Preparation method and application of bone repair scaffold

Abstract

The invention discloses a preparation method and application of a bone repair scaffold, and belongs to the field of bone tissue engineering and regenerative medicine. The preparation method of the bone repair scaffold comprises the following steps: pretreating a PCL raw material to obtain PCL particles; preparing a 3D printed PCL stent, and performing 3D printing on the PCL particles to obtain the PCL stent; an IONPs solution is prepared; iONPs is loaded by using a time gradient pulse method; and carrying out post-treatment on the stent. The loading is carried out in a time gradient pulse loading system in a standing and shaking table alternate pulse mode. According to the preparation method of the bone repair scaffold provided by the invention, the effects of optimizing the iron ion release behavior and accurately controlling the space-time distribution of the IONPs can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to bone tissue engineering and regenerative medicine, and in particular to a preparation method and application of a bone repair scaffold. BACKGROUND

[0002] Bone defect is a common clinical disease, usually caused by bone disease, tumor resection or trauma, etc. Traditional bone defect repair methods include autologous bone transplantation, allogeneic bone transplantation, synthetic material transplantation, etc. However, these methods have many shortcomings. Autologous bone transplantation has problems such as limited donor site, large surgical trauma, and complications at the donor site; allogeneic bone transplantation faces challenges such as immune rejection, disease transmission risk, and donor shortage; synthetic material transplantation avoids donor problems, but its biological activity and integration ability with host bone tissue are limited, making it difficult to completely repair bone defects.

[0003] In recent years, with the development of biomaterial science and tissue engineering technology, 3D printing technology has been widely applied in the field of bone tissue engineering. 3D printing technology can accurately manufacture personalized scaffolds according to the specific shape and size of bone defects, providing a good three-dimensional microenvironment for bone tissue regeneration. Polycaprolactone (PCL) is a high molecular weight material with good biocompatibility and biodegradability, and is widely used in the preparation of bone tissue engineering scaffolds. However, PCL material itself has certain limitations, such as strong surface hydrophobicity and lack of biological active ligands, which leads to poor cell adhesion and proliferation, affecting the effect of bone tissue repair.

[0004] In order to overcome these defects of PCL material, researchers have tried to modify its surface. Polydopamine (PDA) is a material with good biocompatibility and surface modification ability, and surface modification of PCL with PDA can significantly improve its hydrophilicity and cell adhesion performance. In addition, iron oxide nanoparticles (IONPs) are widely used in the field of biomedicine due to their unique physical and chemical properties and good biocompatibility. IONPs have good pro-osteogenic properties and can significantly promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), thereby accelerating the regeneration and repair of bone tissue. Studies have shown that IONPs can promote the expression of osteogenesis-related genes such as RUNX2 and COL-1 by regulating intracellular signaling pathways such as the MAPK signaling pathway, thereby enhancing the formation ability of bone tissue. In addition, IONPs can also improve the microenvironment of cells, promote cell adhesion and proliferation, and provide more favorable conditions for bone tissue regeneration.

[0005] However, there are still many limitations in the current loading of IONPs: (1) IONPs distribution is not uniform: static long-term soaking can easily lead to physical adsorption of IONPs on the surface layer of PDA, and the amount of IONPs entering the deep pores decreases exponentially with the increase of distance, resulting in a "shell-core" distribution of surface layer oversaturation and deep layer underloading, which reduces the effective volume; (2) the release behavior is uncontrollable: the surface layer oversaturation area will have a burst release of IONPs in the early stage of implantation, and the ion release rate exceeds 70% within 5 days, however, the high local iron ion concentration may cause cytotoxicity; and the deep layer underloading area cannot provide sustained osteogenic signals in the later stage.

[0006] In summary, the traditional static adsorption relies on simple concentration diffusion, the solution concentration is constant, the soaking time is long, and there is a lack of driving force gradient and time dimension control. If the concentration is simply increased or the soaking time is prolonged, the surface layer oversaturation will be more serious; if ultrasonic or stirring assistance is used, the fine pore structure of the PCL scaffold will be easily damaged. SUMMARY

[0007] The purpose of the present application is to provide a preparation method of a bone repair scaffold with optimized iron ion release behavior and precise control of IONPs spatial-temporal distribution.

[0008] Another purpose of the present application is to provide a use of the above-mentioned bone repair scaffold in cell inoculation and culture.

[0009] Technical scheme: The preparation method of the bone repair scaffold according to the present application comprises the following steps:

[0010] (1) preparing PCL particles;

[0011] (2) 3D printing the PCL particles to obtain a PCL scaffold blank;

[0012] (3) preparing an IONPs solution;

[0013] (4) loading the IONPs solution on the PCL scaffold blank by time gradient pulse method;

[0014] (5) post-treating the PCL scaffold blank loaded with the IONPs solution to obtain a bone repair scaffold.

[0015] Preferably, the concentration of the IONPs solution used in step (4) is 230-920 μg / mL.

[0016] Preferably, the time gradient pulse method in step (4) is carried out in the form of alternating pulses of static and shaking bed in a time gradient pulse loading system.

[0017] Preferably, the loading time in step (4) is 2-5 days, and more preferably 3 days.

[0018] Preferably, the alternating pulse condition is to stand still and shake, and the total cycle is 2-5 times, and further preferably 3 times; the standing time is 5-7h, and the shaking time is 1-3h.

[0019] Preferably, the pretreatment operation in step (1) is as follows: the PCL raw material is dissolved in tetrahydrofuran and subjected to negative pressure suction in a water bath, and then it is cut into PCL particles.

[0020] Preferably, the IONPs solution in step (3) is prepared by a dextran modification method: FeCl3, FeCl2 and ammonium hydroxide are added to a dextran aqueous solution, and then the solution is purified by magnetic stirring and ultrafiltration.

[0021] Preferably, the post-treatment method in step (5) is as follows: the PCL particles are irradiated with ultraviolet light, disinfected, and then rinsed with PBS.

[0022] Invention principle: the PCL scaffold is prepared by 3D printing technology, the PCL scaffold is surface-modified by PDA, and then IONPs are loaded on the PDA-modified PCL scaffold by time gradient pulse loading method. The scaffold improves the surface hydrophilicity and cell adhesion performance, promotes the proliferation and osteogenic differentiation of BMSCs, and thus accelerates the regeneration and repair of bone tissue.

[0023] Among them, the PCL scaffold serves as a basic framework structure, provides necessary mechanical support and cell attachment sites, and is prepared by 3D printing technology, so that it has a uniform pore structure, provides a three-dimensional porous growth environment for cells, and is conducive to the growth of cells and the formation of bone tissue. The PDA coating is formed on the surface of the PCL scaffold through a self-polymerization reaction, which can improve the hydrophilicity and cell adhesion performance of the scaffold, promote the attachment and growth of cells. The present application innovatively uses time gradient pulse loading method to load IONPs on the PDA coating, so that IONPs are gradiently distributed in the PDA coating in a radial density decreasing manner, with high concentration in the outer layer region and decreasing layer by layer inward; the gradient structure has no additional interface or stratification, has good biocompatibility and osteogenic performance, and can promote the proliferation and osteogenic differentiation of BMSCs.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention adopts a time gradient pulse loading method to prepare a bone repair scaffold, so that IONPs are distributed in a radial gradient in the PDA layer, optimizing the iron ion release behavior, avoiding early burst toxicity, and significantly prolonging the duration of the osteogenic signal. On the 21st day, it can still maintain a stable release of 2%; (2) The present invention proposes a time gradient pulse loading method, which realizes the radial gradient distribution of IONPs in the PDA layer through a process of time division, variable concentration, and intermittent immersion, without completely changing the structure and material system of the bone repair scaffold, and provides a first fast and then slow, continuous and stable iron ion release curve; (3) The gradient distributed IONPs quickly release iron ions in the early stage (i.e., the high concentration area in the outer layer), quickly activating the osteogenic signal; and then (i.e., the low concentration area in the inner layer), continuously releasing iron ions, maintaining long-term osteogenic stimulation, achieving a "first fast and then slow" temporal activation effect, and accelerating bone tissue regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 1 is a comparison chart of the ion release curves of the bone repair scaffolds prepared in Example 1 and Comparative Example 1;

[0026] Figure 2 The results of cell culture of the bone repair scaffold prepared in Example 1 are as follows;

[0027] Figure 3 This is the cell culture result of the bone repair scaffold prepared in Comparative Example 1. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0029] Example 1

[0030] The method for preparing the bone repair scaffold of the present invention comprises the following steps:

[0031] (1) Preparation of PCL particles

[0032] Polycaprolactone (PCL, Sigma-Aldrich, USA) with a molecular weight of 80,000 was selected as the main raw material. The PCL raw material was weighed and dissolved in tetrahydrofuran to prepare a solution with a mass-to-volume ratio of 25%. The PCL solution was placed in a rotary evaporator and vacuum-evacuated in an 80°C water bath to remove the tetrahydrofuran solvent. After the solvent completely evaporated, the resulting PCL mixture was shredded into PCL particles with a diameter of approximately 500 μm.

[0033] (2) 3D printing of PCL particles to obtain PCL scaffold blanks

[0034] A 3D printer (HTS-750, Shanghai Fufan Technology) was used to print the PCL particles in the extrusion cavity of the printer. The printing temperature was set to 110°C ± 5°C. During the printing process, the nozzle moving speed was set to 4 mm / s. A CAD software was used to design a PCL porous scaffold model with a radius of 2.5 mm, a height of 1 mm, and a pore angle of 90°. The designed model was imported into the slicing software of the 3D printer, and the printing layer thickness was set to 0.2 mm. After printing, a PCL scaffold blank with uniform pore structure was obtained.

[0035] (3) Preparation of IONPs solution

[0036] The IONPs were prepared by dextran modification method: 200 mg of dextran was dissolved in 10 mL of deionized water, 6.0 mg of FeCl3 and 30 mg of FeCl2 were added, followed by the addition of 1 g of 28% (w / v) ammonium hydroxide, and strong mechanical stirring was performed. The colloidal mixture was heated to 80°C by adjusting the magnetic field strength and kept for 1 h. Finally, the IONPs solution with a concentration of 23 mg / mL was obtained by ultrafiltration purification of deionized water using a 100 kDa membrane for 6 times.

[0037] (4) Loading of IONPs solution on PCL scaffold blank by time gradient pulse method

[0038] The PDA modified PCL scaffold was placed in the time gradient pulse loading system: on the first day, the IONPs solution with a concentration of 460 μg / mL was used for static-shaking alternating pulse, with a static time of 6 h and a shaking time of 2 h, and the cycle was repeated for three times. On the second day, the IONPs solution with a concentration of 230 μg / mL was used for repeated pulse, with the same alternating pulse conditions as the first day. On the third day, the IONPs solution with a concentration of 920 μg / mL was used for pulse again, with the same alternating pulse conditions as the first day. Finally, a radial density decreasing IONPs distribution was formed on the surface of the scaffold, and a scaffold precursor was obtained.

[0039] (5) Post-treatment of scaffold precursor

[0040] The scaffold precursor obtained in step (4) was irradiated with ultraviolet light for 2 h for disinfection, to ensure that the surface of the scaffold was sterile. The disinfected scaffold was washed with phosphate buffered saline (PBS) for three times to remove the residual disinfectant, and a bone repair scaffold was obtained.

[0041] The application of the bone repair scaffold in cell inoculation and culture comprises the following steps:

[0042] The bone repair scaffold is placed in a low adhesion well plate, and rabbit bone marrow mesenchymal stem cells (rBMSCs) cultured to the third generation are uniformly inoculated on the surface of the scaffold at a number of 5×10^4 per well; the scaffold is placed in an incubator for 4h to enable the cells to adhere to the scaffold more quickly, and then a certain amount of culture medium is added for continuous culture to observe the growth of the cells. The live and dead staining diagram of rBMSCs cells adhering and growing on the bone repair scaffold is shown in Figure 2 .

[0043] Comparative Example 1

[0044] The same as Example 1 is not repeated, and the difference is that the loading process of step (4) is as follows: the prepared PCL scaffold is placed in a 460μg / mL IONPs solution, and the scaffold is placed in a shaker together with the solution for soaking for three days; after the soaking is completed, the scaffold is washed with deionized water three times to remove the unabsorbed IONPs, thereby obtaining the IONPs-loaded bone repair scaffold. The live and dead staining diagram of rBMSCs cells adhering and growing on the bone repair scaffold is shown in Figure 3 .

[0045] The bone repair scaffolds prepared in Example 1 and Comparative Example 1 are subjected to iron ion release testing, and the curve comparison diagram shown in Figure 1 is obtained.

[0046] The test method is as follows: the bone repair scaffold is immersed in a physiological simulation solution at a constant temperature of 37℃, and samples are taken on the 1st, 2nd, 3rd, 4th, …, 21st day, and the iron ion concentration in the solution is determined by inductively coupled plasma mass spectrometry (ICP-MS), and the daily release rate is calculated. The equipment and consumables used include: Agilent 7700X or similar ICP-MS instrument, constant temperature oscillator, 50rpm, 50mL light-proof centrifuge tube, 0.22μm inert needle filter equipped with PES membrane, pH7.4±0.1 Hank's balanced salt solution, 10mL polypropylene centrifuge tube soaked in 10% HNO3 overnight and rinsed with ultrapure water, 1000mgL-1 Fe single-element standard solution.

[0047] The calculation formula of the daily release rate is as follows:

[0048] v=[(C i -C i-1 )×V total ]÷Fe total ×100.

[0049] Wherein,

[0050] C i : the iron ion concentration (mg / L) measured in the medium on the ith day;

[0051] Ci-1 : Iron ion concentration in the medium measured on day i-1 (mg / L);

[0052] V total : The total volume of the system after each sampling (after taking out 1 mL of sample each time, 1 mL of fresh HBSS was immediately added, so V total fixed at 20 mL);

[0053] Fe total : Total iron mass of IONPs in the scaffold (mg). Figure 1 It can be seen that the ion release rates of the two loading methods are different. The bone repair scaffold loaded with IONPs using the simple immersion method in the existing technology has an ion release rate of up to 30% on the first day, while the bone repair scaffold loaded with IONPs using the innovative time-gradient pulse method of the present invention has an ion release rate of only 18% on the first day, effectively avoiding the defect of early burst toxicity. In addition, the bone repair scaffold prepared using the method of the present invention can maintain a steady release of approximately 2%-3% per day from day 7 to 21, which can significantly prolong the duration of the osteogenic signal.

[0054] like Figure 2 and Figure 3 As shown in the figure, the green portion represents living cells adhering and growing on the scaffold. The more green and brighter the color, the better the cell growth and adhesion. Therefore, compared with the traditional immersion method, the bone repair scaffold prepared using this method has better adhesion, more cell growth, and lower toxicity.

Claims

1. A method for preparing a bone repair scaffold, characterized in that: The following steps are involved: (1) Preparation of PCL particles; (2) 3D printing PCL particles to obtain PCL scaffold blanks; (3) preparing IONPs solution; (4) The IONPs solution was loaded onto the PCL scaffold blank using a time gradient pulse method to obtain a scaffold precursor; (5) Post-processing the scaffold precursor to obtain a bone repair scaffold.

2. The preparation method according to claim 1, characterized in that The concentration of the loading IONPs solution in step (4) is 230-920 μg / mL.

3. The preparation method according to claim 1, characterized in that The time gradient pulse method described in step (4) is carried out in a time gradient pulse loading system in the form of alternating static and shaking pulses.

4. The preparation method according to claim 1, characterized in that The loading time in step (4) is 2-5 days.

5. The preparation method according to claim 3, characterized in that The alternating pulse condition is to first let it stand and then shake it, for a total of 2-5 cycles.

6. The preparation method according to claim 5, characterized in that The standing time is 5-7 hours, and the shaking time is 1-3 hours.

7. The preparation method according to claim 1, characterized in that The pretreatment operation described in step (1) is as follows: the PCL raw material is dissolved in tetrahydrofuran and vacuum-extracted in a water bath, and then chopped to obtain PCL particles.

8. The preparation method according to claim 1, characterized in that The IONPs solution described in step (3) is prepared by a dextran modification method: FeCl3, FeCl2, and ammonium hydroxide are added to the dextran aqueous solution, and then ultrafiltration is performed after magnetic stirring.

9. The preparation method according to claim 1, characterized in that The post-processing method described in step (5) is: irradiating with ultraviolet light, disinfecting, and then rinsing with PBS.

10. Use of the bone repair scaffold according to any one of claims 1 to 9 in cell inoculation and culture.

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