CN114470336A - Co-extrusion and co-molding mixed hydrogel and 3D printing method of hydrogel support - Google Patents
Co-extrusion and co-molding mixed hydrogel and 3D printing method of hydrogel support Download PDFInfo
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Abstract
A mixed hydrogel capable of being co-extruded and co-molded and a method for 3D printing a hydrogel stent belong to the technical field of biomedical high polymer materials. The method comprises the following steps: placing the mixed hydrogel with the two proportions in a needle cylinder of a 3D printer to form a core-shell pattern distributed biological ink material; extruding the bio-ink material distributed with the preset pattern in the needle cylinder onto a low-temperature printing platform under the preset printing parameters, and preliminarily solidifying the bio-ink material distributed with the preset pattern into a preset support structure; and carrying out calcium ion crosslinking on the preliminarily cured scaffold, and carrying out calcium ion crosslinking and curing on the scaffold structure to form the heterogeneous hydrogel scaffold. According to the mixed hydrogel disclosed by the invention, the coextrudability of the mixed hydrogel is ensured through the high-viscosity sodium alginate hydrogel, the coextrudability of the mixed hydrogel is improved by adding the gelatin or the methacrylamide gelatin, and the material pattern distribution before and after the extrusion molding of the bio-ink material is well maintained.
Description
Technical Field
The invention belongs to the technical field of biomedical high polymer materials, and particularly relates to a mixed hydrogel capable of being co-extruded and co-molded and a method for 3D printing of a hydrogel support.
Background
The clinical availability of artificial tissues and organs remains limited and the advent of tissue engineering has created a hope for research in this area, involving two different technical routes: the in vivo synthesis technology route is that living cells gradually grow into the stent after the stent is implanted into the body; while the in vitro culture type technology route is that the scaffold is combined with living cell tissue culture through cells/growth factors before being implanted into the body. Scaffolds serve on the one hand as carriers for cells and on the other hand provide for new tissue growth. As new tissue develops, the scaffold gradually degrades away. Because different tissues have different functions, the scaffolds are different, and the research on the scaffold materials and structures for repairing different tissues is needed.
The 3D printing technology adopts a discrete/stacking idea to prepare a structure, on one hand, feasibility is provided for personalized bracket manufacturing, a growth microenvironment can be provided, cell adhesion, positioning and field planting can be guided, reasonable spatial distribution of cells on the bracket can be provided, and cell climbing and growth can be guided. In particular the printed scaffold biomechanical and biochemical properties can be customized. On the other hand, 3D printing is easy to realize various mixing and gradient changes of material components, an effective means is provided for regulating and controlling the property of the scaffold, and the regulation and control research of cell behaviors in a certain range and the preparation of functionalized tissues are promoted.
The hydrogel material is widely used as a biological ink material for 3D printing, and the extrusion molding process of the hydrogel material needs to meet the requirements of (1) good shear thinning and thixotropic property, so that the extrudability and the formability of the biological ink material are ensured; (2) biophysical and biochemical properties suitable for cell adhesion, proliferation and differentiation; (3) good biocompatibility and biodegradability. In order for the stent to simultaneously satisfy good biophysical and biochemical characteristics, the stent material should be a composite material composed of two or more different materials. However, the traditional method is realized by mixing materials or chemically modifying, and the mechanical property, the biochemical property and the like of the customized bracket are limited.
The controllable distribution of the material in the 3D printing needle cylinder and the fluid laminar flow state with low Reynolds number are not easy to mix, and feasibility is provided for forming the fiber pattern with the controllable distribution of the material. The gelatin, the methacrylamide gelatin and the sodium alginate hydrogel have good biocompatibility, the gelatin or the methacrylamide gelatin hydrogel has temperature-sensitive curing characteristics, and the sodium alginate hydrogel can realize physical crosslinking curing by combining calcium ions. The formability of the sodium alginate hydrogel is poor, and the forming precision of the bracket can be improved by introducing gelatin or methacrylamide gelatin into the sodium alginate hydrogel. Although the mixed hydrogel material of sodium alginate and gelatin or methacrylamido gelatin has been used for 3D printing, how to regulate and control the mixed hydrogel components distributed in a preset pattern in a syringe for 3D printing makes the mixed hydrogel components meet the requirements of co-extrusion and co-molding, and realizes the manufacture of a stent with higher precision is a problem to be solved in the bio-ink material distributed in the preset pattern for 3D printing.
Disclosure of Invention
The invention aims to solve the problems that sodium alginate and gelatin cannot be co-extruded and co-molded and the like, and provides a method for preparing a 3D printing hydrogel bracket and a co-extruded and co-molded mixed hydrogel.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a co-extrusion and co-molding mixed hydrogel is composed of 100mL of a solvent, 1-5 g of sodium alginate and 3.75-12.5 g of gelatin or methacrylamide gelatin, wherein the solvent is water or PBS.
The high concentration sodium alginate hydrogel has high viscosity, which affects the extrudability of the bio-ink material with preset pattern distribution, but the low concentration sodium alginate hydrogel has poor coextrudability and moldability. When the hydrogel support is manufactured through 3D printing, the low-concentration sodium alginate hydrogel is not easy to form, and the 3D printing precision is poor. The co-molding property of the low-concentration sodium alginate hydrogel distributed in a preset pattern is improved by adding gelatin or methacrylamide-based gelatin, the sodium alginate hydrogel is rapidly subjected to temperature control curing molding by introducing the temperature-sensitive property of the gelatin or methacrylamide-based gelatin hydrogel, and the heterogeneous hydrogel support is formed by further performing calcium ion crosslinking curing after molding.
Further, based on 100ml of mixed hydrogel, the following two conditions are included: (1) 3-4 g of sodium alginate and 5-7.5 g of gelatin or methacrylamide gelatin; (2) 3-5 g of sodium alginate and 3.75-10 g of gelatin or methacrylamide gelatin.
The addition amount of gelatin or methacrylamido gelatin is less affected by the concentration of sodium alginate, because the gelatin or methacrylamido gelatin component regulates the temperature-sensitive curing of the mixed hydrogel. In addition, the addition amount of gelatin or methacrylamido gelatin is too small, and the mixed hydrogel is difficult to form; the addition amount of gelatin or methacrylamido gelatin is too much, and the nozzle is easily blocked in the printing process.
Further, the viscosity of the sodium alginate hydrogel is not less than 3000 Pa.s. The mixed hydrogel with the viscosity can ensure good hydrogel support forming precision.
A method for preparing a 3D-printed hydrogel stent by using the above mixed hydrogel, the method comprising the steps of:
the method comprises the following steps: placing the mixed hydrogel with the two proportions in a needle cylinder of a 3D printer to form a biological ink material distributed in a preset pattern;
step two: extruding the bio-ink material distributed with the preset pattern in the needle cylinder onto a low-temperature printing platform under the preset printing parameters, and preliminarily solidifying the bio-ink material distributed with the preset pattern into a preset support structure;
step three: and carrying out calcium ion crosslinking on the preliminarily cured scaffold, and carrying out calcium ion crosslinking and curing on the scaffold structure to form the heterogeneous hydrogel scaffold. The diameter of the prepared hydrogel scaffold is 0.45-0.75 mm.
Further, in the first step, the two mixture ratios of the mixed hydrogel comprise the following three combinations:
(1) outer layer: sodium alginate 3g and gelatin or methacrylamide gelatin 7.5g, inner layer: 3g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin;
(2) outer layer: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin, wherein the inner layer comprises: 4g of sodium alginate and 5g of gelatin or methacrylamide gelatin;
(3) outer layer: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin, wherein the inner layer comprises: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin.
The mixed hydrogel with any combination of the two proportions can form a bio-ink material distributed in a preset pattern in a 3D printing needle cylinder, and can ensure good coextrudability and coextrudability.
Further, in the first step, the preparation of the bio-ink material with the preset pattern distribution is realized by customizing a mold and introducing a coating material.
The outer layer and the inner layer of the biological ink material distributed in the preset pattern have two characteristics of temperature sensitivity and ionic crosslinking. According to the printing method, the viscosity difference of sodium alginate and gelatin or methacrylamide gelatin at the printing temperature of the needle cylinder is utilized, the two mixed hydrogels in the ratio are co-extruded, and the hydrogel support is printed through two-step curing. Specifically, extrusion molding hydrogel fibers with preset pattern distribution are extruded to a low-temperature printing platform to be solidified to form a preset three-dimensional structure of the scaffold, and finally, a calcium chloride solution is introduced to form the stable hydrogel scaffold by utilizing the chelation of calcium ions and alginate ions.
The printing method provided by the invention can ensure high-precision 3D printing of the hydrogel material with different proportions and preset pattern distribution, and can print out the biological scaffold with different material pattern distribution by modifying the preset pattern distribution and introducing biochemical factors.
Further, in step two, the temperature at the syringe is controlled at 37 ℃, which can facilitate the survival of cells in the 3D printing process. Meanwhile, the viscosity of gelatin or methacrylamide gelatin in the mixed hydrogel in the needle cylinder is far lower than that of sodium alginate, so that the coextrudability of the biological ink material distributed in the preset pattern with the concentration difference of the gelatin or methacrylamide gelatin is ensured; the temperature of the low-temperature printing platform is 0-4 ℃, and the temperature-controlled printing platform enables the mixed hydrogel printed on the low-temperature printing platform to be solidified, so that the 3D printing precision is improved; the printing parameters include: the extrusion air pressure of the needle cylinder is 100-300 kPa, the printing speed is 6-12 mm/s, and the inner diameter of a nozzle of the 3D printer is 0.4-0.6 mm.
Further, in the third step, the calcium ions come from a calcium chloride solution, and the concentration of the calcium chloride solution is 2-5% (w/v). w/v refers to solute mass (g)/solvent volume (ml). 2-5% (w/v) means that the solute is 2-5 g calcium chloride (solute) and then dissolved in 100mL water (solvent).
Further, the printed sample is immersed in the calcium chloride solution for crosslinking for 3-5 min, and then washed with phosphate buffered saline for 3-5 times.
Compared with the prior art, the invention has the beneficial effects that:
(1) the formability of the sodium alginate hydrogel is improved by adding gelatin or methacrylamide gelatin, the coextrusion characteristic of the bio-ink material with the preset pattern distribution at a given syringe temperature is not influenced by the addition of the gelatin or the methacrylamide gelatin, and the balance between the coextrusion and the co-forming of the bio-ink material with the preset pattern distribution is well maintained.
(2) According to the printing method, the mixed hydrogel with two proportions is placed in the needle cylinder to form the biological ink material distributed in the preset pattern. The introduction of an appropriate volume of biochemical factor does not significantly alter the viscosity of the mixed hydrogel. Therefore, the material distribution of the formed hydrogel bracket can be regulated and controlled by introducing biochemical factors and combining with the preset pattern distribution of the biological ink material in the needle cylinder, and the material distribution is applied to tissue engineering research.
(3) The printing method provided by the invention is not only suitable for 3D printing of cell-free biological ink materials, but also suitable for biological 3D printing of biological ink loaded with cells, considering that the temperature in the needle cylinder for 3D printing is favorable for cell survival. In addition, the used hydrogel material has low cost and is easy to popularize.
Drawings
FIG. 1 is a schematic view of a predetermined material distribution within the syringe of the present invention;
figure 2 is a diagram of a 3D printed hydrogel scaffold prepared in example 1;
fig. 3 is a diagram of a 3D printed hydrogel scaffold prepared in comparative example 1;
figure 4 is a drawing of a 3D printed hydrogel scaffold prepared in example 2;
FIG. 5 is a CFD fluid simulation result diagram of the bio-ink material forming process with a predetermined pattern distribution according to example 3;
figure 6 is a drawing of a 3D printed hydrogel scaffold prepared in example 3;
fig. 7 is a diagram of a 3D printed hydrogel scaffold prepared in comparative example 2.
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and the embodiments, but not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The preset material distribution of bio-ink material in the 3D printing cylinder referred to in the following examples is a core-shell distribution with the dimensional parameters as shown in fig. 1, where a is 6mm, b is 9.59mm, and c is 20 mm.
Example 1:
(1) mixing sodium alginate and gelatin to prepare aqueous solution, and obtaining mixed hydrogel solution. The concentration of the sodium alginate in the mixed hydrogel solution of the shell layer and the core layer is 3 percent (w/v), and the concentration of the gelatin is 7.5 percent (w/v);
(2) placing the mixed hydrogel in a needle cylinder of a 3D printer, controlling the temperature in the needle cylinder to be 37 ℃ and the temperature of a printing platform to be 0 ℃, and extruding fibers from the mixed hydrogel into filaments;
the printing parameters are as follows: extrusion pressure: 180kPa, printing speed: 10mm/s, needle size: 22G (inner diameter 0.41mm), the grid support size is 10mm x 1.23mm, and the inter-line spacing is 1 mm.
(3) Extruding the hydrogel onto a printing platform for temperature control solidification to preliminarily obtain a grid support structure;
(4) and (3) immersing the grid support structure of the printing platform into 4% (w/v) calcium chloride solution, crosslinking for 3min to enable the filamentous hydrogel to generate ion crosslinking curing with calcium ions, and washing for 3 times by using phosphate buffered saline to form the hydrogel support. The printed hydrogel scaffold is shown in fig. 2, and the printing effect is better. Subsequently, based on the components of the original mixed hydrogel solution, different regulating factors (such as biochemical factors) or cells are respectively introduced into the core layer and the shell layer, and when the ratio of the sodium alginate to the gelatin in the mixed hydrogel solution of the shell layer and the core layer is kept unchanged after the components are introduced, the hydrogel support with better appearance can be formed.
Comparative example 1:
(1) sodium alginate is prepared into water solution to obtain hydrogel solution. The concentration of the sodium alginate in the hydrogel solution of the shell layer and the core layer is 3% (w/v);
(2) placing the mixed hydrogel in a needle cylinder of a 3D printer, controlling the temperature in the needle cylinder to be 37 ℃ and the temperature of a printing platform to be 0 ℃, and extruding fibers from the mixed hydrogel into filaments;
the printing parameters are as follows: extrusion pressure: 150kPa, printing speed: 10mm/s, needle size: 22G (inner diameter 0.41mm), the grid support size is 10mm x 1.23mm, and the inter-line spacing is 1 mm.
(3) Extruding hydrogel onto a printing platform to preliminarily obtain a grid support structure;
(4) and (3) immersing the grid support structure of the printing platform into 4% (w/v) calcium chloride solution, crosslinking for 3min to enable the filamentous hydrogel to generate ion crosslinking curing with calcium ions, and washing for 3 times by using phosphate buffered saline to form the hydrogel support. The printed hydrogel stent is shown in fig. 3, and it can be seen that the printed hydrogel stent has poor appearance without the introduction of gelatin. In view of the fact that the 3D printing morphology is determined by hydrogel rheological properties (shear thinning, thixotropy and the like) and printing process parameters, different regulatory factors (such as biochemical factors) or cells are respectively introduced based on the existing core layer and shell layer materials, and a hydrogel scaffold with a good morphology is difficult to form.
Example 2:
(1) mixing sodium alginate and gelatin to prepare aqueous solution, and obtaining mixed hydrogel solution. The concentration of the sodium alginate in the mixed hydrogel solution of the shell layer and the core layer is 4% (w/v), and the concentration of the gelatin is 7.5% (w/v);
(2) placing the mixed hydrogel in a needle cylinder of a 3D printer, controlling the temperature in the needle cylinder to be 37 ℃ and the temperature of a printing platform to be 0 ℃, and extruding fibers from the mixed hydrogel into filaments;
the printing parameters are as follows: extrusion pressure: 250kPa, printing speed: 8mm/s, needle size: 22G (inner diameter 0.41mm), the grid support size is 10mm x 1.23mm, and the inter-line spacing is 1 mm.
(3) Extruding the hydrogel onto a printing platform for temperature control solidification to preliminarily obtain a grid support structure;
(4) and (3) immersing the grid support structure of the printing platform into 4% (w/v) calcium chloride solution, crosslinking for 3min to enable the filamentous hydrogel to generate ion crosslinking curing with calcium ions, and washing for 3 times by using phosphate buffered saline to form the hydrogel support. The printed hydrogel scaffold is shown in fig. 4, and it can be seen that the printing effect is better. Subsequently, based on the components of the original mixed hydrogel solution, different regulating factors (such as biochemical factors) or cells are respectively introduced into the core layer and the shell layer, and when the ratio of the sodium alginate to the gelatin in the mixed hydrogel solution of the shell layer and the core layer is kept unchanged after the components are introduced, the hydrogel support with better appearance can be formed.
Example 3:
(1) sodium alginate and gelatin are mixed to prepare aqueous solution, and mixed hydrogel solution with two proportions is obtained. The concentration of the sodium alginate in the shell layer mixed hydrogel solution is 4% (w/v), and the concentration of the gelatin is 7.5% (w/v); the concentration of the sodium alginate in the nuclear layer mixed hydrogel solution is 4% (w/v), and the concentration of the gelatin is 5% (w/v);
(2) and respectively placing the two mixed hydrogels with different proportions in a shell layer and a core layer in a needle cylinder of a 3D printer to form the biological ink material distributed by the core-shell material. Controlling the temperature in the needle cylinder to be 37 ℃ and the temperature of the printing platform to be 0 ℃ so that the mixed hydrogel extrudes fibers into filaments;
the printing parameters are as follows: extrusion pressure: 250kPa, printing speed: 8mm/s, needle size: 22G (inner diameter 0.41mm), the grid support size is 10mm x 1.23mm, and the inter-line spacing is 1 mm.
(3) Extruding the hydrogel onto a printing platform for temperature control solidification to preliminarily obtain a grid support structure;
(4) and (3) immersing the grid support structure of the printing platform into 4% (w/v) calcium chloride solution, crosslinking for 3min to enable the filamentous hydrogel to generate ion crosslinking curing with calcium ions, and washing for 3 times by using phosphate buffered saline to form the hydrogel support. The CFD fluid simulation result of the 3D printed bio-ink material forming process with the preset pattern distribution is shown in fig. 5, and the formed fiber can be seen, so that a core-shell type distribution pattern similar to the preset bio-ink material distribution is formed. The printed hydrogel scaffold is shown in fig. 6, and it can be seen that the printing effect is better. Based on the material ratio of the shell layer to the core layer, the formed hydrogel scaffold has enough mechanical strength on one hand to ensure the integrity of the scaffold structure, and on the other hand, the rigidity is close to the internal tissue, so that the unsatisfactory tissue repair or regeneration effect caused by the stress shielding effect generated after the hydrogel scaffold is implanted into the body is avoided. And then based on the components of the original mixed hydrogel solution, different regulating factors (such as biochemical factors) or cells are respectively introduced into the core layer and the shell layer, and the shell layer and the core layer are kept in the mixed hydrogel solution after introduction, so that the hydrogel scaffold with better appearance can be formed when the ratio of sodium alginate to gelatin is unchanged.
Comparative example 2:
(1) sodium alginate and gelatin are mixed to prepare aqueous solution, and mixed hydrogel solution with two proportions is obtained. The concentration of the sodium alginate in the shell layer mixed hydrogel solution is 3% (w/v), and the concentration of the gelatin is 7.5% (w/v); the concentration of the sodium alginate in the nuclear layer mixed hydrogel solution is 3% (w/v), and the concentration of the gelatin is 5% (w/v);
(2) and respectively placing the two mixed hydrogels with different proportions in a shell layer and a core layer in a needle cylinder of a 3D printer to form the biological ink material distributed by the core-shell material. Controlling the temperature in the needle cylinder to be 37 ℃ and the temperature of the printing platform to be 0 ℃ so that the mixed hydrogel extrudes fibers into filaments;
the printing parameters are as follows: extrusion pressure: 180kPa, printing speed: 10mm/s, needle size: 22G (inner diameter 0.41mm), the grid support size is 10mm x 1.23mm, and the inter-line spacing is 1 mm.
(3) Extruding the hydrogel onto a printing platform for temperature control solidification to preliminarily obtain a grid support structure;
(4) and (3) immersing the grid support structure of the printing platform into 4% (w/v) calcium chloride solution, crosslinking for 3min to enable the filamentous hydrogel to generate ion crosslinking curing with calcium ions, and washing for 3 times by using phosphate buffered saline to form the hydrogel support. As shown in fig. 7, the hydrogel stent printed by the mixture ratio of the shell layer material and the core layer material has a poorer appearance than that of the hydrogel stent printed by the embodiment 3, that is, when the mixture ratio of the core layer material and the core layer material is different, the following mixture ratios are selected to achieve a better molding effect: the concentration of sodium alginate is 4% (w/v), the concentration of gelatin is 7.5% (w/v); the concentration of sodium alginate in the core layer mixed hydrogel solution was 4% (w/v) and the concentration of gelatin was 5% (w/v).
Claims (9)
1. A coextrudable, coformable, hybrid hydrogel, characterized in that: the mixed hydrogel is composed of 100mL of solvent, 1-5 g of sodium alginate and 3.75-12.5 g of gelatin or methacrylamide gelatin, and the solvent is water or PBS.
2. A coextrudable, coform hybrid hydrogel according to claim 1, characterized in that: based on 100ml of mixed hydrogel, the following two conditions are included: (1) 3-4 g of sodium alginate and 5-7.5 g of gelatin or methacrylamide gelatin; (2) 3-5 g of sodium alginate and 3.75-10 g of gelatin or methacrylamide gelatin.
3. A coextrudable, coformable, hybrid hydrogel according to claim 1 or 2, characterized in that: the viscosity of the sodium alginate hydrogel is not lower than 3000 Pa.s.
4. A method for preparing a 3D printed hydrogel scaffold by using the mixed hydrogel of any one of claims 1 to 3, wherein the method comprises the following steps: the method comprises the following steps:
the method comprises the following steps: placing the mixed hydrogel with the two proportions in a needle cylinder of a 3D printer to form a biological ink material distributed in a preset pattern;
step two: extruding the bio-ink material distributed with the preset pattern in the needle cylinder onto a low-temperature printing platform under the preset printing parameters, and preliminarily solidifying the bio-ink material distributed with the preset pattern into a preset support structure;
step three: and carrying out calcium ion crosslinking on the preliminarily cured scaffold, and carrying out calcium ion crosslinking and curing on the scaffold structure to form the heterogeneous hydrogel scaffold.
5. The method of claim 4, wherein the method comprises the steps of: in the first step, the mixed hydrogel with two proportions comprises the following three combinations:
(1) outer layer: sodium alginate 3g and gelatin or methacrylamide gelatin 7.5g, inner layer: 3g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin;
(2) outer layer: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin, wherein the inner layer comprises: 4g of sodium alginate and 5g of gelatin or methacrylamide gelatin;
(3) outer layer: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin, wherein the inner layer comprises: 4g of sodium alginate and 7.5g of gelatin or methacrylamide gelatin.
6. The method for preparing 3D printed hydrogel scaffold using mixed hydrogel according to claim 4 or 5, wherein: in the first step, the preparation of the bio-ink material with preset pattern distribution is realized by customizing a mould and introducing a coating material.
7. The method of claim 4, wherein the method comprises the steps of: in the second step, the temperature of the needle cylinder is controlled to be 37 ℃; the temperature of the low-temperature printing platform is 0-4 ℃; the printing parameters include: the extrusion air pressure of the needle cylinder is 100-300 kPa, the printing speed is 6-12 mm/s, and the inner diameter of a nozzle of the 3D printer is 0.4-0.6 mm.
8. The method of claim 4, wherein the method comprises the steps of: in the third step, the calcium ions come from a calcium chloride solution, and the concentration of the calcium chloride solution is 2-5% (w/v).
9. The method for preparing 3D printed hydrogel scaffold using mixed hydrogel according to claim 4 or 8, wherein: and immersing the printed sample into the calcium chloride solution for crosslinking for 3-5 min, and then washing for 3-5 times by using phosphate buffered normal saline.
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