CN115970054B - 3D printed porous bone scaffold loaded with silicon nitride and preparation method and application thereof - Google Patents

Abstract

The invention discloses a 3D printing porous bone scaffold loaded with silicon nitride, and a preparation method and application thereof, belonging to the technical field of prosthesis capable of being transplanted to bones or prosthesis materials. The biological ink aqueous dispersion is prepared by mixing gelatin aqueous solution with the concentration of 4-10% g/mL and silk fibroin aqueous solution with the concentration of 2-5% g/mL according to the volume ratio of 1:1 to form a composite solution, adding silicon nitride into the composite solution according to the mass-volume ratio of 1-8%g/mL of silicon nitride to the composite solution, and constructing the porous bracket by adopting a low-temperature forming 3D printing device and an organic combination physical crosslinking method. The invention has controllable porous structure, and can slowly release silicon ions to promote the differentiation of mesenchymal stem cells to osteoblasts, enhance the healing of bone defects and realize personalized bone defect treatment.

Description

3D printed porous bone scaffold loaded with silicon nitride and preparation method and application thereof

Technical Field

The present invention belongs to the technical field of prostheses that can be transplanted into bones, or prosthetic materials, and specifically relates to a 3D printed porous bone scaffold loaded with silicon nitride, and a preparation method and application thereof.

Background technique

The failure of bone tissue function is one of the main reasons for the decline in quality of life. The repair of large-sized bone defects has always been a clinical problem. The treatment strategies of autologous bone transplantation or allogeneic bone transplantation have largely limited their therapeutic effects due to the shortcomings of donor disease and limited donors. It is of great significance to develop ideal bone scaffold grafts. Silicon nitride, as a non-bioactive ceramic, has the advantage of promoting bone regeneration, and is widely used as a processing material in the preparation of non-degradable orthopedic medical devices such as spinal fusion devices and joint prostheses. However, it has not been found that it can be processed into nanoparticles as a bioactive substance that can release silicon ions and combined with natural materials such as silk fibroin and gelatin to form a degradable bioactive scaffold for the treatment of bone defect regeneration. Although the traditional scaffold preparation process has certain advantages in processing and molding, it is not suitable for irregular wounds due to the lack of adaptability to the defect size and cannot meet the clinical customization needs. In view of the widespread application of 3D printing technology in customized orthopedic medical products, by creatively preparing silicon nitride, silk fibroin and gelatin into 3D printing bio-ink, and optimizing the labor ratio and exploring the printing process, it is expected to obtain customized bone scaffolds that can release silicon ions to improve the repair effect of bone defects.

Summary of the invention

The present invention aims to solve the problems of the current 3D printed bone scaffolds, such as the difficulty in personalized preparation of irregular bone defects and insufficient biological activity, and provides a 3D printed porous bone scaffold loaded with silicon nitride and its preparation method and application.

The present invention is specifically achieved through the following technical solutions.

A 3D printed porous bone scaffold loaded with silicon nitride, wherein the bio-ink dispersion in the printer feeding tube is a composite solution formed by mixing a gelatin aqueous solution with a concentration of 4-10% g/mL and a silk fibroin aqueous solution with a concentration of 2-5% g/mL in a volume ratio of 1:1, and then silicon nitride is added to the composite solution at a mass volume ratio of 1-8% g/mL of silicon nitride to the composite solution, and the composite solution is prepared by low-temperature 3D printing equipment.

A preparation method of a 3D printed porous bone scaffold loaded with silicon nitride comprises the following steps: a gelatin aqueous solution with a concentration of 4-10% g/mL and a silk fibroin aqueous solution with a concentration of 2-5% g/mL are mixed in a volume ratio of 1:1 to form a composite solution, silicon nitride is then added to the composite solution at a mass volume ratio of 1-8% g/mL of silicon nitride to the composite solution to form a bio-ink, the bio-ink dispersion is prepared by stirring at 37°C, the obtained gel is pre-cooled, placed in a 3D printer, and printed layer by layer from bottom to top by extrusion under the pressure of a printing nozzle to form a multi-layer scaffold semi-finished product, which is then soaked in anhydrous ethanol, washed 5 times with 0.01 mol/L phosphate buffer, and freeze-dried to obtain a 3D printed porous bone scaffold.

In the preparation method, the precooling treatment temperature is 4°C and the time is 30 minutes.

In the preparation method, the temperature of the printing nozzle is set to 21°C, balanced for 5 minutes, and the temperature of the printer receiving platform is set to 4°C.

In the preparation method, the diameter of the printing nozzle is 0.4-0.8 mm, the pressure applied to the 3D printing nozzle is 60-500 kPa, and the filament spacing is 200-1000 μm.

The preparation method has 5 to 10 layers of printing layer by layer and a printing speed of 40 to 60 mm/s.

In the preparation method, the biological ink dispersion liquid must be uniform and free of bubbles.

Application of a 3D printed porous bone scaffold loaded with silicon nitride in the preparation of bone defect repair materials.

Compared with the prior art, the present invention has the following beneficial effects:

In view of the problem that traditional porous scaffolds are difficult to prepare in an individualized manner and have insufficient biological activity in irregular bone defects, the present invention adopts 3D printing technology to prepare bioactive porous scaffolds, which can release silicon ions, promote osteogenic differentiation of mesenchymal stem cells, and enhance the healing of bone defects, so as to achieve personalized bone defect treatment. The specific advantages are as follows:

(1) In terms of material selection, biodegradable natural silk fibroin and gelatin have good biocompatibility. Gelatin has the characteristics of low-temperature molding, which is conducive to low-temperature 3D printing of materials. Silk fibroin has good mechanical properties and can enhance the mechanical strength of gelatin.

(2) In terms of preparation technology, the use of 3D printing technology can achieve personalized preparation of the scaffold; the physical cross-linking method is used to solidify the scaffold, which has the characteristics of mild preparation conditions.

(3) In terms of product function, the prepared scaffold has a uniform and controllable pore structure and can release silicon ions, which promotes the differentiation of mesenchymal stem cells into osteoblasts, enhances the healing of bone defects, and is beneficial to the repair and regeneration of bone defects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an observation diagram of the macroscopic morphology A and the microscopic morphology B of the porous bone scaffold of the present invention;

FIG2 is a graph showing the silicon ion release behavior of the porous bone scaffold of the present invention;

FIG3 is a diagram showing the effect of the porous bone scaffold of the present invention on promoting osteogenic differentiation of rBMSCs;

FIG4 is a diagram showing the effect of printed porous bone scaffolds on promoting bone regeneration evaluated by hematoxylin & eosin staining;

Figure 5 is a diagram showing the effect of printed porous bone scaffolds in promoting bone regeneration evaluated by Masson's trichrome staining.

Detailed ways

In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available products unless otherwise specified.

The inventive concept of the present invention:

In order to solve the problems that traditional porous scaffolds are difficult to prepare in an individualized manner in irregular bone defects and have insufficient biological activity, the present invention uses silicon nitride as the bioactive substance, silk fibroin and gelatin as bio-ink, and constructs a porous scaffold through low-temperature 3D printing equipment combined with a physical cross-linking method.

The present invention is specifically described below by the following examples and data.

Example 1

A method for preparing a 3D printed porous bone scaffold loaded with silicon nitride comprises the following steps:

(1) Preparation of bio-ink: Prepare a gelatin aqueous solution with a concentration of 5% g/mL and a silk fibroin aqueous solution (SF solution) with a concentration of 2.5% g/mL, and mix the two in a volume ratio of 1:1 to form a composite solution; then, add silicon nitride to the composite solution at a mass volume ratio of 8% g/mL to form bio-ink. Place the ink in a 37°C water bath and stir thoroughly to evenly disperse the silicon nitride, then transfer it to the printer feed tube for later use.

(2) Preparation of 3D printed porous bone scaffolds loaded with silicon nitride: Place the ink cartridge into the print head, set the head temperature to 4°C to pre-cool the ink for 30 minutes, then set the head temperature to 21°C, and set the temperature of the print receiving platform to 4°C at the same time, and print after 5 minutes of balancing. During the printing process, print layer by layer from bottom to top, with 8 layers, a printing speed of 50 mm/s, and a printing needle diameter of 0.6 mm; the pressure applied to the 3D printing head is 100 kPa, and the filament spacing is 500 μm.

Example 2

A method for preparing a 3D printed porous bone scaffold loaded with silicon nitride comprises the following steps:

(1) Preparation of bio-ink: Prepare a gelatin aqueous solution with a concentration of 6% g/mL and a SF solution with a concentration of 3% g/mL, and mix the two in a volume ratio of 1:1 to form a composite solution; then, add silicon nitride to the composite solution at a mass volume ratio of 4% g/mL to the composite solution to form bio-ink. Place the ink in a 37°C water bath and stir thoroughly to evenly disperse the silicon nitride, then transfer it to the printer feed tube for later use.

(2) Preparation of 3D printed porous bone scaffolds loaded with silicon nitride: Place the ink cartridge into the print head, set the temperature of the print head to 4°C to pre-cool the ink for 30 minutes, then set the temperature of the print head to 21°C, and at the same time set the temperature of the print receiving platform to 4°C, and print after 5 minutes of balancing. During the printing process, print layer by layer from bottom to top, the number of printing layers is 10, the printing speed is 50mm/s, and the printing needle diameter is 0.6mm; the pressure applied to the 3D printing nozzle is 150kPa, and the filament spacing is 400μm.

Example 3

A method for preparing a 3D printed porous bone scaffold loaded with silicon nitride comprises the following steps:

(1) Preparation of bio-ink: Prepare a gelatin aqueous solution with a concentration of 8% g/mL and a SF solution with a concentration of 4% g/mL, and mix the two in a volume ratio of 1:1 to form a composite solution; then, add silicon nitride to the composite solution at a mass volume ratio of 1% g/mL to the composite solution to form bio-ink. Place the ink in a 37°C water bath and stir thoroughly to evenly disperse the silicon nitride, then transfer it to the printer feed tube for later use.

(2) Preparation of 3D printed porous bone scaffolds loaded with silicon nitride: Place the ink cartridge into the print head, set the head temperature to 4°C to pre-cool the ink for 30 minutes, then set the head temperature to 21°C, and set the temperature of the print receiving platform to 4°C at the same time, and print after 5 minutes of balancing. During the printing process, print layer by layer from bottom to top, the number of printing layers is 5, the printing speed is 60mm/s, and the printing needle diameter is 0.8mm; the pressure applied to the 3D printing nozzle is 200kPa, and the filament spacing is 800μm.

The performances of the stents prepared in the above-mentioned Examples 1 to 3 are similar, and the structure and performance of the stent are described below by taking Example 3 as an example.

Specifically, the performance test of the silicon nitride loaded 3D printed porous bone scaffold prepared in the above Example 3 was carried out as follows:

(1) The macroscopic morphology of the 3D printed porous bone scaffold loaded with silicon nitride was photographed, and the microstructure of the scaffold was observed using a scanning electron microscope, as shown in Figure 1. As can be seen from Figure 1, the scaffold has a reticular porous structure with pores running through it, and the pore size structure is 643.96±59.25 μm.

(2) Testing of stent silicon ion release performance:

350 mg of the scaffold was weighed and added to centrifuge tubes containing 20 mL of PBS. The centrifuge tubes were fixed to a vertical mixer for shaking. The entire device was placed at 37°C for experiment. 12 mL of release solution was taken out at specific time points (1st, 3rd, 5th, 7th, 10th, 14th, 21st and 28th days), and 12 mL of fresh PBS was added. The release solution was diluted and filtered with a 0.45 μm filter membrane. The Si ion concentration in the release solution was determined by ICP-OES instrument. The results are shown in Figure 2.

The results in Figure 2 show that the scaffold has the ability to release Si ions. The concentration of Si ions released by the scaffold on the first day was 8.90±0.18ppm, and on the 28th day, the concentration of released Si ions was 62.98±00.13ppm, indicating that the scaffold can release Si for up to 28 days, providing a guarantee for the scaffold's osteogenic bioactivity.

(3) Detection of scaffold osteogenic activity in vitro:

1 mL of 0.1% gelatin was added to each well of a 6-well cell culture plate, and the plate was shaken to cover the bottom of the plate. After standing for 30 min, the gelatin was discarded and dried for later use. Bone marrow mesenchymal stem cells (rBMSCs) from a large source were seeded into the 6-well plate at a density of 150,000/well. After culturing in normal culture medium for 24 hours, the culture medium was replaced with osteogenic induction culture medium, and the sterilized scaffold was added to co-culture with the cells. Fresh culture medium was replaced every three days. Alkaline phosphatase staining was performed after 7 days of induction, and Alizarin red staining was performed after 14 days to evaluate the osteogenic induction effect of the scaffold on rBMSCs. The results are shown in Figure 3.

The results in Figure 3 show that the scaffold group containing silicon nitride can have significant positive staining for alkaline phosphatase and alizarin red, indicating that the scaffold group containing silicon nitride can promote the production of alkaline phosphatase in the early stage of osteogenesis and the deposition of calcium matrix in the late stage of osteogenesis.

(4) Detection of scaffold-induced bone repair in vivo:

SPF male SD rats weighing 280-320g were used to establish a femoral defect of 3mm in diameter and 3mm in depth, and were divided into 3 groups, including a blank control group without any scaffold implanted, a scaffold group without silicon nitride added, and a scaffold group containing silicon nitride. Sodium penicillin was injected intramuscularly for 3 consecutive days after surgery to prevent infection. The samples were collected at 4 weeks and 8 weeks, and after fixation with 4% paraformaldehyde for 48 hours, decalcification was performed for 6 weeks. Subsequently, paraffin embedding and sectioning were performed, and the sections were stained with hematoxylin & eosin and Masson's trichrome to evaluate the bone defect repair. The results are shown in Figures 4 and 5.

The results in Figures 4 and 5 show that: 4 weeks after defect repair, the defect area of the scaffold group containing silicon nitride was reduced, and there was obvious collagen production; as the repair time increased, at the 8th week, the defect area of the scaffold group containing silicon nitride healed further, and the new bone was more complete and rich in collagen, indicating that the scaffold group containing silicon nitride can significantly promote the regeneration of bone defects.

Claims (8)

1. A porous bone scaffold of 3D printing of load silicon nitride, its characterized in that: the biological ink aqueous dispersion in the printer feeding tube is prepared by mixing gelatin aqueous solution with the concentration of 4-10% g/mL and silk fibroin aqueous solution with the concentration of 2-5% g/mL according to the volume ratio of 1:1 to form a composite solution, adding silicon nitride into the composite solution according to the mass volume ratio of 1-8%g/mL of silicon nitride to the composite solution, and preparing the biological ink aqueous dispersion by using low-temperature 3D printing equipment.

2.A preparation method of a 3D printing porous bone scaffold loaded with silicon nitride is characterized by comprising the following steps: mixing gelatin water solution with the concentration of 4-10% g/mL and silk fibroin water solution with the concentration of 2-5% g/mL according to the volume ratio of 1:1 to form a composite solution, adding silicon nitride into the composite solution according to the mass volume ratio of 1-8%g/mL of silicon nitride to form biological ink, stirring at 37 ℃ to prepare biological ink aqueous dispersion, pre-cooling, placing the obtained gel in a 3D printer, printing layer by layer under the pressure of a printing nozzle by extrusion, forming a multi-layer bracket semi-finished product, soaking by absolute ethyl alcohol, washing for 5 times by using 0.01mol/L of phosphoric acid buffer solution, and freeze-drying to obtain the 3D printing porous bone bracket.

3. The preparation method according to claim 2, characterized in that: the pre-cooling treatment is carried out at 4 ℃ for 30min.

4. The preparation method according to claim 2, characterized in that: the print head temperature was set at 21 ℃, equilibrated for 5min, and the printer receiving platform temperature was set at 4 ℃.

5. The preparation method according to claim 2, characterized in that: the diameter of the printing nozzle is 0.4-0.8mm, the pressure applied to the 3D printing nozzle is 60-500kPa, and the filament outlet distance is 200-1000 mu m.

6. A method of preparation according to claim 3, characterized in that: the number of the printing layers is 5-10, and the printing speed is 40-60mm/s.

7. The method of manufacture of claim 2, wherein; the aqueous bio-ink dispersion should be uniform and bubble free.

8. Use of a silicon nitride loaded 3D printed porous bone scaffold according to claim 1 for the preparation of bone defect repair materials.