CN113198051B

CN113198051B - Preparation method of three-dimensional composite porous scaffold and three-dimensional composite porous scaffold

Info

Publication number: CN113198051B
Application number: CN202110469865.2A
Authority: CN
Inventors: 刘义; 王云; 鲁越
Original assignee: Sichuan University of Science and Engineering
Current assignee: Sichuan University of Science and Engineering
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2022-06-28
Anticipated expiration: 2041-04-28
Also published as: CN113198051A

Abstract

The invention discloses a preparation method of a three-dimensional composite porous scaffold and the three-dimensional composite porous scaffold, which relate to the technical field of biological scaffolds and comprise the following steps of S1: dissolving polycaprolactone and cellulose nanocrystal in an organic solvent, uniformly mixing, and then carrying out electrostatic spinning to obtain a nanofiber membrane; s2: mechanically stirring and crushing the nanofiber membrane in a mixed solvent, sieving to obtain short nanofiber filaments, and performing suction filtration to obtain a wet material; s3: dispersing the wet material in a mixed solution of absolute ethyl alcohol and deionized water, carrying out thermal self-aggregation reaction for 5min at the temperature of 50-57 ℃ to obtain aggregates, and then carrying out freeze drying in a vacuum freeze dryer to obtain the three-dimensional composite porous scaffold. The invention discloses a preparation method of a three-dimensional composite porous scaffold and the three-dimensional composite porous scaffold, wherein the composite porous scaffold is prepared by adopting a thermal self-aggregation method, so that the porous scaffold with high porosity and good interoperability is obtained.

Description

Preparation method of three-dimensional composite porous scaffold and three-dimensional composite porous scaffold

Technical Field

The invention relates to the technical field of tissue engineering materials, in particular to a preparation method of a three-dimensional composite porous scaffold and the three-dimensional composite porous scaffold.

Background

In recent years, bone tissue engineering has been rapidly developed, which provides new technical possibilities for bone defect repair. Tissue engineering is the process of combining cell, material and processing method to prepare the rack as three-dimensional micro environment for cell adhesion, proliferation, differentiation and formation of extracellular matrix. And then by matching appropriate biochemical and physicochemical factors, the microenvironment determined by the physical and chemical properties of the scaffold plays a crucial role in cell function and subsequent tissue regeneration, thereby improving or replacing the original biological tissue.

In the extracellular matrix of natural bone tissue, collagen fibers having diameters of several tens to several hundreds of nanometers are a main component. In tissue engineering, how to prepare a nanofiber scaffold which has interpenetrating macropores and high porosity, better simulates a natural extracellular matrix structure, can greatly improve the possibility of long-term survival of cells in vitro culture, and finally generates functionalized tissues remains a significant technical challenge. Three-dimensional scaffolds provide a large specific surface area and pore structure, which can enhance the function of cells and tissues to support the adhesion and growth of a large number of cells. Porosity provides sufficient space to allow cells to suspend and penetrate three-dimensional structures. The porous structure also promotes extracellular matrix production, transport of nutrients from the nutrient medium, and waste drainage. Thus, proper pore size and a uniformly distributed and interconnected pore structure are critical to easier cell distribution throughout the scaffold. Preparation method of scaffold in the past decades, extensive research has been conducted on scaffolds for tissue engineering, and various materials and techniques have been applied in the field of tissue engineering. The preparation methods of various three-dimensional porous scaffolds comprise multilayer alternate electrospinning, mechanical extrusion molding, a template method, addition of a pore-forming agent and the like, but the prepared scaffolds lack interconnected and intercommunicated pore structures and may have the defects of residual pore-forming agent and the like.

Disclosure of Invention

The application aims to overcome the problem that a three-dimensional support prepared by the prior art is lack of an interconnected and intercommunicated layered pore structure, and provides a simple and effective preparation method of a three-dimensional composite porous support.

The application also discloses a three-dimensional composite porous scaffold.

In order to achieve the above object, the present application provides the following technical solutions: one of the steps includes:

s1: dissolving polycaprolactone and cellulose nanocrystal in an organic solvent, uniformly mixing, and then performing electrostatic spinning to obtain a nanofiber membrane;

s2: mechanically stirring and crushing the nanofiber membrane in a mixed solvent, sieving to obtain short nanofiber filaments, and performing suction filtration to obtain a wet material;

s3: weighing a proper amount of wet materials, dispersing the wet materials in a mixed solution of absolute ethyl alcohol and deionized water, carrying out thermal self-aggregation reaction for 3-10min at the temperature of 50-57 ℃ to obtain aggregates, and then carrying out freeze drying in a vacuum freeze dryer to obtain the three-dimensional composite porous scaffold.

In the technical scheme, the broken and sieved nano-fiber membrane and polycaprolactone are subjected to thermal self-aggregation reaction in hot water to obtain the three-dimensional composite porous scaffold. Meanwhile, in the technical scheme, the wet material for the thermal self-aggregation reaction is prepared by adopting a method of firstly carrying out electrostatic spinning and then mechanically crushing, the preparation method is simple, the preparation conditions are easy to achieve, and the popularization and the utilization are convenient. In addition, in the preparation method, the crushed short nano fibers are filtered firstly and then are subjected to thermal self-aggregation reaction in a mixed solution of absolute ethyl alcohol and deionized water, so that the short nano fibers can be suspended in the mixed solution fully and uniformly to form a scaffold structure with high porosity.

Further, the organic solvent is a mixed solvent of dichloromethane and dimethylamide.

Suitable solvents are one of the key factors in forming smooth, non-beaded electrospun nanofibers. Generally, two factors are considered in selecting a solvent. Firstly, the preferred solvents for the electrospinning process have completely soluble polymers, and secondly, the solvent should have a moderate boiling point mainly determining the volatility of the solvent affecting the formation of the fibers. Dichloromethane and N, N-dimethylformamide can be used as solvents of PCL, but the dielectric constant of single dichloromethane is only 9.1(25 ℃) and the conductivity is poor, while the dielectric constant of N, N-dimethylformamide can reach 36.7(25 ℃), but the solubility to PCL is poor, so that a smooth and non-beaded nano fiber film is difficult to obtain by using a single solvent. Therefore, in the technical scheme, dichloromethane and N, N-dimethylformamide are used as a mixed solvent, the morphology of the nanofiber membrane obtained by electrostatic spinning is improved, and the porosity and cell compatibility of the obtained three-dimensional composite porous scaffold are improved.

Preferably, in the step S2, the broken short nano fiber filaments are mechanically stirred and sieved by a 20-mesh sieve, so as to obtain short nano fiber filaments with the length not exceeding 0.85 mm.

Further, the volume ratio of the dichloromethane to the dimethyl amide is 1: 4-3: 2.

Further, in step S2, the mechanical stirring and breaking is intermittent accelerated stirring, the stirring speed is 20000 to 25000rpm, preferably 22000rpm, each stirring time is 5min, the intermittent stirring is 15min, the process is repeated for 3 to 10 times until the fiber fragments are stirred and broken into short nano-fiber filaments, preferably 4 times.

Further, in step S3, in the mixed solution formed by the absolute ethyl alcohol and the deionized water, the mass concentration of the absolute ethyl alcohol is 10-18 wt%; preferably, in step S3, the mass concentration of the absolute ethyl alcohol in the mixed solution is 12 to 16%, and more preferably, the mass concentration of the absolute ethyl alcohol in the mixed solution is 15%.

Further, in the step S3, the mass concentration of the wet material in the mixed solution is 0.075-0.2 g/mL; meanwhile, the mass concentration of the short nano-fiber filaments in the mixed solution is 0.03-0.15 g/mL; preferably, the mass concentration of the wet material in the mixed solution is 0.1 g/mL.

Further, the mixed solvent adopted in the step S2 is absolute ethyl alcohol and deionized water with a volume ratio of 1: 1-2, and the volume ratio of the absolute ethyl alcohol to the deionized water in the mixed solvent is preferably 1: 1.

Further, in step S1, the mass ratio of the added cellulose nanocrystals to the polycaprolactone is 3: 100; the sum of the mass concentrations of the polycaprolactone and the cellulose nanocrystal in the organic solvent is 14-20 wt%; preferably, the sum of the mass concentrations of the polycaprolactone and the cellulose nanocrystal in the organic solvent is 16-18 wt%; further preferably, the sum of the mass concentrations of the polycaprolactone and the cellulose nanocrystal in the organic solvent is 18 wt%.

Further, in step S1, the voltage of electrostatic spinning is 16 to 24kV, the sample injection speed is 0.4 to 1.2mL/h, the distance between the needle and the receiver is 20cm, the needle is 20G, that is, the inner diameter of the needle is 0.6 mm; further preferably, the voltage of the electrostatic spinning is 18kV, and the feeding speed is preferably 0.8 mL/h.

The invention also discloses a three-dimensional composite porous support prepared by the preparation method, and the three-dimensional composite porous support has high porosity and good interoperability.

Compared with the prior art, the invention has the following beneficial effects: the invention discloses a preparation method of a three-dimensional composite porous scaffold, which comprises the step of carrying out thermal self-aggregation reaction on a broken and sieved nano-fiber membrane and polycaprolactone in hot water to obtain the three-dimensional composite porous scaffold. Meanwhile, in the technical scheme, the wet material for the thermal self-aggregation reaction is prepared by adopting a method of firstly carrying out electrostatic spinning and then mechanically crushing, the preparation method is simple, the preparation conditions are easy to achieve, and the popularization and the utilization are convenient. In addition, in the preparation method, the crushed short nano fibers and micro fragments are filtered firstly and then are subjected to thermal self-aggregation reaction in a mixed solution of absolute ethyl alcohol and deionized water, so that the short nano fibers can be suspended in the mixed solution fully and uniformly to form a scaffold structure with high porosity.

Drawings

FIG. 1 is a scanning electron micrograph of a nanofiber membrane obtained in example 1 of the present disclosure;

FIG. 2 is a scanning electron micrograph of a nanofiber membrane obtained in example 3 of the present disclosure;

FIG. 3 scanning electron micrographs of nanofiber membranes obtained in some of examples 2 of the present disclosure;

FIG. 4 scanning electron micrographs of nanofiber membranes were obtained in some comparative examples 4 of the present disclosure;

FIG. 5 nanofiber membrane nanofiber diameter profile of the disclosed example 1;

FIG. 6 is a scanning electron micrograph of the three-dimensional composite porous scaffold obtained in comparative example 6 of the present disclosure;

FIG. 7 is a scanning electron micrograph of a three-dimensional composite porous scaffold obtained in example 1 of the disclosure;

FIG. 8 is a scanning electron micrograph of the three-dimensional composite porous scaffold obtained in comparative example 7 of the disclosure of the present invention;

FIG. 9 is a product map of a three-dimensional composite porous scaffold obtained in some embodiments of the present disclosure;

FIG. 10 is a comparison graph of cell viability assay results for three-dimensional composite porous scaffolds obtained in some embodiments of the present disclosure;

FIG. 11 is a comparison graph of ALP activity measurements for three-dimensional composite porous scaffolds obtained in some embodiments of the present disclosure;

FIG. 12 is a graphical representation of alizarin stained 14d, 21d results for three-dimensional composite porous scaffolds obtained in some embodiments of the disclosure; wherein, the ratio of PCL-2D, b is from left to right and from top to bottom: PCL-CNC-2D, c: PCL-CNC-3D, d PCL-3D, e osteogenic medium;

Figure 13 is a comparison of alizarin staining semi-quantitative analysis results for three-dimensional composite porous scaffolds obtained in some embodiments of the present disclosure.

Detailed Description

The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter of the present invention is not limited to the following examples, and any technique realized based on the contents of the present invention is within the scope of the present invention.

In the following examples, Polycaprolactone (PCL) was obtained from Qingdao Dianshi science and technology, having an average molecular weight of 80000.

The cellulose nanocrystal is purchased from the technical company of the quick science, and is of a type ScienceK CNC;

dichloromethane, N-dimethylformamide, and absolute ethanol were purchased from kyotong chemicals, ltd, and analyzed.

Example 1

S1: taking dichloromethane and N, N-dimethylformamide according to a volume ratio of 4:1, uniformly mixing dichloromethane and N, N-dimethylformamide to obtain an organic solvent, then weighing PCL and CNC according to a mass ratio of 16% of the organic solvent to the sum of the masses of Polycaprolactone (PCL) and Cellulose Nanocrystalline (CNC), stirring and mixing the PCL and CNC with the organic solvent to dissolve the PCL and CNC, and carrying out electrostatic spinning according to electrostatic spinning parameters of 18kV voltage, 0.8mL/h sampling speed, 20cm distance between a needle head and a receiver and 20G needle head to obtain a nanofiber membrane with the diameter of 200-600 nm; wherein, the mass ratio of the added CNC to the PCL is 3: 100;

S2: mixing according to the volume ratio of 1:1 to obtain a mixed solvent of absolute ethyl alcohol and deionized water, then weighing 4g of the nanofiber membrane obtained in the step S1, adding the nanofiber membrane into 200mL of the mixed solvent, mechanically stirring and crushing the nanofiber membrane into intermittent accelerated stirring, wherein the stirring speed is 22000rpm, stirring is performed for 5-7 min every time, the intermittent stirring is performed for 15min, stirring is repeated for four times, short nanofiber filaments are obtained by sieving with a 20-mesh sieve, and wet materials are obtained by suction filtration;

s3: uniformly mixing absolute ethyl alcohol and deionized water to obtain a mixed solution with the concentration of the absolute ethyl alcohol being 15wt%, weighing 0.3g of wet material, dispersing the wet material in 2mL of the mixed solution, carrying out thermal self-aggregation reaction for 5min at the temperature of 55 ℃ to obtain aggregates, and then carrying out freeze drying in a vacuum freeze dryer for 24h to obtain a product A.

Example 2

S1: taking dichloromethane and N, N-dimethylformamide according to the volume ratio of 3:2, uniformly mixing dichloromethane and N, N-dimethylformamide to obtain an organic solvent, then weighing PCL and CNC according to the mass ratio of the organic solvent to the sum of the masses of Polycaprolactone (PCL) and Cellulose Nanocrystalline (CNC) of 20%, stirring and mixing the PCL and CNC with the organic solvent to dissolve the PCL and CNC, and carrying out electrostatic spinning according to electrostatic spinning parameters of 18kV voltage, 0.8mL/h sampling speed, 20cm distance between a needle head and a receiver and 20G needle head to obtain a nanofiber membrane with the diameter of 200-600 nm;

S2: mixing according to the volume ratio of 1:2 to obtain a mixed solvent of absolute ethyl alcohol and deionized water, then weighing 4g of the nanofiber membrane obtained in the step S1, adding the nanofiber membrane into 200mL of the mixed solvent, mechanically stirring and crushing the nanofiber membrane into intermittent accelerated stirring, wherein the stirring speed is 22000rpm, every stirring time is 5min, the intermittent stirring time is 15min, stirring is repeated for 4 times, and the obtained short nanofiber filaments are subjected to suction filtration to obtain a wet material;

s3: uniformly mixing absolute ethyl alcohol and deionized water to obtain a mixed solution with the absolute ethyl alcohol concentration of 15wt%, weighing 0.3g of wet material, dispersing the wet material in 2mL of the mixed solution, carrying out thermal self-aggregation reaction at 55 ℃ for 5-7 min to obtain an aggregate, and then carrying out freeze drying in a vacuum freeze dryer for 24h to obtain a product B.

Example 3

In step S1, the mass ratio of the organic solvent to the sum of the mass of PCL and CNC is 14%, and the other preparation steps are substantially the same as in example 1, to obtain product C.

Example 4

In step S1, the parameters of electrostatic spinning are: voltage 16kV, sample injection speed 0.4mL/h, distance between the needle and the receiver 20cm, needle 20G, and other preparation steps are basically the same as example 1, and product D is obtained.

Example 5

In step S1, the parameters of electrostatic spinning are: voltage 24kV, sample injection speed 1.2mL/h, distance between the needle and the receiver 20cm, needle 20G, and other preparation steps are basically the same as example 1, to obtain product E.

Example 6

In step S3, the concentration of absolute ethanol in the mixed solution was 18% by weight, and the other preparation steps were substantially the same as in example 1, to obtain a product F.

Example 7

In step S3, the volume of the mixed solution was taken to be 4mL, and the other preparation steps were substantially the same as in example 1 to obtain product G.

Comparative example 1

The mixed solution obtained by mixing the absolute ethyl alcohol and the deionized water in the step S3 in the example 1 is replaced by the mixed solution with the volume ratio: 4:2:1 mixed solution formed by deionized water, gelatin and absolute ethyl alcohol, weighing 0.3g of wet material, dispersing in 2mL of mixed solution formed by deionized water, gelatin and absolute ethyl alcohol, carrying out thermal self-aggregation reaction at 55 ℃ for 5-7 min to obtain aggregates, and then carrying out freeze drying in a vacuum freeze dryer for 24h, wherein other preparation steps are basically the same as those in example 1, so as to obtain a comparative product 1.

Comparative example 2

In step S3 of example 1, thermal auto-aggregation reaction was carried out at 45 ℃ for 15min, and the other preparation steps were substantially the same as in example 1, to obtain comparative product 2.

Comparative example 3

In step S3 of example 1, thermal self-assembly reaction was carried out at 60 ℃ for 5min, and the other preparation steps were substantially the same as in example 1 to obtain comparative product 3.

Comparative example 4

In step S1 of example 1, the mass ratio of the organic solvent to the sum of the masses of PCL and CNC was 12%, and the other preparation steps were substantially the same as those of example 1, to obtain comparative product 4.

Comparative example 5

In step S3, the concentration of absolute ethanol in the mixed solution was 20% by weight, and the other production steps were substantially the same as in example 1 to obtain comparative product 5.

Comparative example 6

In step S3, the concentration of absolute ethanol in the mixed solution was 5% by weight, and the other production steps were substantially the same as in example 1, to obtain comparative product 6.

Comparative example 7

In step S1 of example 1, CNC was not added to the organic solvent, PCL was added in an amount of 16%, and other preparation steps were substantially the same as those of example 1, to obtain comparative product 7.

Comparative example 8

According to the raw material ratio of example 1, wet materials are obtained according to the steps S1 and S2 of example 1, 0.3g of the wet materials are dispersed in 2mL of a mixed solution obtained by uniformly mixing 15wt% of absolute ethyl alcohol and deionized water, the mixed solution is kept stand for 6min, and then freeze-dried in a vacuum freeze-dryer for 24h, so that a comparative product 8 is obtained.

Comparative example 9

On the basis of comparative example 8, CNC was not added to the organic solvent, and the other preparation steps were substantially the same as in comparative example 8, to obtain comparative product 9.

Comparative example 10

In step S2 of example 1, the nanofiber membrane obtained in step S1 was ground into short nanofiber filaments and nanofiber fragments under liquid nitrogen, and then the short nanofiber filaments were added to a mixed solution of absolute ethanol and deionized water, which was uniformly mixed, and had an absolute ethanol concentration of 15wt%, and a thermal self-assembly reaction was performed, and other preparation steps were substantially the same as those of example 1, to obtain a comparative product 10.

The nanofiber membranes obtained in example 1, example 2, example 3 and comparative example 4 are subjected to electrostatic spinning by using a Scanning Electron Microscope (SEM), the morphology of the obtained nanofiber membrane is shown in figure 1, wherein figure 1 is an SEM image of the nanofiber membrane obtained in example 1; FIG. 2 is an SEM photograph of the nanofiber membrane obtained in example 3; FIG. 3 is an SEM image of the nanofiber membrane obtained in example 2; fig. 4 is an SEM image of the nanofiber membrane obtained in comparative example 4. As can be seen from FIG. 1, the mass ratio of the sum of the mass of PCL and CNC in the organic solvent is 14-20%, and a filamentous nanofiber membrane can be obtained through electrostatic spinning; when the mass ratio of the sum of the mass of PCL and CNC in the organic solvent is 12%, a lot of granular polymers appear, and it is difficult to form uniform filamentous fibers into a nanofiber membrane. Wherein, when the mass ratio of the PCL to the CNC in the organic solvent is 16%, the diameter of the filamentous fiber of the nanofiber membrane is most uniform, and the shape is smooth. The random selection of holes for each SEM image obtained in example 1 was used to measure diameter size, and the results are shown in fig. 5. As can be seen from FIG. 5, the diameter of the nanofiber membrane obtained in the example is mainly concentrated at about 400nm, the uniformity is good, and the distribution is narrow.

The porosity of the products obtained in examples 1 to 7 and comparative examples 1 to 7 was measured by a liquid displacement method, and the measurement results are shown in table 1:

serial number	Porosity (%)
		Example 1	95.81±0.50
Example 2	94.60±1.01
		Example 3	93.19±0.71
Example 4	94.13±1.35
		Example 5	94.23±0.43
Example 6	92.13±0.35
		Example 7	84.13±1.21
Comparative example 1	86.39±1.16
		Comparative example 2	1.45±0.48
Comparative example 3	54.32±1.28
		Comparative example 4	92.13±0.51
Comparative example 5	91.31±1.53
		Comparative example 6	76.54±2.36
Comparative example 7	93.43±1.23

As can be seen from table 1, the porosity of the product prepared from the three-dimensional composite porous Scaffold prepared by the preparation method disclosed by the invention can reach about 93%, which is higher than the porosity of the three-dimensional porous Scaffold prepared by the prior art by adopting multi-layer alternate electrospinning, mechanical extrusion molding, template method, pore-foaming agent addition and other methods (the porosity of the three-dimensional porous Scaffold prepared by the methods is about 90%, such as 92.5% obtained by adding salt pore-forming agent disclosed in patent/article a Manual for Biomaterials/Scaffold fabric Technology, but the pore diameter is about 100 μm), and the porosity of the product a obtained in example 1 is the highest, and can reach 95%. As can be seen from table 1, in step S3, the concentration of absolute ethanol is not too high, nor too low, and an excessively high concentration of absolute ethanol may cause the short nanofibers and the fine fragments to be uniformly dispersed in the mixed solution, but may also cause the PCL to melt, resulting in incomplete and unstable scaffold; and if the concentration of absolute ethyl alcohol is too low, the short nano fibers and the small fragments are difficult to disperse in the mixed solution incompletely to form a conglomerate structure, so that the porosity of the generated three-dimensional composite porous scaffold is reduced, and if the mixed solvent of 15% absolute ethyl alcohol is adopted, the formed three-dimensional composite porous scaffold has high porosity, good intercommunity among pores and complete whole scaffold, which is shown in fig. 6-7. Wherein FIG. 7 is a scanning electron micrograph of the product obtained in example 1; and wherein FIG. 6 is a scanning electron micrograph of the product obtained in comparative example 6; FIG. 8 is a scanning electron micrograph of a product obtained in comparative example 7.

As can also be seen from table 1, the porosity of the product obtained in comparative example 2 is low because at 45 ℃, due to the low temperature, the short nanofibers and the tiny fragments are in a dispersed state and do not self-aggregate, so that they do not form a three-dimensional porous scaffold structure. The porosity of the product obtained in comparative example 3 was also low because PLC in the short nanofibers and micro chips also melted during self-aggregation at 60 ℃, resulting in difficulty in maintaining the stable three-dimensional porous structure of the obtained product, and also it became black and hard, affecting the elasticity and cell compatibility thereof, see fig. 9. FIG. 9(a) schematic representation of the product obtained in comparative example 3; FIG. 9(b) is a schematic diagram of the product obtained in example 1.

It can also be seen from table 1 that the breaking of the nanofiber membrane by grinding under liquid nitrogen condition results in a product with rather lower porosity, because under liquid nitrogen condition the nanofiber membrane is broken into a small number of short fiber filaments and a large number of fiber fragments, resulting in a long time required for the product to self-aggregate during the self-aggregation reaction, while a large number of fiber fragments are melted by the long time thermal self-aggregation reaction, thus resulting in a decrease in product porosity. In addition, the grinding and crushing are carried out under the condition of liquid nitrogen, the crushing condition is harsh, the operation is complex, the cost is high, and the popularization and the utilization are difficult.

The cell culture was performed on the products obtained in example 1 and comparative examples 8 to 10 in an osteogenic medium, and the cell viability of each product was measured by the MTT assay after 24 hours and 72 hours, respectively, as shown in FIG. 10. As can be seen from fig. 10, the cell viability of the three-dimensional porous scaffold structure is significantly higher than that of the two-dimensional scaffold, and the cell viability of the three-dimensional composite porous scaffold formed after the CNC is added is also significantly improved compared with that of the single PCL three-dimensional porous scaffold.

Cell differentiation was evaluated on the products obtained in example 1 and comparative examples 8 to 10 by a method of detecting alkaline phosphatase (ALP) activity, which was specifically as follows: the products obtained in example 1 and comparative examples 8 to 10 and cells were respectively replaced in osteogenic medium (90% α -MEM medium, 10% fetal bovine serum, 1% diabody (100 μ g/mL streptomycin +100U/mL penicillin), 50 μ g/mL ascorbic acid, 10mM β -glycerophosphate) at the 7 th, 14 th, and 21 th day after completion of the time points; ALP activity was determined by measuring the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate (pNPP) and osteogenic medium was used as a blank. The measurement results are shown in FIG. 11. As can be seen from fig. 11, the ALP activity of the three-dimensional porous scaffold structure was always significantly higher than that of the two-dimensional scaffold, and the ALP activity of the three-dimensional composite porous scaffold formed after CNC addition was also significantly improved compared to that of the single PCL three-dimensional porous scaffold.

Alizarin red is a dye salt with selective binding force to calcium. In the application, after the products obtained in example 1 and comparative examples 8-10 are respectively inoculated with cells 14 and 21d, alizarin red staining is carried out on the products obtained in example 1 and comparative examples 8-10, and the formation of calcifications is detected. In the case of the scaffold for bone tissue engineering, whether it can be differentiated to form bone tissue depends mainly on the structure and properties of the scaffold material. Therefore, the electrostatic spinning polymer nanofiber with the bioactive substances can further improve the bionic property of the nanofiber scaffold and enhance cell attachment, proliferation and differentiation. Alizarin red staining is carried out after

differentiation culture

14d and 21d of MC3T3-E1 is induced by osteogenic differentiation culture medium respectively, and the staining effect is shown in figure 12. As can be seen from fig. 12, the deeper the color of the dyed scaffold obtained after the longer the induced differentiation time, the more calcium nodules are formed, which indicates that the differentiation capability of the scaffold is stronger, and meanwhile, the comparison of the shades of the scaffold colors before and after the scaffold is doped with CNC shows that compared with the PCL scaffold alone, the scaffold containing CNC has the advantages of uniform alizarin red dyeing, obvious dyeing effect, high dyeing speed, deeper color and more calcium nodules being formed, which indicates that the osteogenesis induction and conduction capability of the scaffold material is improved by adding CNC. Further semi-quantitative analysis was performed based on the staining results of alizarin red to evaluate the effect of the prepared scaffold on the differentiation ability of osteoblasts, which is a further evaluation of the osteogenic differentiation ability of the scaffold, and the results are shown in fig. 13. As can be seen from the figure, the calcification degree of the three-dimensional porous scaffold structure is always obviously higher than that of the two-dimensional scaffold, and the calcification degree of the three-dimensional composite porous scaffold formed after the CNC is added is also obviously improved compared with that of a single PCL three-dimensional porous scaffold.

In fig. 10 to 13, the data obtained were examined using GraphPad prism8.0 software, and the data were expressed as mean ± Standard Deviation (SD), and the significance P value of each group difference was calculated: p is greater than 0.05, which indicates that the difference is not significant; 0.01

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

1. The preparation method of the three-dimensional composite porous scaffold is characterized by comprising the following steps of:

s1: dissolving polycaprolactone and cellulose nanocrystal in an organic solvent, uniformly mixing, and then carrying out electrostatic spinning to obtain a nanofiber membrane; the mass ratio of the cellulose nanocrystals to the polycaprolactone is 3: 100; the sum of the mass concentrations of the polycaprolactone and the cellulose nanocrystal in the organic solvent is 14-20 wt%; the organic solvent is dichloromethane and N, N-dimethylformamide in a volume ratio of 1: 4-3: 2;

s2: mechanically stirring and crushing the nanofiber membrane in a mixed solvent, sieving to obtain short nanofiber filaments, and performing suction filtration to obtain a wet material; the mechanical stirring and crushing are intermittent stirring, and the stirring speed is 20000-25000 rpm; stirring for 5min each time, pausing for 15min, repeating for 3-10 times until the nanofiber membrane is broken into short nanofiber filaments;

S3: weighing a proper amount of wet materials, dispersing the wet materials in a mixed solution formed by absolute ethyl alcohol and deionized water, carrying out thermal self-aggregation reaction for 3-10min at the temperature of 50-57 ℃ to obtain aggregates, and then carrying out freeze drying in a vacuum freeze dryer to obtain a three-dimensional composite porous scaffold; in the mixed solution, the mass concentration of the absolute ethyl alcohol is 10-18 wt%.

2. The preparation method according to claim 1, wherein in the step S3, the mass concentration of the wet material in the mixed solution is 0.075-0.2 g/mL.

3. The preparation method according to claim 1, wherein the mixed solvent used in step S2 is absolute ethanol and deionized water in a volume ratio of 1: 1-2.

4. The method of claim 1, wherein in step S1, the voltage of the electrostatic spinning is 16-24 kV, the sample injection speed is 0.4-1.2 mL/h, the distance between the needle and the receiver is 20cm, and the needle is 20G.

5. A three-dimensional composite porous scaffold obtained by the preparation method according to any one of claims 1 to 4.