CN109010924A - The preparation method of BMP2-PLA/HAP composite material bone repairing support - Google Patents

Abstract

The invention discloses a preparation method of a BMP2-PLA/HAP composite material bone repair bracket. The method first mixes nano-scale hydroxyapatite powder with a mass ratio of 10% to 20%:1 and medical-grade PLA plastic powder, and after melting, extrudes and draws to obtain PLA/HAP consumables for 3D printing, and then sets 3D printing parameters, control the temperature of the printing nozzle to 200-220°C, the temperature of the printing base to 20-50°C, the printing speed to 20-40mm/s, the cooling fan speed to 2000-2500rpm, import the 3D model of the bone repair bracket and start printing. The PLA/HAP composite bone repair scaffold is obtained, and finally the scaffold is soaked in 0.25-2 mg/mL BMP2 chitosan acetate solution to obtain the BMP2‑PLA/HAP composite bone repair scaffold. The invention adopts 3D printing technology, uses nano-scale hydroxyapatite powder and medical-grade PLA plastic powder as raw materials, loads BMP2 bone growth factor with high biological induction, and prepares hydroxyapatite/PLA composite material with high Strength, high hardness and impact strength, and has good antibacterial properties, good biocompatibility and osteoinductivity.

Description

Preparation method of BMP2-PLA/HAP composite bone repair scaffold

technical field

The invention belongs to the technical field of medical orthopedic materials, and relates to a preparation method of a BMP2-PLA/HAP composite bone repair support, in particular to a preparation method of a BMP2-PLA/HAP composite bone repair support based on 3D printing technology.

Background technique

Hydroxyapatite (Hydroxyapatite, HAP) (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) is the main inorganic component of human and animal bones, and has good biological activity and osteoconductivity. After HAP is implanted into the human body, Ca ²⁺ and P ³⁺ will dissociate from the surface of HAP, thereby being absorbed by body tissues and growing new tissues. The structure of human bone apatite is about 40-60nm in length and 20nm in width, and the main basic unit is needle-like or rod-like apatite crystals.

Polylactic acid (PLA) plastic is synthesized from lactic acid as a monomer, and has good mechanical properties and biocompatibility. PLA has excellent impact strength, good mechanical strength, hardness and certain wear resistance. At the same time, it has good bioabsorbability, can be degraded into lactic acid monomers in the body, and can be further metabolized into carbon dioxide and water with the lactic acid cycle. It is an ideal material for preparing bone repair scaffolds.

Bone morphogenetic protein 2 (BMP2) is a potent inducer of osteogenesis in bone marrow mesenchymal stem cells (MSCs). The routine application of BMP2 involves direct incorporation of BMP2 protein or ex vivo BMP2 gene transfer into stem cells before their transplantation (Xue J, Lin H, Bean A, et al. One-Step Fabrication of Bone Morphogenetic Protein-2 Gene-Activated Porous Poly-L-Lactide Scaffold for Bone Induction. [J]. Molecular Therapy Methods & Clinical Development, 2017, 7(1):50-59.). In promoting the repair of bone defects, its effect is almost similar to that of autologous bone. Under certain conditions, it can induce the transformation of undifferentiated mesenchymal cells into bone-derived cells, and promote the growth and proliferation of bone cells (Han L, Wang M , Sun H, et al. Porous titanium scaffolds with self-assembled micro/nano hierarchical structure for dual functions of bone regeneration and anti-infection. [J]. Journal of Biomedical Materials Research Part A, 2017: 1-14.).

3D printing, also known as additive manufacturing (AM), rapid prototyping (RP), is the process of layering and printing three-dimensional model layers by computer. 3D printing has many advantages: ① save materials, improve material utilization and reduce costs; ② can achieve high precision and high complexity, and can manufacture very complex parts that cannot be manufactured using traditional methods; ③ does not require traditional tools, fixtures, Machine tools or any tool can generate real-world products directly from any 3D CAD graphics; ④ can automatically, quickly, directly and accurately convert the computer's 3D design into a physical model, and even directly manufacture parts or molds, thus effectively shortening product development cycle; ⑤ 3D printing does not require centralized, fixed manufacturing workshops and distributed production; ⑥ can print assembled products, thereby reducing assembly costs, and even challenge large-scale production methods.

Contents of the invention

The purpose of the present invention is to provide a preparation method of BMP2-PLA/HAP composite material bone repair scaffold. Based on 3D printing technology, the method uses HAP and PLA as raw materials and loads BMP2 to prepare a bone repair scaffold with excellent mechanical properties and good biocompatibility.

The technical scheme that realizes the object of the present invention is as follows:

The preparation method of BMP2-PLA/HAP composite material bone repair support comprises the following steps:

Mix nano-scale hydroxyapatite powder with a mass ratio of 10% to 20%: 1 and medical-grade PLA plastic powder. After melting, extrude and draw to obtain PLA/HAP consumables for 3D printing, and then set 3D printing parameters. Control the temperature of the printing nozzle to 200-220°C, the temperature of the printing base to 20-50°C, the printing speed to 20-40mm/s, the cooling fan speed to 2000-2500rpm, import the 3D model of the bone repair bracket and start printing to obtain PLA/HAP The composite bone repair scaffold is soaked in 0.25-2 mg/mL BMP2 chitosan acetate solution to obtain the BMP2-PLA/HAP composite bone repair scaffold.

Preferably, both the nanoscale hydroxyapatite powder and the medical grade PLA plastic powder pass through a 200-mesh sieve.

Preferably, the melting temperature is 200-220°C.

Preferably, the temperature of the printing nozzle is controlled at 210°C, the temperature of the printing base is controlled at 30°C, the printing speed is controlled at 30mm/s, and the cooling fan speed is controlled at 2000rpm.

Preferably, the nanoscale hydroxyapatite powder is prepared by co-precipitation.

In the chitosan-acetic acid solution of BMP2, the BMP2 concentration is 1 mg/mL, the acetic acid concentration is 0.02 g/mL, and the chitosan is 0.01 mg/mL.

Compared with the prior art, the present invention has the following advantages:

The invention adopts 3D printing technology, uses nano-scale hydroxyapatite powder and medical-grade PLA plastic powder as raw materials, and loads BMP2 to prepare the BMP2-PLA/HA composite bone repair material, which has high strength, high hardness and impact resistance. And it has good antibacterial properties and good biocompatibility. The bone repair bracket prepared by the invention can be degraded in the human body, greatly reduces the probability of inflammation at the bone defect, and is a good implant material. Due to the high impact strength and hardness of the material itself, it meets a certain supporting performance of the bone defect in the human body. Due to the osteoinductive property of the loaded BMP2 material, the self-repair effect of the human body is improved.

Description of drawings

Fig. 1 is the XRD diffraction pattern of the hydroxyapatite prepared by the co-precipitation method in Example 1.

FIG. 2 is a transmission electron micrograph of hydroxyapatite prepared by co-precipitation method in Example 1. FIG.

FIG. 3 is a scanning electron microscope picture of hydroxyapatite prepared by co-precipitation method in Example 1.

Fig. 4 is the infrared spectrogram of the hydroxyapatite prepared by the co-precipitation method in Example 1.

FIG. 5 is a Raman spectrum of hydroxyapatite prepared by the co-precipitation method in Example 1. FIG.

FIG. 6 is a physical picture of the PLA/HAP consumables with a mass ratio of 10% prepared in Example 1.

Fig. 7 is a sample diagram of a BMP-PLA/HAP composite bone repair scaffold.

Figure 8 shows the dumbbell-shaped test and rectangular test samples printed by PLA/HAP composite materials in the mechanical strength test.

Fig. 9 is a graph showing the change trend of impact strength of PLA/HAP composites with different mass ratios.

Fig. 10 is a graph showing the change trend of fracture strength of PLA/HAP composites with different mass ratios.

Fig. 11 is a graph showing the change trend of elongation at break of PLA/HAP composites with different mass ratios.

Fig. 12 is a graph showing the changing trend of elastic modulus of PLA/HAP composites with different mass ratios.

Fig. 13 is a graph of X-ray data after one month of animal experiments implanted with 10% PLA/HAP composite material by mass ratio.

Fig. 14 is a diagram of X-ray data after 2 months of animal experiments implanted with 10% PLA/HAP composite material by mass ratio.

Fig. 15 is a graph showing the growth of bone cells on the surface of PLA/HAP composite material with a mass ratio of 10% 3 days after cell inoculation.

Fig. 16 is a graph showing the growth of bone cells on the surface of PLA/HAP composite material with a mass ratio of 10% 7 days after cell inoculation.

Fig. 17 is a graph showing the inhibition effect of the PLA/HAP composite material with a mass ratio of 10% soaked in 5g/L vancomycin solution on the E. coli plate.

Fig. 18 is a graph showing the inhibition effect of the PLA/HAP composite material with a mass ratio of 10% soaked in a 5g/L vancomycin solution on a Staphylococcus aureus plate.

Fig. 19 is a graph showing the inhibition effect of PLA/HAP composite material with a mass ratio of 10% soaked in 5 g/L levofloxacin ethanol solution on the E. coli plate.

Fig. 20 is a graph showing the inhibition effect on the Staphylococcus aureus plate after soaking PLA/HAP composite material with a mass ratio of 10% in 5g/L levofloxacin ethanol solution.

Fig. 21 is a scanning electron micrograph of a PLA/HAP composite material with a mass ratio of 10%.

Fig. 22 is a scanning electron micrograph of a PLA/HAP composite material with a mass ratio of 10% soaked in SBF solution for 1 day.

Fig. 23 is a scanning electron micrograph of a PLA/HAP composite material with a mass ratio of 10% soaked in SBF solution for 3 days.

Fig. 24 is the energy spectrum of the PLA/HAP composite material with a mass ratio of 10% soaked in SBF solution for 3 days.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings.

The medical-grade PLA plastic used in the following examples is an imported medical-grade material purchased from Nature Works and certified by the FDA.

Embodiment 1 (the PLA/HAP composite material bone repair support that mass ratio is 10%: 1)

Preparation of hydroxyapatite by co-precipitation method: 185.4g of calcium chloride and 380g of sodium phosphate dodecahydrate were dissolved in 1L of water respectively. Under the condition of 90°C water bath and stirring, add the sodium phosphate solution to the calcium chloride solution, adjust the pH to 9-11 with sodium hydroxide solution and hydrochloric acid, and keep stirring for 4 hours. Stand still and age for 12 hours. After aging, wash to remove the by-product sodium chloride, and dry to obtain nano-sized hydroxyapatite.

The hydroxyapatite and PLA plastics were pulverized into 200 mesh fine powder. Mix the fine powder of hydroxyapatite and the fine powder of PLA with a mass ratio of 10%:1, add to an internal mixer and mix for 10 minutes at 200°C. Put the mixed material into the extruder for wire drawing, control the melting temperature to 200-230°C, and the mold temperature to 105°C to form consumables that can be used for 3D printing.

Load the PLA/HAP consumables into the FDM 3D printer, and adjust the parameters so that the temperature of the print head during the printing of the bone repair scaffold should be controlled at 200-220°C; the temperature of the printing plate should be controlled at 20-50°C; the printing speed should be controlled at 20-40mm/s; the cooling fan speed should be controlled at 2000rpm, import the 3D model of the bone repair bracket and start printing. After printing, soak the scaffold in 0.25-2 mg/mL chitosan acetate solution of BMP2 for 2 hours. Obtain BMP2-PLA/HAP composite bone repair scaffold.

Embodiment 2 (the PLA/HAP composite material bone repair support that mass ratio is 20%: 1)

This example is basically the same as Example 1, the only difference is that the mass ratio of hydroxyapatite fine powder to PLA fine powder is 20%:1.

Comparative example 1

This comparative example is basically the same as Example 1, the only difference is that the mass ratio of hydroxyapatite fine powder to PLA fine powder is 5%:1.

Fig. 1 is the XRD diffraction pattern of the hydroxyapatite prepared by the co-precipitation method in Example 1. It can be seen from the figure that almost all peaks in the XRD spectrum are consistent with the standard HA (JCPDS No.09-0432), and the diffraction peaks of the HA sample are the same as those of pure HA with 2θ values of 26.1, 32.1, 33.0, and 40.1 The consistent (002), (211), (300), (310), (222), (213) and (004) planes correspond to 47.0, 49.7 and 53.4, respectively. Strong peaks around 2Θ=26 and 2Θ=33 prove that these samples are mainly HA. FIG. 2 is a transmission electron micrograph of the hydroxyapatite prepared by the co-precipitation method in Example 1, and FIG. 3 is a scanning electron micrograph of the hydroxyapatite prepared by the co-precipitation method in Example 1. It can be seen from the figure that hydroxyapatite presents a rod-like structure with an average length of 52-58nm. Fig. 4 is the FTIR spectrogram of the hydroxyapatite prepared by the co-precipitation method in Example 1. The absorption peaks at 1089, 1024 and 962 cm can be attributed to the υ1 and ^υ3 phosphate modes. Absorption peaks at 560 and 600 cm were attributed to the ^υ4 phosphate mode. The antisymmetric stretching vibrations of CO(υ3) in the range of 1500-1400cm ^-1 and υ2 vibration of CO ₃ ^2- at 875cm ^-1 indicate that natural HA contains CO ₃ ^2- . FIG. 5 is a Raman spectrum of hydroxyapatite prepared by the co-precipitation method in Example 1. FIG. In the figure, the characteristic peaks at 420 and 578cm ^-1 can be attributed to υ2 and υ4 modes, respectively. There are some peaks around 1037cm ^-1 corresponding to the asymmetric stretching vibration υ3. The strongest symmetrical stretching υ1 mode of PO ₄ ^3- ion in HA is at 956cm ^-1 , and OH stretching is at 3567cm ^-1 . The sample has low OH peaks, matching the FTIR results.

Fig. 6 is a physical picture of the BMP-PLA/HAP composite material consumable with a mass ratio of 20% obtained in Example 1. It can be seen from the figure that the BMP-PLA/HAP material has a white color similar to natural bone and has a good gloss effect.

Figure 7 is a sample diagram of the BMP-PLA/HAP composite bone repair scaffold. It can be seen that there are gaps in the bone repair scaffold with a pore size of about 300 microns, which conforms to the characteristics of bone regeneration and has a good bone regeneration effect.

Fig. 8 is a dumbbell-shaped test sample and a rectangular test sample with BMP-PLA/HAP mass ratios of 5%, 10%, and 20% in the mechanical strength test. It can be seen from the figure that PLA/HAP materials with different mass ratios are put into the 3D printer and printed into dumbbell-shaped and cuboid-shaped splines. Dumbbell-shaped splines are used for mechanical tensile tests, and cuboid-shaped splines are used for mechanical impact tests.

Figure 9 is a trend diagram of the impact strength of BMP-PLA/HAP composites with different mass ratios. As the mass ratio of hydroxyapatite increases, the overall impact strength of the composite decreases. Fig. 10 is a trend diagram of the fracture strength of BMP-PLA/HAP composites with different mass ratios. As the mass ratio of hydroxyapatite increases, the overall fracture strength of the composite decreases. Figure 11 is a graph showing the change trend of the elongation at break of BMP-PLA/HAP composites with different mass ratios. As the mass ratio of hydroxyapatite increases, the overall elongation at break of the composite decreases. Figure 12 is a trend diagram of the elastic modulus of BMP-PLA/HAP composites with different mass ratios. As the mass ratio of hydroxyapatite increases, the overall elastic modulus of the composite increases.

Table 1 shows the blood cell detection data of experimental rabbits 1 month after implantation of BMP-PLA/HAP bone nails. The inflammatory response is judged by indicators such as lymphocytes and neutrophils. The biocompatibility of bone nails needs to analyze the composition of white blood cells. The reason for the slightly high white blood cell indicators in Table 1 may be the increase in postoperative stress, which is a normal phenomenon .

Table 1 The blood cell detection data of experimental rabbits implanted with BMP-PLA/HAP bone nail 1 month later

Table 2 The blood cell detection data of experimental rabbits implanted with BMP-PLA/HAP bone nails 2 months later

Table 2 shows the blood cell detection data of experimental rabbits 2 months after implantation of BMP-PLA/HAP bone nails. The data showed that all the experimental rabbits used were normal.

Figure 13 and Figure 14 are the X-ray data graphs after the animal experiment was carried out for 1 month and 2 months respectively. It can be seen from the figure that there is an obvious bone defect in the femur of the experimental rabbit, the fusion of the bone nail and the leg bone is good, and there are some fine bone hyperplasia on the leg bone, indicating that the PLA/HAP bone nail can promote the proliferation of bone cells and fusion effects.

Figure 15 is a diagram of the growth of osteoblasts on the surface of the BMP-PLA/HAP composite material after 3 days of cell inoculation, indicating that the BMP-PLA/HAP material is used as a repair scaffold to penetrate into the human bone defect, and it has a tendency to fuse with human bone, and The fusion works well. Figure 16 is a diagram of the growth of bone cells on the surface of the BMP-PLA/HAP composite material 7 days after cell inoculation, indicating that the BMP-PLA/HAP material is used as a repair scaffold to penetrate into human bone defects and can be well integrated with human bone.

Fig. 17 is a graph showing the inhibition effect of the BMP-PLA/HAP composite material with a mass ratio of 10% soaked in a 5g/L vancomycin solution on an E. coli plate. Fig. 18 is a graph showing the inhibition effect of the BMP-PLA/HAP composite material with a mass ratio of 10% soaked in a 5g/L vancomycin solution on a Staphylococcus aureus plate. Fig. 19 is a graph showing the inhibition effect on the E. coli plate after soaking the BMP-PLA/HAP composite material with a mass ratio of 10% in 5g/L levofloxacin ethanol solution. Fig. 20 is a graph showing the inhibition effect on the Staphylococcus aureus plate after soaking the BMP-PLA/HAP composite material with a mass ratio of 10% in 5g/L levofloxacin ethanol solution. It can be seen from the inhibition effect diagram that the soaked PLA/HAP composite material has obvious inhibitory effect on Escherichia coli and Staphylococcus aureus, and has good antibacterial properties.

Figures 21, 22, and 23 are the comparison charts of the BMP-PLA/HAP composite material with a mass ratio of 10% soaked in FBS for 0 day, 1 day, and 3 days. It can be seen that as the soaking time prolongs, the surface of the material emerges rough material.

Figure 24 is the energy spectrum of the BMP-PLA/HAP composite material soaked in FBS for 3 days. It can be seen that the rough material on the surface of the material is a CaP analogue.

In summary, the BMP2-PLA/HA composite material of the present invention has excellent mechanical properties and biocompatibility, can adapt to the complex body fluid environment inside the human body, and can resist strong external impact. Both vancomycin and levofloxacin endow BMP2-PLA/HA composites with antibacterial properties, but the antibacterial performance of levofloxacin tablets is significantly better than that of vancomycin, which means that BMP2-PLA/HA composites are also more efficient in absorbing and retaining fat-soluble antibiotics . After animal experiments, the BMP2-PLA/HA composite material bone repair scaffold prepared by the present invention has good fusion with bone and excellent supporting effect.

Claims (6)

1. The preparation method of BMP2-PLA/HAP composite material bone repair support, is characterized in that, comprises the following steps: Mix nano-scale hydroxyapatite powder with a mass ratio of 10% to 20%: 1 and medical-grade PLA plastic powder. After melting, extrude and draw to obtain PLA/HAP consumables for 3D printing, and then set 3D printing parameters. Control the temperature of the printing nozzle to 200-220°C, the temperature of the printing base to 20-50°C, the printing speed to 20-40mm/s, the cooling fan speed to 2000-2500rpm, import the 3D model of the bone repair bracket and start printing to obtain PLA/HAP The composite bone repair scaffold is soaked in 0.25-2 mg/mL BMP2 chitosan acetate solution to obtain the BMP2-PLA/HAP composite bone repair scaffold. 2. The preparation method according to claim 1, characterized in that, both the nanoscale hydroxyapatite powder and the medical grade PLA plastic powder pass through a 200-mesh sieve. 3. The preparation method according to claim 1, characterized in that, the melting temperature is 200-220°C. 4. The preparation method according to claim 1, characterized in that, the temperature of the printing nozzle is controlled at 210°C, the temperature of the printing base is controlled at 30°C, the printing speed is controlled at 30mm/s, and the cooling fan speed is controlled at 2000rpm. 5. The preparation method according to claim 1, characterized in that, the nano-scale hydroxyapatite powder is prepared by co-precipitation. 6. preparation method according to claim 1 is characterized in that, in the chitosan acetic acid solution of described BMP2, BMP2 concentration is 1mg/mL, and acetic acid concentration is 0.02g/mL, and chitosan is 0.01mg/mL. mL.