CN111166933B

CN111166933B - 3D prints composite support of degradable polymer support and photocrosslinking aquogel

Info

Publication number: CN111166933B
Application number: CN202010027576.2A
Authority: CN
Inventors: 杨熙; 崔文国; 余嘉; 刘星志; 柯东旭
Original assignee: Suzhou Nuopu Regenerative Medicine Co ltd
Current assignee: Suzhou Nuopu Regenerative Medicine Co ltd
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-09-04
Anticipated expiration: 2040-01-10
Also published as: CN111166933A

Abstract

The invention relates to a 3D printed degradable polymer scaffold and photo-crosslinking hydrogel composite scaffold, which comprises a 3D printed degradable polymer scaffold, wherein the inside of the 3D printed degradable polymer scaffold comprises crosslinked photo-crosslinking hydrogel with high substitution degree and low substitution degree, and preferably a Polycaprolactone (PCL) scaffold and methacrylic acid anhydrization gelatin (GelMA) with different substitution degrees are subjected to crosslinking compounding. In the composite scaffold, the degradable polymer scaffold printed by 3D has good mechanical property; the photo-crosslinking hydrogel with high substitution degree has high crosslinking degree, can form a fiber network and micropores, and well supports cells; the photo-crosslinking hydrogel with low substitution degree has a plurality of active sites, is beneficial to cell adhesion and growth, and can absorb a large amount of nutrient solution. The inner layer and the outer layer of the composite scaffold are suitable for cell growth and vascularization through the cooperation of the three, and when the scaffold is used for medical human body repair, the scaffold is integrated to promote new tissue regeneration.

Description

3D prints composite support of degradable polymer support and photocrosslinking aquogel

Technical Field

The invention relates to the field of tissue engineering scaffolds, in particular to a 3D printed degradable polymer scaffold and a composite scaffold of photo-crosslinking hydrogel with different degrees of substitution and crosslinking.

Background

The tissue engineering scaffold material is a material which can be combined with tissue living cells and can be implanted into organisms. The scaffold material is beneficial to being used as a carrier of cells and promoting cell propagation and differentiation. In addition, the material needs to have certain mechanical strength, and the stent is degraded into harmless metabolites.

The tissue engineering scaffold material is divided into a natural scaffold material and an artificial synthetic scaffold material. The natural scaffold material comprises collagen, gelatin, fibrin, chitosan, alginate, hyaluronic acid and the like, and is characterized by high degradation speed, biological active sites and suitability for cell growth; the artificially synthesized scaffold material comprises polycaprolactone, polylactic acid, polyglycolic acid, polyethylene glycol and the like, and is characterized by good mechanical property, slow degradation speed and few bioactive sites. For example, Polycaprolactone (PCL) is a synthetic polymer material, has good biocompatibility, degradability, toughness and strength, is suitable for being used as a scaffold material for tissue engineering, but has no bioactivity, smooth surface, strong hydrophobicity and is not suitable for cell adhesion and growth. Gelatin is a natural biological macromolecular material, is a product of partial degradation of collagen, has good degradability, biocompatibility and bioactivity, is beneficial to cell adhesion, and can promote the growth of cells; but the mechanical property is poor, and the paint is easy to be corroded by bacteria and is not water-resistant.

The composite stent of natural stent material and artificially synthesized stent material is prepared, and the stent with excellent performance can be obtained by combining the properties of the natural stent material and the artificially synthesized stent material. For example, CN102242463B discloses a method for preparing a gelatin/polycaprolactone composite nanofiber membrane by electrospinning, which comprises preparing a gelatin/polycaprolactone mixed solution into a spinning solution, and obtaining a gelatin/polycaprolactone composite nanofiber membrane by electrospinning, wherein the gelatin/polycaprolactone composite nanofiber membrane has good properties and is also beneficial to cell growth. However, the scaffold material obtained by the method has a single pore diameter, and the polycaprolactone and the gelatin are completely mixed, so that the mechanical property of the polycaprolactone can be reduced, and the promotion effect of the gelatin on cell growth can be hindered.

The 3D printing technology is one of rapid prototyping technologies, is widely used for printing various supports, and can accurately print the required support aperture and fiber diameter. The 3D printing of PCL scaffolds has been reported in the literature, for example CN110327495A discloses the preparation of tissue engineering auricle morphology composite scaffolds by 3D printing of PCL substrates, and the use of the scaffolds to construct accurate human ear morphology cartilage in vitro. However, the above documents only use a single degradable polymer scaffold material, which is not favorable for the adhesion and growth of cells.

The natural polymer is modified by the photo-crosslinking group, and the modified natural polymer can be used for preparing photo-crosslinking hydrogel, common 3D printing and support materials, such as photo-crosslinking gelatin, photo-crosslinking hyaluronic acid, photo-crosslinking chitosan and the like. For example, methacrylated gelatin (GelMA), in which methacryl groups are introduced to make it photosensitive, can permanently convert a colloidal liquid to be cured into a solid under uv light. GelMA has good biocompatibility, and can form a certain pore size after cross-linking, thereby being beneficial to the entry of cells, the entry of nutrients and the removal of metabolites and providing a good environment for the proliferation of cells. In part of the literature, GelMA is used as a substrate for 3D printing, for example, CN109821075A discloses the preparation of a biomaterial, wherein a photoinitiator is added into a GelMA solution, and then the GelMA solution is subjected to 3D printing and ultraviolet irradiation to obtain a GelMA photo-crosslinking material.

Based on the technical problems in the prior art, the invention provides a 3D-printed degradable polymer scaffold and a composite scaffold of photo-crosslinked hydrogel with different degrees of substitution, which has good mechanical properties and can promote cell growth.

Disclosure of Invention

The first objective of the present invention is to provide a composite scaffold of a degradable polymer scaffold and a photo-crosslinked hydrogel, which is characterized by comprising a 3D printed degradable polymer scaffold, wherein the photo-crosslinked hydrogel is compounded inside the 3D printed degradable polymer scaffold, the photo-crosslinked hydrogel includes a first-degree-of-substitution photo-crosslinked hydrogel and a second-degree-of-substitution photo-crosslinked hydrogel that are cross-linked with each other, and the degree of cross-linking of the first-degree-of-substitution photo-crosslinked hydrogel is higher than that of the second-degree-of-substitution photo-crosslinked hydrogel.

The preparation method of the composite scaffold comprises the following steps: and 3D printing to obtain the degradable high-molecular scaffold, firstly soaking the degradable high-molecular scaffold into a photo-crosslinking hydrogel raw material solution with a high substitution degree, then performing ultraviolet crosslinking, and then soaking the degradable high-molecular scaffold into a photo-crosslinking hydrogel raw material solution with a low substitution degree, and then performing ultraviolet crosslinking to obtain the photo-crosslinking hydrogel and degradable high-molecular composite scaffold with different substitution degrees.

The second purpose of the invention is to improve the bonding tightness of the surface of the degradable high-molecular scaffold and the photo-crosslinking hydrogel, improve the crosslinking of the photo-crosslinking hydrogel raw material on the surface of the degradable high-molecular scaffold, perform the treatment by means of solution soaking treatment, grafting modification and the like on the degradable high-molecular scaffold, and then perform the subsequent steps of soaking the photo-crosslinking hydrogel raw material solution and crosslinking.

The third purpose of the invention is to select the degradable polymer and the photocrosslinking hydrogel according to the types, and the 3D printed degradable polymer scaffold and the composite scaffold of the photocrosslinking hydrogel with different degrees of substitution are used as various tissue composite scaffolds for culturing cells and tissues in vitro; or made into various products for medical human tissue repair, such as GelMA/PCL composite scaffold used as bone tissue engineering scaffold, in vitro bone tissue culture, or GelMA/PCL composite scaffold implanted into human body for medical human bone defect repair.

1.3D prints degradable polymer support

The 3D printing comprises the following steps: the support printing model is designed, degradable polymers are added into the 3D printer, corresponding parameters are set, extrusion is carried out through a fine spray head, and the support is formed through superposition. The 3D of degradable polymer support prints fibre diameter 0.1 ~ 0.5mm, and the fibre interval is 0.3 ~ 1.0 mm.

In the process of 3D printing of the degradable polymer stent, the diameter and the fiber distance of the control fiber can be controlled by adjusting the size of the nozzle and printing a model, and the aperture and the mechanical property of the stent can be controllably adjusted. Meanwhile, the degradable high-molecular scaffold forms a large hole, when the scaffold is crosslinked after being soaked in the photocrosslinking hydrogel raw material solution, the crosslinked hydrogel can form a hydrogel fiber microporous structure in the large hole, and the adhesion growth of cells can be promoted more effectively due to the matching of the micropores and the large hole.

In addition, the degradable polymer scaffold prepared by electrostatic spinning can also achieve most of the technical effects of the invention, but the diameter and pore size of the fiber cannot be controlled, and the degradable polymer scaffold is not a preferred technical scheme.

2. Preparation method of photo-crosslinking hydrogel raw material

The raw material of the photo-crosslinking hydrogel is a natural macromolecular compound modified by a photo-crosslinking group.

Further, the raw material of the photo-crosslinking hydrogel can be selected from one or more of photo-crosslinking gelatin, photo-crosslinking collagen, photo-crosslinking fibrin, photo-crosslinking hyaluronic acid, photo-crosslinking chitosan, photo-crosslinking dextran and photo-crosslinking alginate.

Further, the photo-crosslinking group can be selected from one or more of methacrylate group, coumarin group, cinnamic acid group and azide group.

Further, GelMA is preferably used as a raw material of the photo-crosslinked hydrogel.

Further, the first substitution degree photo-crosslinking hydrogel means that the substitution degree of the raw material of the photo-crosslinking hydrogel is 60-90%, and preferably 80%; the second substitution degree photo-crosslinking hydrogel is that the substitution degree of the photo-crosslinking hydrogel raw material is 10-60%, and preferably 50%; the substitution degree of the photo-crosslinking hydrogel raw material refers to the percentage of the substitution number of photo-crosslinking groups in the substitutable groups of the photo-crosslinking hydrogel raw material; for example, the degree of substitution of GelMA refers to the number of methacrylate group substitutions as a percentage of the total amino groups of the gelatin.

The method comprises the steps of modifying a natural high molecular compound by using a photo-crosslinking group compound to obtain a crosslinking hydrogel raw material, and controlling the process parameters such as the dosage of the photo-crosslinking group compound, the reaction time and the like to obtain the crosslinking hydrogel raw materials with different degrees of substitution.

The invention takes GelMA with high degree of substitution and GelMA with low degree of substitution as examples, the specific method for preparation is as follows: preparing a solution with the pH value of 9 from a certain amount of gelatin, heating the solution to 50 ℃ in a water bath, dropwise adding a certain amount of methacrylamide into the solution, stirring at a high speed to start a substitution reaction, adding PBS (phosphate buffer solution) after the reaction is finished to adjust the pH value of the solution to 7.4 to stop the substitution reaction, filtering the solution after the reaction through filter paper, dialyzing, and freezing and freeze-drying the final solution for later use.

Furthermore, in order to improve the substitution degree of methacrylate groups of the gelatin and improve the crosslinking effect, the degradable gelatin can be adopted, the molecular weight is lower, more amino groups are exposed, and the crosslinking is more sufficient. The degradable gelatin can be selected as thermally degraded gelatin, and a typical preparation method is that a gelatin solution is heated for a certain time under a neutral condition to be degraded, so that the viscosity and the molecular weight are reduced, and the substitution reaction and the subsequent soaking crosslinking reaction are facilitated. A typical example is that gelatin is dissolved in PBS solution with pH value of 7 and is thermally degraded for 1-2h under the hydrothermal condition of 110-130 ℃ to obtain the gelatin.

The GelMA with high substitution degree has high crosslinking degree, can form a fiber network and micropores on the degradable polymer scaffold, can play a good supporting role on the adhesion of cells, but part of active groups of the GelMA with high substitution degree are covered, so that the GelMA with high substitution degree has fewer active sites and slower degradation speed; and the more complete the crosslinking, the denser the formed network structure is, so that the mechanical property is enhanced and the water absorption capacity is weakened, which is not beneficial to absorbing the nutrient solution. Therefore, the composite scaffold prepared by the GelMA with single high substitution degree and the degradable polymer scaffold is not beneficial to the growth of cells.

The GelMA with low substitution degree has more active sites, can be used as nutrition for cell adhesion growth, has high swelling rate and can adsorb a large amount of nutrient solution; gels can also be formed by appropriate crosslinking. However, if only a single low degree of substitution GelMA is used, the degree of crosslinking is low, the strength is low and the degradation is too rapid, which is detrimental to the continued proliferation and differentiation of cells.

The GelMA with high degree of substitution and low degree of substitution is matched with the degradable high polymer scaffold to form a composite scaffold; the degradable polymer scaffold provides good mechanical property and macropores, and the GelMA with high substitution degree forms a fiber network and micropores in the degradable polymer scaffold, so that the degradable polymer scaffold plays a good supporting role in cell adhesion; the GelMA with low substitution degree has more bioactive sites and strong capability of swelling and absorbing nutrient substances, and after the scaffold is implanted into the injured tissue, elements for promoting tissue regeneration, such as peripheral nutrient components, stem cells and the like, can be directly recruited into the scaffold to induce the functional reconstruction of the tissue. With the growth of cells, GelMA with low degree of substitution is firstly degraded, a growth space is reserved, and at the moment, GelMA with high degree of substitution also basically keeps a fiber network and micropores; the cells continue to grow, the degradable high-molecular scaffold is partially degraded along with the degradation of the cross-linked GelMA with high substitution degree, and certain mechanical property can be kept; finally, the scaffold is completely degraded, cells grow, differentiate, sink and the like, and new bone tissues are generated.

3. Composite crosslinking of degradable high-molecular scaffold and photo-crosslinking hydrogel raw material

The degradable polymer is artificially synthesized, and can be selected from one or more of Polycaprolactone (PCL), polylactic acid (PLA), Polyglycolide (PGA), polylactic acid-glycolide copolymer (PLGA), Polydioxanone (PDS), polylactic acid caprolactone (PLCL), polylactic acid-ethylene glycol-lactic acid copolymer (PLA-PEG-PLA), polyethylene glycol (PEG) and methoxy polyethylene glycol (mPEG), and the PCL is preferably selected. Taking the composite crosslinking of PCL and GelMA with the degree of substitution of 60-90% and GelMA with the degree of substitution of 10-60% as an example, the method specifically comprises the following steps:

(1) adding the prepared GelMA with high substitution degree into a PBS (phosphate buffer solution), heating and dissolving the GelMA in water bath at 50-70 ℃, and then adding a photoinitiator with the mass of 0.005-1% of that of the solution and 10-20% of glycerol to prepare a solution A;

(2) adding the prepared GelMA with low substitution degree into a PBS (phosphate buffer solution), heating and dissolving the GelMA in water bath at 50-70 ℃, adding a photoinitiator accounting for 0.005-1% of the mass of the solution and glycerol accounting for 10-20% of the mass of the solution, and preparing a solution B;

(3) soaking the PCL bracket in the solution A for 5-10min, and carrying out ultraviolet crosslinking for 2-5 min for the first time;

(4) before the second ultraviolet crosslinking, the composite scaffold subjected to the first ultraviolet crosslinking is soaked in the solution B for 1-3 times, and each time lasts for 5-30 min, so that the solution A is replaced.

(5) And soaking the PCL support in the solution B for 5-10min, and performing ultraviolet crosslinking for 1-3 min for the second time.

In the crosslinking method, GelMA with high substitution degree has longer crosslinking time, which is beneficial to the GelMA with high substitution degree to form a fiber network and reserve a large amount of micropores; the crosslinking time adopted by the GelMA with low substitution degree is shorter, which is beneficial to the GelMA with low substitution degree to fill and swell.

4. Improving the crosslinking of the photo-crosslinking hydrogel raw material on the surface of the degradable polymer scaffold

Most degradable polymer scaffolds are hydrophobic or have too few surface active groups, and the photo-crosslinking hydrogel raw materials are hydrophilic, so that the photo-crosslinking hydrogel raw materials are difficult to adsorb and crosslink on the surfaces of the degradable polymer scaffolds, and a step of improving the surfaces of the degradable polymer scaffolds is included before the degradable polymer scaffolds are soaked in the crosslinking hydrogel raw material solution.

Taking a PCL support as an example, in the aspect of improving hydrophilicity, NaOH can be used for soaking the support to moderately degrade and carboxylate the surface of the PCL support, so that the surface hydrophilicity of the 3D printing support is improved, and photo-crosslinking hydrogel raw materials are assembled and combined on the surface of the PCL support subjected to carboxylation in an electrostatic manner, so that excellent interface combination is realized, and the cell adsorption function and the structural stability of the 3D printing support are improved. A typical example is to soak the PCL scaffold in 1M NaOH solution for 10 min. Other treatments to improve hydrophilicity include plasma treatment of the composite scaffold or treatment with hexamethylenediamine, etc.

In addition, the polypeptide is a degradation product of a protein high-molecular raw material, has high hydrophilicity, is grafted on the surface of the PCL, and can also improve the affinity between the cross-linked hydrogel raw material and the surface of the PCL. One typical example is: the PCL scaffold was activated with a 10% (w/w) solution of 1,6-hexanediamine/isopropanol (DEA/IPA hexanediamine/isopropanol) to formulate the polypeptide: EDC: and (3) putting the PCL scaffold into the mixed solution, wherein the molar ratio of NHS is 4:4:1, and grafting the polypeptide. Further, the polypeptide can be a polypeptide obtained by completely degrading gelatin, and can also be a small molecule active peptide, such as erythropoietin EPO and RGD polypeptide.

In addition, when the GelMA/PCL bone composite scaffold is prepared, the GelMA can be uniformly coated on the surface of the PCL scaffold by adding glycerol, so that the cell adhesion of the PCL surface is improved.

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

1. in the composite scaffold, the degradable polymer scaffold printed by 3D has good mechanical property and macropores; the photo-crosslinking hydrogel with high substitution degree has higher crosslinking degree, can form micropores and fiber networks in the macropores of the degradable polymer scaffold, has low swelling rate and slightly slow degradation rate, plays a supporting role in the macropores, and is suitable for the adhesion growth of cells; the low-substitution photo-crosslinking hydrogel has low crosslinking degree, but has a plurality of active sites, good swelling effect, can absorb a large amount of components for promoting tissue regeneration, has high degradation speed, can be rapidly degraded along with the growth of cells, and plays a role in nutrition and initial support in macropores.

2. Through surface hydrophilic treatment such as NaOH soaking and plasma treatment on the degradable polymer scaffold, or grafting polypeptide on the degradable polymer scaffold, the surface affinity of the photo-crosslinking hydrogel raw material and the degradable polymer scaffold can be improved, and the crosslinking and bonding compactness of the photo-crosslinking hydrogel raw material on the surface of the scaffold are improved.

3. The 3D printing degradable polymer and photo-crosslinking hydrogel composite stent prepared by the invention is suitable for cell growth and vascularization from the inner layer to the outer layer, and when the stent is used for medical human body repair, the stent is integrated to promote new tissue regeneration.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a pictorial view of a composite stent of example 1;

FIG. 2 is a scanning electron micrograph of the microstructure of the composite scaffold of example 1: (a) is a top view, and (b) is a longitudinal sectional view.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Preparing a PCL support by 3D printing:

the printer used for 3D printing is an OPUS seven-channel biological printing system, the molecular weight of the adopted PCL is 43000, the diameter of the fiber used for 3D printing is 200 μm, the distance between the fibers is 200 μm, and the printing speed is 90 ℃, 600Psi and 250 mm/min. The specific steps of 3D printing are as follows: designing a support printing model, setting corresponding parameters, extruding through a fine nozzle, and laminating fibers to form the PCL support.

Preparing GelMA with low degree of substitution and high degree of substitution:

1. preparation of 0.25M carbonate-bicarbonate buffer (CB buffer): 0.3975g of sodium carbonate and 0.7325g of sodium bicarbonate were added to deionized water, after which the pH of the solution was measured and HCl was added if the pH was less than 9.0 and NaOH was added if the pH was greater than 9.0 to reach a final pH of 9.0.

Preparation reaction of GelMA: 8.0g of gelatin was added to a flask containing 50mL of CB buffer, mixed in an oil bath at 50-60 ℃ under high-speed stirring for 60min and plugged with a rubber plug, and then a syringe needle was inserted into the rubber plug to vent. The solution was injected into another syringe with 0.3mL or 0.5mL of methacrylate at a rate of 0.1mL/h, and after the injection, stirring was continued for 3h in the dark, and then 80mL of PBS was added to stop the reaction. The addition of 0.3mL of the reaction produced a low degree of substitution GelMA (50% degree of substitution) and the addition of 0.5mL of the reaction produced a high degree of substitution GelMA (80% degree of substitution).

3. And (3) dialysis: soaking the 3.5kDa dialysis tube in hot water for 30min, cleaning the tube with deionized water after soaking, and fastening the bottom of the tube. The reacted GelMA solution was filled into a dialysis tube, and then the upper part was tightly bound and dialyzed in deionized water at 37 ℃ for 5 days, with the deionized water being changed 3 times a day.

4. And (3) post-treatment and preservation: pouring out the solution after dialysis, freezing for one day at-20 ℃, taking out, freeze-drying, and finally storing in a vacuum environment at-20 ℃ for later use.

③ the final preparation method of the GelMA and PCL composite stent with different degrees of substitution comprises the following steps:

(1)3D prints PCL support, includes the following step: designing a support printing model, adding PCL into a 3D printer, setting corresponding parameters, extruding through a fine nozzle, and superposing to form a support; soaking the prepared PCL bracket in 1M NaOH1h for hydrolysis treatment;

(2) adding the prepared GelMA with high substitution degree into a PBS solution, heating and dissolving the GelMA with the amount of 5 percent of the mass of the PBS solution in a water bath at 50 ℃, and then adding an Irgacure 2959 photoinitiator with the mass of 0.01 percent of the mass of the solution and 10 percent of glycerol to prepare a solution A;

(3) adding the prepared GelMA with low substitution degree into the PBS solution, heating and dissolving the GelMA with the amount of 5 percent of the mass of the PBS solution in a water bath at 50 ℃, and then adding 0.01 percent of Irgacure 2959 photoinitiator and 10 percent of glycerol based on the mass of the solution to prepare a solution B;

(4) soaking the PCL bracket in the solution A for 5min, and performing first ultraviolet crosslinking for 5 min;

(5) before the second ultraviolet crosslinking, soaking the composite scaffold subjected to the first ultraviolet crosslinking by using the solution B for 3 times, 5min each time, so as to replace the solution A;

(6) then soaking in the solution B for 5min, and carrying out ultraviolet crosslinking for 3min for the second time.

(7) And finally, freeze-drying and storing the sample to obtain the GelMA and PCL composite scaffold with different degrees of substitution and crosslinking.

As can be seen in fig. 1, the composite scaffold has a small size and the composite scaffold is uniformly wrapped with GelMA. As can be seen from FIG. 2, the photo-crosslinked GelMA is tightly combined with the PCL scaffold, and inside the macropores of the PCL scaffold, the photo-crosslinked GelMA also forms a large number of micropores, which is beneficial to the absorption of nutrient solution by the composite scaffold and the growth of cells. The GelMA with low substitution degree can better promote the growth of cells, and the GelMA with high substitution degree has slower degradation speed and is beneficial to the support of the cells.

Example 2

Preparing a PLA stent by 3D printing:

the printer used for 3D printing is an OPUS seven-channel biological printing system, the average molecular weight of the PLA used is 10 ten thousand, the diameter of the fiber for 3D printing is 200 mu m, the distance between the fibers is 200 mu m, the pressure of 100Psi is used for printing, and the printing speed is 250 mm/min. The specific steps of 3D printing are as follows: designing a support printing model, setting corresponding parameters, extruding through a fine nozzle, and laminating fibers to form the PLA support.

Secondly, according to the existing documents, modifying Hyaluronic Acid (HA) by Glycidyl Methacrylate (GMA) to obtain hyaluronic acid with low substitution degree (30%) and high substitution degree (70%);

thirdly, the final preparation method of the composite scaffold of hyaluronic acid and PLA with different degrees of substitution and crosslinking comprises the following steps:

(1)3D prints PLA support, includes the following step: designing a support printing model, adding PLA into a 3D printer, setting corresponding parameters, extruding through a fine nozzle, and superposing to form a support; treating the prepared PLA bracket by adopting nitrogen plasma;

(2) adding the prepared hyaluronic acid with high substitution degree into PBS solution, heating in water bath for dissolving, wherein the using amount of the hyaluronic acid is 3% of the mass of the PBS solution, the heating temperature is 40 ℃, and then adding Irgacure 2959 photoinitiator of which the mass is 0.01% of the mass of the solution to prepare solution A;

(3) adding the prepared GelMA with low substitution degree into PBS solution, heating in water bath for dissolving, wherein the using amount of the GelMA is 3% of the mass of the PBS solution, the heating temperature is 40 ℃, and then adding Irgacure 2959 photoinitiator which is 0.01% of the mass of the solution to prepare solution B;

(4) soaking the PLA bracket in the solution A for 5min, and performing ultraviolet crosslinking for 4min for the first time;

(6) then soaking in the solution B for 5min, and carrying out ultraviolet crosslinking for 2min for the second time.

(7) And finally, freeze-drying and storing the sample to obtain the hyaluronic acid and PLA composite scaffold with different substitution degrees and crosslinking degrees.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite scaffold of a degradable polymer scaffold and photo-crosslinking hydrogel is characterized by comprising a 3D printed degradable polymer scaffold, wherein the photo-crosslinking hydrogel is compounded into the 3D printed degradable polymer scaffold; the photocrosslinking hydrogel comprises a first-degree-of-substitution photocrosslinking hydrogel and a second-degree-of-substitution photocrosslinking hydrogel which are crosslinked with each other, wherein the degree of crosslinking of the first-degree-of-substitution photocrosslinking hydrogel is higher than that of the second-degree-of-substitution photocrosslinking hydrogel.

2. The composite scaffold according to claim 1, wherein the first degree of substitution photo-crosslinked hydrogel is a photo-crosslinked hydrogel with a degree of substitution of 60-90% of the raw material; the second substitution degree photo-crosslinking hydrogel is that the substitution degree of the photo-crosslinking hydrogel raw material is 10-60%; the substitution degree of the photo-crosslinking hydrogel raw material refers to the percentage of the substitution number of photo-crosslinking groups in the substitutable groups of the photo-crosslinking hydrogel raw material.

3. The composite scaffold according to claim 2, wherein the first degree of substitution photo-crosslinked hydrogel means that the degree of substitution of the raw material of the photo-crosslinked hydrogel is 80%; the second substitution degree of the photocrosslinking hydrogel means that the substitution degree of the photocrosslinking hydrogel raw material is 50%; the substitution degree of the photo-crosslinking hydrogel raw material refers to the percentage of the substitution number of photo-crosslinking groups in the substitutable groups of the photo-crosslinking hydrogel raw material.

4. The composite scaffold according to claim 2 or 3, wherein the photo-crosslinking hydrogel material is a natural polymer compound modified with a photo-crosslinking group.

5. The composite scaffold according to claim 2 or 3, wherein the photo-crosslinked hydrogel material is one or more of photo-crosslinked gelatin, photo-crosslinked collagen, photo-crosslinked fibrin, photo-crosslinked hyaluronic acid, photo-crosslinked chitosan, photo-crosslinked dextran, and photo-crosslinked alginate.

6. The composite scaffold according to claim 2, wherein the photo-crosslinking group is one or more of methacrylate group, coumarin group, cinnamate group, azide group.

7. The composite stent of claim 6, wherein the photo-crosslinked hydrogel material is GelMA.

8. The composite stent of claim 7, wherein the GelMA preparation method is a methacrylate group substituted gelatin method comprising the steps of: preparing a solution with pH =9 from a certain amount of gelatin, heating the solution to 50 ℃ in a water bath, dripping a certain amount of methacrylamide into the gelatin solution, stirring the solution to start a substitution reaction, adding PBS (phosphate buffer solution) after the reaction is finished to adjust the pH of the solution to 7.4 to stop the substitution reaction, filtering the solution after the reaction by filter paper, dialyzing the solution, and freezing and freeze-drying the solution for later use.

9. The composite stent of claim 8, wherein gelatin used for preparing the GelMA is thermally degraded gelatin, and the thermally degraded gelatin is prepared by dissolving gelatin in PBS (phosphate buffer solution) with pH =7 and thermally degrading the gelatin for 1-2h under a hydrothermal condition of 110-130 ℃.

10. The composite stent of claim 1, wherein the degradable polymer is a synthetic degradable polymer.

11. The composite stent of claim 1, wherein the degradable polymer is one or more of Polycaprolactone (PCL), polylactic acid (PLA), Polyglycolide (PGA), polylactic acid-glycolide copolymer (PLGA), Polydioxanone (PDS), polylactic acid caprolactone (PLCL), polylactic acid-ethylene glycol-lactic acid copolymer (PLA-PEG-PLA), polyethylene glycol (PEG), methoxy polyethylene glycol (mPEG).

12. The composite stent of claim 1, wherein the degradable polymer is PCL.

13. A method for preparing the composite scaffold of any one of claims 1 to 12, wherein the degradable polymer scaffold is a PCL scaffold, and the raw materials of the photo-crosslinking hydrogel are high-substitution GelMA with a substitution degree of 60 to 90% and low-substitution GelMA with a substitution degree of 10 to 60%, and the method comprises the following steps:

(1)3D prints PCL support, includes the following step: designing a support printing model, adding PCL into a 3D printer, setting corresponding parameters, extruding through a fine nozzle, and superposing to form a support; soaking the prepared PCL bracket in 1-5M NaOH for 1-24h for hydrolysis;

(2) adding the prepared GelMA with high substitution degree into a PBS (phosphate buffer solution), heating and dissolving the GelMA in water bath at 50-70 ℃, and then adding a photoinitiator with the mass of 0.005-1% of that of the solution and 10-20% of glycerol to prepare a solution A;

(3) adding the prepared GelMA with low substitution degree into a PBS (phosphate buffer solution), heating and dissolving the GelMA in water bath at 50-70 ℃, and then adding a photoinitiator accounting for 0.005-1% of the mass of the solution and glycerol accounting for 10-20% of the mass of the solution to prepare a solution B;

(4) soaking the PCL bracket in the solution A for 5-10min, and carrying out ultraviolet crosslinking for 2-5 min for the first time; and then soaking in the solution B for 5-10min, and carrying out secondary ultraviolet crosslinking for 1-3 min.

14. The method according to claim 13, wherein in the step (4), before the second uv crosslinking, the first uv crosslinked composite scaffold is soaked with the solution B for 1 to 3 times, each time for 5 to 30min, to replace the solution a.

15. Use of the composite scaffold of any one of claims 1 to 12 as a scaffold for tissue engineering, for culturing cells and tissues in vitro.

16. A composite scaffold according to any one of claims 1 to 12 for use as a medical product for the repair of human tissue.

17. A composite scaffold according to any one of claims 1 to 12 for use as a medical product for bone repair in humans.