阅读说明：本技术 一种可持续释放无机活性成分的大孔骨组织工程支架及其制备方法 (Macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients and preparation method thereof ) 是由 刘来俊 王富军 张天添 李超婧 王璐 张宇 周俊泽 于 2020-04-23 设计创作，主要内容包括：本发明公开了一种可持续释放无机活性成分的大孔骨组织工程支架及其制备方法。大孔骨组织工程支架由静电纺丝一步制得的微米级串珠和纳米级纤维的复合结构组成,纳米级纤维中的无机活性物质优先释放,待串珠有机基体逐步降解后,串珠中的无机活性物质随后释放。制备方法：将分散液逐滴加入聚合物溶液中,制得含无机活性成分的静电纺丝溶液；将静电纺丝溶液进行静电纺丝,将得到的静电纺纤维膜真空干燥,然后进行环氧乙烷处理,最终得到富含无机活性成分且具备微米串珠及纳米纤维复合结构的大孔骨组织工程支架。串珠的存在,会增大支架的孔径及表面粗糙度,利于细胞的浸润生长及成骨分化。本发明所述的制备方法简单,流程短,适合大批量生产。(The invention discloses a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients and a preparation method thereof. The macroporous bone tissue engineering scaffold is composed of a composite structure of micron-sized beads and nano-sized fibers prepared by electrostatic spinning in one step, inorganic active substances in the nano-sized fibers are preferentially released, and the inorganic active substances in the beads are subsequently released after the organic matrix of the beads is gradually degraded. The preparation method comprises the following steps: dropwise adding the dispersion liquid into the polymer solution to prepare an electrostatic spinning solution containing inorganic active ingredients; and (3) performing electrostatic spinning on the electrostatic spinning solution, performing vacuum drying on the obtained electrostatic spinning fibrous membrane, and then performing ethylene oxide treatment to finally obtain the macroporous bone tissue engineering scaffold which is rich in inorganic active ingredients and has a micron bead string and nanofiber composite structure. The existence of the beads can increase the aperture and the surface roughness of the stent, and is beneficial to the infiltration growth and osteogenic differentiation of cells. The preparation method is simple, short in flow and suitable for mass production.)

1. The macroporous bone tissue engineering scaffold capable of continuously releasing inorganic active ingredients is characterized by comprising a composite structure of micron-sized beads and nano-sized fibers prepared by electrostatic spinning in one step, wherein inorganic active substances in the nano-sized fibers are preferentially released, and the inorganic active substances in the beads are subsequently released after an organic matrix of the beads is gradually degraded.

2. The macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to claim 1, wherein the micro-sized beads have an organic/inorganic two-phase structure, and the diameter of the beads is 1 to 10 μm; the nano-scale fiber is of an organic/inorganic two-phase structure, and the diameter of the fiber is 50-500 nm.

3. The macroporous bone tissue engineering scaffold capable of sustainably releasing an inorganic active ingredient as claimed in claim 1, wherein the organic material in the micro-beads/nano-fibers is at least one of a natural degradable material and a synthetic degradable material; the inorganic material in the micron-scale bead/nano-scale fiber is an inorganic active component.

4. The macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients as claimed in claim 3, wherein the natural degradable material comprises at least one of collagen, chitosan, gelatin, silk fibroin and hyaluronic acid; the synthetic degradable material polymer comprises at least one of PCL, PLA, PLLA, PGA, PLGA, PGS and PHB; the inorganic active component comprises at least one of hydroxyapatite, tricalcium phosphate, bioactive glass, silicon dioxide and metal oxide according to the requirement.

5. The method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to any one of claims 1 to 4, comprising the steps of:

step 1): dissolving a degradable polymer in a solvent, and magnetically stirring for 1-12 hours to obtain a polymer solution;

step 2): adding an inorganic active ingredient into the solvent same as the solvent in the step 1), and performing ultrasonic dispersion for 3-6 hours to uniformly disperse the inorganic active ingredient;

step 3): dropwise adding the dispersion liquid prepared in the step 2) into the polymer solution prepared in the step 1), and stirring by magnetic force while dropwise adding to prepare an electrostatic spinning solution containing inorganic active ingredients;

step 4): and (3) carrying out electrostatic spinning on the electrostatic spinning solution containing the inorganic active ingredient prepared in the step 3), carrying out vacuum drying on the obtained electrostatic spinning fibrous membrane, and then carrying out ethylene oxide treatment to finally obtain the macroporous bone tissue engineering scaffold which is rich in the inorganic active ingredient and has a micron bead string and nanofiber composite structure.

6. The method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients as claimed in claim 5, wherein the solvent in step 1) comprises any one or a combination of deionized water, absolute ethanol, dichloromethane, trichloromethane, tetrahydrofuran, hexafluoroisopropanol, methanol, formic acid, acetic acid, dimethyl sulfoxide, N-dimethylformamide and acetone.

7. The method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to claim 5, wherein the mass percentage of the degradable polymer in the electrospinning solution obtained in the step 3) is 10-80 g/L; the mass ratio of the degradable polymer to the inorganic active ingredient in the electrostatic spinning solution is (5-20): 1.

8. the method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to claim 5, wherein the process parameters of the electrospinning in the step 4) are as follows: a flat plate receiving device is adopted, the spinning voltage is 12-16 kV, the receiving distance is 10-15 cm, the propelling speed is 1-2 mL/h, the diameter of a spinning nozzle is 0.5-0.8 mm, the temperature is 20-30 ℃, and the relative humidity is 30-60%.

9. The method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to claim 5, wherein the vacuum drying time in the step 4) is 6-24 h, and the temperature is 30-50 ℃; the ethylene oxide treatment time was 12h or 24 h.

10. The method for preparing a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients according to claim 5, wherein the porosity of the macroporous bone tissue engineering scaffold obtained in the step 4) is 70-90%, and the average pore diameter is 5-20 μm.

Technical Field

The invention relates to a macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients and a preparation method thereof, belonging to the field of biomedical materials.

Background

The treatment of critical-sized bone tissue defects caused by trauma, tumors and congenital diseases remains a major clinical challenge. It is estimated that over 3000 million cases worldwide require bone repair via clinical grafting means every year. Due to good osteointegration, osteoconductivity, and osteoinductivity, auto/allograft transplantation remains the gold standard for the treatment of this disease. However, the limited number of grafts, damage to donor sites, and potential cross-contamination have limited the clinical use of auto/allografts. With the development of Bone Tissue Engineering (BTE), scaffold-based tissue engineering offers an option to replace auto/allografts to meet the growing demand for grafts to repair and regenerate bone tissue. Generally, a scaffold for bone tissue engineering with excellent properties should have excellent biocompatibility, bioactivity, and mechanical properties, and have a porous structure to support bone tissue ingrowth and integration. Among the various techniques for the fabrication of scaffolds for bone tissue engineering, electrospinning has attracted increasing interest because of its ability to convert a variety of materials (natural and synthetic organic materials) into fibers that are morphologically similar to the structure of the natural extracellular matrix (ECM). However, as a scaffold for bone tissue engineering, only scaffolds made of natural and synthetic organic materials do not have biological activity of promoting bone tissue regeneration. Therefore, the blending and spinning of inorganic active ingredients (such as hydroxyapatite and tricalcium phosphate) to prepare organic-inorganic composite scaffolds with biological activity and two-phase structure simulating natural bone has been widely studied. However, the conventional organic-inorganic blend spinning enables the inorganic active ingredients to be coated by the organic matrix, and the characteristics of the inorganic active ingredients cannot be exerted in the early stage of stent implantation, so that the bone tissues are delayed to heal. Secondly, acidic products of organic matrix degradation are not well neutralized at the early stage of implantation, which is likely to cause tissue inflammation at the site of implantation. Therefore, the design and preparation of the electrostatic spinning bone tissue engineering scaffold with the sustainable release of the inorganic active ingredients have important significance.

In the existing research, ① patent with publication number CN107320787A introduces a porous fiber membrane material for periodontal repair and a preparation method thereof, after a mixed solution of chitosan and hydroxyapatite is dripped on the surface of an electrostatic spinning fiber membrane, the mixed solution is frozen and dried to prepare a composite membrane material for periodontal repair, but the technology is relatively complicated in process, and the problem that early scaffold bioactivity is insufficient due to the fact that the hydroxyapatite is coated by the chitosan still exists, ② patent with publication number CN105148321B introduces a bone substitute material of a bionic natural bone structure constructed by mineralized nanofibers and a preparation method thereof, the nanofiber yarns prepared by electrostatic spinning are woven into a nanofiber fabric, the nanofiber fabric is mineralized in a simulated body fluid which is 1-1.5 times that of the nanofiber is put into, and a mineralized layer is formed on the surface of the scaffold to improve the scaffold bioactivity²⁺A ③ article titled, namely, a preparation method of calcium phosphate electrospun fiber is introduced, the hydroxyapatite particles and PLGA are blended and spun, and then the PLGA is calcined to obtain the pure inorganic calcium phosphate electrospun fiberThey have apparent small pores of submicron size due to the accumulation of conventional electrospun nano-or micro-fibers, lacking large pores necessary for cell penetration and tissue regeneration. Therefore, the design and preparation of the macroporous bone tissue engineering scaffold capable of continuously releasing inorganic active ingredients have important significance.

Disclosure of Invention

The technical problem solved by the invention is as follows: a macroporous bone tissue engineering scaffold capable of continuously releasing inorganic active ingredients.

In order to solve the problems, the invention provides a macroporous bone tissue engineering scaffold capable of continuously releasing inorganic active ingredients, which is characterized by consisting of a composite structure of micron-sized beads and nano-sized fibers prepared by electrostatic spinning in one step, wherein inorganic active substances in the nano-sized fibers are preferentially released, and the inorganic active substances in the beads are subsequently released after an organic matrix of the beads is gradually degraded. The bone tissue engineering scaffold simulates a two-phase structure of natural bone tissue in terms of components, and has a macroporous structure which has the function of continuously releasing inorganic active components and is beneficial to cell permeation and tissue ingrowth.

Preferably, the micron-sized beads are of an organic/inorganic two-phase structure, and the diameter of the beads is 1-10 μm; the nano-scale fiber is of an organic/inorganic two-phase structure, and the diameter of the fiber is 50-500 nm.

Preferably, the organic material in the micro-sized beads/nano-sized fibers is at least one of natural degradable material and synthetic degradable material; the inorganic material in the micron-scale bead/nano-scale fiber is an inorganic active component.

More preferably, the natural degradable material comprises at least one of collagen, chitosan, gelatin, silk fibroin and hyaluronic acid; the synthetic degradable material polymer comprises at least one of PCL, PLA, PLLA, PGA, PLGA, PGS and PHB; the inorganic active component comprises at least one of hydroxyapatite, tricalcium phosphate, bioactive glass, silicon dioxide and the like, and optionally comprises metal oxide which is magnesium oxide, zinc oxide and the like according to needs.

The invention also provides a preparation method of the macroporous bone tissue engineering scaffold capable of continuously releasing the inorganic active ingredients, which is characterized by comprising the following steps:

step 1): dissolving a degradable polymer in a solvent, and magnetically stirring for 1-12 hours to obtain a polymer solution;

step 2): adding an inorganic active ingredient into the solvent same as the solvent in the step 1), and performing ultrasonic dispersion for 3-6 hours to uniformly disperse the inorganic active ingredient;

step 3): dropwise adding the dispersion liquid prepared in the step 2) into the polymer solution prepared in the step 1), and stirring by magnetic force while dropwise adding to prepare an electrostatic spinning solution containing inorganic active ingredients;

step 4): and (3) carrying out electrostatic spinning on the electrostatic spinning solution containing the inorganic active ingredient prepared in the step 3), carrying out vacuum drying on the obtained electrostatic spinning fibrous membrane, and then carrying out ethylene oxide treatment to finally obtain the macroporous bone tissue engineering scaffold which is rich in the inorganic active ingredient and has a micron bead string and nanofiber composite structure.

Preferably, the solvent in step 1) includes any one or a combination of several of deionized water, absolute ethyl alcohol, dichloromethane, trichloromethane, tetrahydrofuran, hexafluoroisopropanol, methanol, formic acid, acetic acid, dimethyl sulfoxide, N-dimethylformamide and acetone.

Preferably, the mass percentage of the degradable polymer in the electrostatic spinning solution obtained in the step 3) is 10-80 g/L; the mass ratio of the degradable polymer to the inorganic active ingredient in the electrostatic spinning solution is (5-20): 1.

preferably, the electrostatic spinning process parameters in the step 4) are as follows: a flat plate receiving device is adopted, the spinning voltage is 12-16 kV, the receiving distance is 10-15 cm, the propelling speed is 1-2 mL/h, the diameter of a spinning nozzle is 0.5-0.8 mm, the temperature is 20-30 ℃, and the relative humidity is 30-60%.

Preferably, the vacuum drying time in the step 4) is 6-24 hours, and the temperature is 30-50 ℃; the ethylene oxide treatment time was 12h or 24 h.

Preferably, the porosity of the macroporous bone tissue engineering scaffold obtained in the step 4) is 70-90%, and the average pore diameter is 5-20 μm.

Compared with the prior art, the invention has the beneficial effects that:

(1) the macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients prepared by the invention is a composite structure consisting of micron beads and nano fibers, inorganic active substances in the nano fibers implanted in the early stage are preferentially released, and after the organic matrix of the beads is gradually degraded, the inorganic active substances in the beads are subsequently released, so that the problem of insufficient early biological activity of the conventional electrostatic spinning bone tissue engineering scaffold is greatly improved.

(2) The beads with larger size in the macroporous bone tissue engineering scaffold capable of sustainably releasing inorganic active ingredients prepared by the invention can increase the aperture and the surface roughness of the scaffold, and are beneficial to the infiltration growth and osteogenic differentiation of cells.

(3) The preparation method of the macroporous bone tissue engineering scaffold capable of sustainably releasing the inorganic active ingredients is simple, short in process and suitable for mass production.

Drawings

Fig. 1 and 2 are SEM photographs of the macroporous electrostatic spinning membrane with the micron bead and nanofiber composite structure in example 1 in different proportions;

FIG. 3 is a TEM image of the micro beads of example 1 (in which the particles are tricalcium phosphate, which is an active substance);

fig. 4 is a TEM photograph of the nanofibers of example 1 (in which the particles are the active material tricalcium phosphate).

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

In each example, PMI type pore size tester (CFP-1100-AI) was used to test the pore size distribution and porosity of the macroporous bone tissue engineering scaffolds capable of sustained release of inorganic active ingredients. Confocal microscopy (LSM510) was used in various embodiments of the invention to test the depth of stem cell ingrowth. In the examples of the present invention, inductively coupled plasma (varian vista Pro) was used to test the release of active substances from macroporous scaffolds for bone tissue engineering, which are capable of sustained release of inorganic active ingredients.

The reagents and cells used in each example were as follows: PLLA (Sigma-Aldrich 764698); PCL (national drug group chemical agents ltd 440744); chitosan (national drug group chemical agents ltd 69047436); methylene chloride (national chemical group chemical company, ltd 80047318); chloroform (national chemical group, chemical Co., Ltd 10006818); rat bone marrow mesenchymal stem cells (stem cell bank SCSP-402 of chinese academy of sciences); CCK-8 kit (Bailingwei science and technology Co., Ltd., C2581); DAPI (Sigma-Aldrich 32670-5 MG-F); PBS (Shanghai Yi chemical Co., Ltd. E504-100 mL).