 5 、6 It can be seen that 3 days after cell inoculation, the scaffold showed green fluorescence and exhibited a BMSC spindle-shaped morphology, indicating that the scaffold has good cell compatibility; as the culture time increases to 7 days, the cells rapidly proliferate, spread and migrate on the scaffold.

[0056] (6) qRT-PCR detection of osteogenesis-related gene expression: Bone marrow mesenchymal stem cells (BMSCs) were cultured at a rate of 1×10 5 The cells were seeded on the scaffold at a density of 100 cells / well and cultured in low-glucose DMEM containing 10% FBS. The medium was changed every 3 days. Total cell RNA was extracted on the 7th and 14th days for PCR detection. Figure 7 It can be seen that the expression levels of ALP and OCN in the scaffold group were higher than those in the control group on days 7 and 14.

[0057] (7) Alkaline phosphatase staining and quantitative analysis: Bone marrow mesenchymal stem cells (BMSCs) were cultured at a concentration of 1×10 5 BMSCs were seeded on the scaffold at a density of 100 cells / well and cultured in osteogenic induction medium. Alkaline phosphatase staining was performed on the 3rd and 7th days respectively. The alkaline phosphatase (ALP) activity of BMSCs cultured on the scaffold was measured using an alkaline phosphatase detection kit on the 3rd and 7th days after osteogenic induction. Figure 8 、 9 , 10It can be seen that the ALP activity of the scaffold group was higher than that of the control group on days 3 and 7.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a 3D printed degradable bone defect repair scaffold, It is characterized in that The following steps are involved: Dissolving methacrylated gelatin, sodium alginate, mesoporous bioactive glass and a photoinitiator in water to obtain a 3D printing slurry; The 3D printing slurry is 3D printed to obtain a scaffold semi-finished product, and then the scaffold semi-finished product is subjected to cross-linking reaction in visible light and calcium chloride solution in sequence to obtain the 3D printed degradable bone defect repair scaffold.

2. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that The mass concentration of methacryloyl gelatin in the 3D printing slurry is 10%; the mass ratio of methacryloyl gelatin, sodium alginate, mesoporous bioactive glass and photoinitiator is 10:6:10:0.25; and the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

3. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that The pore size of the mesoporous bioactive glass is 2-50 nm.

4. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that The parameters of the 3D printing are set as follows: using a 0.42 mm aperture needle, printing at a pressure of 0.30 MPa, 10-15° C., and a printing speed of 15 mm / s.

5. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that The wavelength of the visible light is 405 nm; the time for the cross-linking reaction under the visible light condition is 1 min.

6. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that The concentration of the calcium chloride solution is 9 wt% to 11 wt%, and the time for the cross-linking reaction in the calcium chloride solution is 8 to 15 minutes.

7. The method for preparing the 3D printed degradable bone defect repair scaffold according to claim 1, It is characterized in that After the cross-linking reaction is completed, the steps of washing and drying are also included.

8. A 3D printed degradable bone defect repair scaffold prepared by the preparation method according to any one of claims 1 to 7.

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