CN113679884A - Tissue engineering hydrogel scaffold for promoting cell migration and preparation method thereof, and 3D printing slurry and preparation method thereof - Google Patents
Tissue engineering hydrogel scaffold for promoting cell migration and preparation method thereof, and 3D printing slurry and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tissue engineering hydrogel scaffold for promoting cell migration, which comprises 1-2 parts of polyethylene glycol (glycol) diacrylate, 8-32 parts of a photoinitiator, 0.15-1.5 parts of polyphosphate and 1.5-3 parts of sodium alginate. The invention also discloses a preparation method of the tissue engineering hydrogel scaffold for promoting cell migration, and 3D printing slurry for preparing the tissue engineering hydrogel scaffold for promoting cell migration and a preparation method thereof. The tissue engineering hydrogel scaffold disclosed by the invention is good in safety and excellent in mechanical property, and can be used for increasing the extracellular ATP content, promoting the physiological activity of cells and facilitating cell migration.
Description
Technical Field
The invention relates to the field of tissue engineering, in particular to a tissue engineering hydrogel scaffold for promoting cell migration and a preparation method thereof, and 3D printing slurry and a preparation method thereof.
Background
Many diseases and disasters can cause damage to parts of tissues and organs of the human body, and various diseases caused by damage or loss of tissues or organs are the main causes of harm to human health at present. With the increase of clinical transplantation operations, the demand for organs and tissues is rapidly increased, however, the amount of donation of organs is far from satisfying the demand. In the united states, 15% of patients waiting to receive liver and heart transplants die annually due to insufficient donor organs. Although autotransplants and allografts have achieved significant clinical success, this approach has been at the expense of other healthy parts of the patient and the health of the donor and has not met practical requirements. In addition, xenotransplantation (xenotransplantation) tends to result in unregulated long-term immune rejection. In this regard, many biocompatible metals and polymers are used as substitute materials for the repair of damaged organs and tissues. The research application for this aspect is called "tissue engineering" or "regenerative therapeutics".
The tissue engineering scaffold as a carrier of cells plays an important role in the key part of tissue engineering. The tissue engineering scaffold provides a growing and propagating environment for cells and induces the cells to form a tissue structure with a certain geometric shape, provides proper mechanical properties after being implanted into a human body, and can be degraded along with the propagation of the cells and the generation of new tissues in the human body. Therefore, the required scaffold structure and performance are quite different for different damaged tissues. At present, many scientific studies are devoted to the preparation of tissue engineering scaffolds that meet the growth of specific tissues. Due to the advantages of high speed, high precision and customizability, 3D printing gradually becomes the mainstream tissue engineering scaffold preparation method at present. Through the development of many years, the 3D printing technology can prepare the tissue engineering scaffold with the structure size similar to that of human organs and extremely complex.
However, with the complexity of tissue engineering scaffolds, due to the lack of an effective material exchange path, cells inside tissues lack sufficient energy sources, physiological activities are affected, the survival rate is low, and the purpose of tissue regeneration is difficult to achieve. Therefore, the preparation of the tissue engineering hydrogel scaffold which can be used for preparing a complex scaffold through 3D printing and does not influence the activity of internal cells is of great significance.
Disclosure of Invention
In order to overcome the above disadvantages and shortcomings of the prior art, an object of the present invention is to provide a tissue engineering hydrogel scaffold for promoting cell migration, which has good safety and excellent mechanical properties, and can increase the extracellular ATP content, promote the physiological activities of cells, and facilitate cell migration.
The second object of the present invention is to provide a method for preparing the above tissue engineering hydrogel scaffold for promoting cell migration.
The invention also aims to provide a preparation method of the 3D printing slurry for preparing the tissue engineering hydrogel scaffold for promoting cell migration.
The fourth purpose of the invention is to provide the 3D printing paste prepared by the preparation method of the 3D printing paste.
The purpose of the invention is realized by the following technical scheme:
the tissue engineering hydrogel bracket for promoting cell migration comprises the following components in parts by weight
Preferably, the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone.
Preferably, the polyphosphate is sodium polyphosphate.
The preparation method of the tissue engineering hydrogel scaffold for promoting cell migration comprises the following steps:
(1) preparing an aqueous solution of polyethylene glycol (glycol) diacrylate;
(2) adding a photoinitiator into the aqueous solution of the polyethylene glycol (glycol) diacrylate obtained in the step (1), and stirring until the photoinitiator is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator solution;
(3) adding polyphosphate into the polyethylene glycol (glycol) diacrylate/photoinitiator solution prepared in the step (2), and stirring until the polyphosphate is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution;
(4) adding sodium alginate into the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution prepared in the step (3), and stirring the mixture until the mixture is completely uniform to obtain polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate composite slurry;
(5) adding the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate composite slurry obtained in the step (4) into a 3D printing material cylinder, ultrasonically removing bubbles, modeling in a 3D printer, and molding the printing slurry into a bracket;
(6) and (3) placing the stent prepared in the step (5) under an ultraviolet lamp for photocrosslinking, then soaking the stent in a calcium chloride solution for ionic crosslinking, and finally cleaning the stent with deionized water to obtain the polyethylene glycol (glycol) diacrylate/polyphosphate/sodium alginate tissue engineering stent, namely a target product.
Preferably, the preparing an aqueous solution of polyethylene glycol (glycol) diacrylate in the step (1) specifically includes:
adding polyethylene glycol (glycol) diacrylate into ultrapure water, and stirring for 20-30 min to fully dissolve the polyethylene glycol (glycol) diacrylate to obtain a polyethylene glycol (glycol) diacrylate solution.
Preferably, the stirring in the step (2) is specifically: stirring for 5-10 min.
Preferably, the stirring in the step (3) is specifically: stirring for 20-30 min.
Preferably, the stirring in the step (4) is specifically: stirring for 2-4 h.
A preparation method of 3D printing paste comprises the following steps:
(1) preparing an aqueous solution of polyethylene glycol (glycol) diacrylate;
(2) adding a photoinitiator into the aqueous solution of the polyethylene glycol (glycol) diacrylate obtained in the step (1), and stirring until the photoinitiator is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator solution;
(3) adding polyphosphate into the polyethylene glycol (glycol) diacrylate/photoinitiator solution prepared in the step (2), and stirring until the polyphosphate is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution;
(4) and (3) adding sodium alginate into the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution prepared in the step (3), and stirring until the mixture is completely uniform to obtain the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate 3D printing slurry.
3D printing slurry is obtained by the preparation method of the tissue engineering hydrogel scaffold for promoting cell migration.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) in the tissue engineering scaffold prepared by the invention, high-energy phosphate bonds in polyphosphate are subjected to enzymolysis under the action of alkaline phosphatase on cell membranes, so that energy is released, energy is supplied to cells, and physiological activities such as cell migration, cell differentiation and the like are promoted; the problem that the activity of cells in the traditional scaffold is low due to lack of sufficient energy sources is solved, and the migration of the cells can be effectively promoted; solves the problem that the complex tissue engineering scaffold has influence on the physiological activity of cells due to lack of an effective substance exchange way.
(2) A double-network system, namely a covalent crosslinking network of poly (ethylene glycol) diacrylate (PEGDA) and Sodium Alginate (Sodium Alginate, SA) -Ca, is introduced into hydrogel2+The two networks have synergistic effect to enhance the mechanical property of the hydrogel and can be flexibly controlled.
(3) In the invention, a small amount of Ca is added into hydrogel before printing2+Pre-crosslinking is formed, so that the printing performance of the hydrogel is improved, and the hydrogel can be used for printing a complex hollow tissue engineering scaffold.
(4) The tissue engineering scaffold prepared by the invention has good osteogenesis property and bone repair capability, and can effectively achieve the effect of bone defect repair.
(5) The tissue engineering scaffold prepared by the invention overcomes the defect of poor mechanical property of the traditional sodium alginate scaffold, and the prepared scaffold has good mechanical property.
(6) The preparation method has the advantages of simple and easy operation, wide material source, high material bioactivity and wide application prospect in the fields of regenerative medicine, tissue repair and the like.
(7) The invention adopts the biological 3D printing technology, is convenient to form, can control the shape of the material and obtain higher porosity, has accurate and easily-controlled process and stable product quality, and can realize personalized customization.
Drawings
FIG. 1 is a compressive stress-strain curve of a tissue engineering scaffold of a comparative example of the present invention and a pure sodium alginate hydrogel.
Fig. 2 is a graph of the rheological behavior of the tissue engineering scaffold of example 1 of the present invention.
FIG. 3 is a graph comparing the extracellular ATP concentrations of the tissue engineering scaffold of comparative example of the present invention and the tissue engineering scaffold of example 1.
FIG. 4 is a graph comparing the extracellular ATP concentrations of the tissue engineering scaffold of comparative example of the present invention and the tissue engineering scaffold of example 2.
FIG. 5 is a confocal microscope image (scale: 100 μm) of the whole cells of the tissue engineering scaffold according to the comparative example of the present invention.
FIG. 6 is a confocal microscope (scale: 100 μm) of the laser scanning of the cells inside the tissue engineering scaffold according to the comparative example of the present invention.
FIG. 7 is a confocal microscope (scale: 100 μm) of the whole cells of the tissue engineering scaffold of example 2.
FIG. 8 is a confocal microscope (scale: 100 μm) of the laser scanning of the cells inside the tissue engineering scaffold of example 2.
Fig. 9 is a graph of compressive stress-strain curves for all tissue engineering scaffold samples of the test examples of the present invention.
FIG. 10 is a histogram of the compressive modulus of all tissue engineering scaffold samples of the test example of the present invention.
FIG. 11 is a bar graph of ALP activity for 7 and 14 days on different tissue engineering scaffold samples of the test example of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Comparative example 1
(1) Adding 1g of polyethylene glycol (glycol) diacrylate (PEGDA) into 30ml of ultrapure water, stirring for 30min to fully dissolve the mixture to obtain a polyethylene glycol (glycol) diacrylate solution;
(2) adding 8mg of photoinitiator I-2959 into polyethylene glycol (glycol) diacrylate solution, and stirring for 10min until the photoinitiator I-2959 is completely dissolved to obtain polyethylene glycol (glycol) diacrylate/I-2959 solution;
(3) adding 3g of sodium alginate into a polyethylene glycol (glycol) diacrylate/I-2959 solution, and stirring for 4 hours till the mixture is completely uniform to obtain polyethylene glycol (glycol) diacrylate/I-2959/sodium alginate composite slurry;
(4) adding the polyethylene glycol (glycol) diacrylate/I-2959/sodium alginate composite slurry obtained in the step (3) into a 3D printing material cylinder, ultrasonically removing bubbles, modeling in a 3D printer, and forming the printing slurry into a bracket, wherein the size of the bracket is set to 10mm by 4mm, the diameter of a printing needle is 0.41mm, the printing speed is 20mm/s, and the printing pressure is 2 bar.
(5) And (3) placing the scaffold obtained in the step (4) under an ultraviolet lamp for photocrosslinking for 6min, then soaking the scaffold in 3 wt% of calcium chloride solution for ionic crosslinking, and finally cleaning the scaffold with deionized water to obtain the polyethylene glycol (glycol) diacrylate/sodium alginate tissue engineering scaffold, namely the target product.
The compressive strength of this comparative example is shown in figure 1, which is higher than pure sodium alginate.
Example 1
The preparation method of the tissue engineering hydrogel scaffold for promoting cell migration in this example is as follows:
(1) adding 2g of polyethylene glycol (glycol) diacrylate (PEGDA) into 30ml of ultrapure water, and stirring for 30min to fully dissolve the mixture to obtain a polyethylene glycol (glycol) diacrylate solution;
(2) adding 32mg of photoinitiator I-2959 (2-hydroxy-4 '- (2-hydroxyethoxy) -2-methyl propiophenone) into a polyethylene glycol (glycol) diacrylate solution, and stirring for 10min until the photoinitiator I-2959 (2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone) is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/I-2959 solution;
(3) slowly adding 0.3g of sodium polyphosphate into the polyethylene glycol (glycol) diacrylate/I-2959 solution, and stirring for 30min until the sodium polyphosphate is completely dissolved to obtain the polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate solution;
(4) adding 3g of sodium alginate into a polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate solution, and stirring for 4 hours until the mixture is completely uniform to obtain polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate/sodium alginate composite slurry, namely 3D printing slurry;
(5) adding the polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate/sodium alginate composite slurry obtained in the step 4 into a 3D printing material cylinder, ultrasonically removing bubbles, modeling in a 3D printer, and forming the printing slurry into a bracket, wherein the size of the bracket is set to 10mm by 4mm, the diameter of a printing needle is 0.41mm, the printing speed is 15mm/s, and the printing pressure is 1.5 bar.
(6) And (3) placing the scaffold obtained in the step (5) under an ultraviolet lamp for photocrosslinking for 3min, then soaking the scaffold in a 5% calcium chloride solution for ionic crosslinking, and finally cleaning the scaffold with deionized water to obtain the polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate tissue engineering scaffold, namely the target product.
Fig. 2 is a graph showing the rheological behavior of the tissue engineering scaffold prepared from polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate in this example, which shows that the composite slurry has shear thinning property, which is very important for 3D printing.
The tissue engineering scaffold prepared from ethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate in this example and the tissue engineering scaffold of polyethylene glycol (glycol) diacrylate/sodium alginate in the comparative example were subjected to extracellular ATP content test by the following method: the total amount is 5x104The cells were seeded on hydrogel scaffolds and cultured for 3 days. Thereafter, the extracellular ATP concentration is detected using an ATP detection kit.
FIG. 3 is a graph comparing the extracellular ATP content of the cells cultured by using the polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate tissue engineering scaffold of the present example with that of the polyethylene glycol (glycol) diacrylate/sodium alginate tissue engineering scaffold of the comparative example, and it can be seen that the extracellular ATP content of the cells cultured on the present example is higher, about 1.37 times that of the comparative example. Indicating that the extracellular ATP content can be increased after addition of 0.3g of polyphosphate.
Example 2
(1) Adding 2g of polyethylene glycol (glycol) diacrylate (PEGDA) into 30ml of ultrapure water, and stirring for 30min to fully dissolve the mixture to obtain a polyethylene glycol (glycol) diacrylate solution;
(2) adding 16mg of photoinitiator I-2959 into polyethylene glycol (glycol) diacrylate solution, and stirring for 10min until the photoinitiator I-2959 is completely dissolved to obtain polyethylene glycol (glycol) diacrylate/I-2959 solution;
(3) slowly adding 0.9g of sodium polyphosphate into the polyethylene glycol (glycol) diacrylate/I-2959 solution, and stirring for 30min until the sodium polyphosphate is completely dissolved to obtain the polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate solution;
(4) adding 1.5g of sodium alginate into a polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate solution, and stirring for 2 hours until the mixture is completely uniform to obtain polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate/sodium alginate composite slurry, namely 3D printing slurry;
(5) adding the polyethylene glycol (glycol) diacrylate/I-2959/sodium polyphosphate/sodium alginate composite slurry obtained in the step (4) into a 3D printing material cylinder, ultrasonically removing bubbles, modeling in a 3D printer, and forming the printing slurry into a bracket, wherein the size of the bracket is set to 10mm x 4mm, the diameter of a printing needle is 0.27mm, the printing speed is 20mm/s, and the printing pressure is 3 bar.
(6) And (3) placing the stent in the step (5) under an ultraviolet lamp for photocrosslinking for 3min, then soaking the stent in a 5% calcium chloride solution for ionic crosslinking, and finally cleaning the stent with deionized water to obtain the polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate tissue engineering stent, namely the target product.
The tissue engineering scaffold prepared from ethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate in this example and the tissue engineering scaffold of polyethylene glycol (glycol) diacrylate/sodium alginate in the comparative example were subjected to extracellular ATP content test by the following method: the total amount is 5x104The cells were seeded on hydrogel scaffolds and cultured for 3 days. Thereafter, the extracellular ATP concentration is detected using an ATP detection kit.
FIG. 4 is a graph comparing the extracellular ATP content of the cells cultured by using the polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate tissue engineering scaffold of the present example with that of the polyethylene glycol (glycol) diacrylate/sodium alginate tissue engineering scaffold of the comparative example, and it can be seen that the extracellular ATP content of the cells cultured on the present example is higher, about 1.51 times that of the comparative example. Indicating that the extracellular ATP content can be increased after addition of 0.9g of polyphosphate.
Fig. 5 to 8 show the results of the laser scanning confocal microscope testing on the polyethylene glycol (glycol) diacrylate/sodium polyphosphate/sodium alginate tissue engineering scaffold of the present embodiment and the polyethylene glycol (glycol) diacrylate/sodium alginate tissue engineering scaffold of the comparative example, which shows that the cells migrate deeper into the scaffold after the sodium polyphosphate is added.
Test example:
the samples of the test example were prepared in the same manner as in example 2 except that 0 g, 0.15g, 0.3g, 0.9g and 1.5g of sodium polyphosphate were added.
The compressive stress-strain curves of the different samples of the test example are shown in fig. 9, and it can be seen from the graph that the compressive properties of the samples gradually increase with the increasing polyphosphate content.
The compression modulus of the samples of the test examples is shown in fig. 10, and it can be seen from the graph that the compression resistance of the samples is gradually increased with the increasing polyphosphate content, and the corresponding compression modulus is gradually increased, and the compression modulus of 1.5PolyP (polyphosphate) is about 6 times that of 0 PolyP.
The compressive modulus of 0.5PolyP is higher, but the breaking strain is smaller, and the materials of 0, 0.15, 0.3 and 0.9PolyP are selected for the comprehensive consideration to carry out the biological experiment.
ALP activities on days 7 and 14 of the different samples of this test example are shown in FIG. 11, and it can be seen from the graph that on day 7, the ALP contents of the respective components are not greatly different; by day 14, cells cultured on the hydrogel containing the PolyP had an ALP content significantly higher than 0PolyP, indicating that the addition of polyphosphate may contribute bone properties to the material.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The tissue engineering hydrogel bracket for promoting cell migration is characterized by comprising the following components in parts by weight
2. The tissue engineering hydrogel scaffold for promoting cell migration according to claim 1, wherein the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone.
3. The tissue engineering hydrogel scaffold for promoting cell migration according to claim 1, wherein the polyphosphate is sodium polyphosphate.
4. The method for preparing the tissue engineering hydrogel scaffold for promoting cell migration according to any one of claims 1 to 3, comprising the following steps:
(1) preparing an aqueous solution of polyethylene glycol (glycol) diacrylate;
(2) adding a photoinitiator into the aqueous solution of the polyethylene glycol (glycol) diacrylate obtained in the step (1), and stirring until the photoinitiator is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator solution;
(3) adding polyphosphate into the polyethylene glycol (glycol) diacrylate/photoinitiator solution prepared in the step (2), and stirring until the polyphosphate is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution;
(4) adding sodium alginate into the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution prepared in the step (3), and stirring the mixture until the mixture is completely uniform to obtain polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate composite slurry;
(5) adding the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate composite slurry obtained in the step (4) into a 3D printing material cylinder, ultrasonically removing bubbles, modeling in a 3D printer, and molding the printing slurry into a bracket;
(6) and (3) placing the stent prepared in the step (5) under an ultraviolet lamp for photocrosslinking, then soaking the stent in a calcium chloride solution for ionic crosslinking, and finally cleaning the stent with deionized water to obtain the polyethylene glycol (glycol) diacrylate/polyphosphate/sodium alginate tissue engineering stent, namely a target product.
5. The method for preparing a tissue engineering hydrogel scaffold for promoting cell migration according to claim 4, wherein the step (1) of preparing the aqueous solution of polyethylene glycol (glycol) diacrylate specifically comprises:
adding polyethylene glycol (glycol) diacrylate into ultrapure water, and stirring for 20-30 min to fully dissolve the polyethylene glycol (glycol) diacrylate to obtain a polyethylene glycol (glycol) diacrylate solution.
6. The method for preparing the tissue engineering hydrogel scaffold for promoting cell migration according to claim 4, wherein the stirring in the step (2) is specifically: stirring for 5-10 min.
7. The method for preparing the tissue engineering hydrogel scaffold for promoting cell migration according to claim 4, wherein the stirring in the step (3) is specifically: stirring for 20-30 min.
8. The method for preparing the tissue engineering hydrogel scaffold for promoting cell migration according to claim 4, wherein the stirring in the step (4) is specifically: stirring for 2-4 h.
9. A preparation method of 3D printing paste is characterized by comprising the following steps:
(1) preparing an aqueous solution of polyethylene glycol (glycol) diacrylate;
(2) adding a photoinitiator into the aqueous solution of the polyethylene glycol (glycol) diacrylate obtained in the step (1), and stirring until the photoinitiator is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator solution;
(3) adding polyphosphate into the polyethylene glycol (glycol) diacrylate/photoinitiator solution prepared in the step (2), and stirring until the polyphosphate is completely dissolved to obtain a polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution;
(4) and (3) adding sodium alginate into the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate solution prepared in the step (3), and stirring until the mixture is completely uniform to obtain the polyethylene glycol (glycol) diacrylate/photoinitiator/polyphosphate/sodium alginate 3D printing slurry.
10. 3D printing paste obtained by the preparation method of the tissue engineering hydrogel scaffold for promoting cell migration according to claim 9.
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