CN110755685A - 3D printing guar gum gel bracket and preparation method thereof - Google Patents

Abstract

The present invention provides a 3D printing guar gum hydrogel support, which is prepared by the following steps: S1. At room temperature, guar gum is dissolved in ultrapure water to obtain a guar gum aqueous solution, and methacrylic anhydride is added to fully Stir, adjust the pH value of the system to 8-10, continue to react for 2-6 hours, and obtain guar gum methacrylate after the solution is dialyzed, freeze-dried; S2. In aseptic environment, guar gum methyl Acrylate was dissolved in sterile PBS solution containing photoinitiator to prepare a sol, the sol was evenly mixed with chondrocytes, and a scaffold was formed by extrusion printing using a 3D bioprinter, and the scaffold was irradiated with ultraviolet light to cross-link to obtain a 3D Print the guar gel scaffold. The present invention also provides a preparation method of the 3D printed guar gum hydrogel scaffold. The 3D printed guar gum hydrogel scaffold provided by the present invention is expected to become an ideal cartilage tissue engineering scaffold, and is expected to be applied to the regeneration and repair of cartilage defects in clinic.

Description

A kind of 3D printing guar glue hydrogel scaffold and preparation method thereof

technical field

The invention relates to a 3D printed guar glue hydrogel support and a preparation method thereof.

Background technique

Tissue engineering provides a new treatment method for the regeneration and repair of human tissues and organs, including electrospinning and 3D printing technology and other common material processing and forming technologies. The former mainly provides a two-dimensional microenvironment for cells, which It is far from satisfying the three-dimensional microenvironment of bionic tissues and organs. 3D printing technology prints a variety of functional, individualized tissues or organs by placing cells and biomaterials precisely at specific locations in a preset space, and provides unprecedented capabilities and versatility to bridge engineering It is possible to construct individualized functional cartilage by using metaplastic and native tissues. However, the lack of individualized biochemical and mechanical properties of biomaterials is still the main obstacle limiting the application of 3D printing technology.

3D bioprinting has significantly higher requirements on materials than ordinary printing. According to the application purpose, the following requirements must be considered: printability, biocompatibility, degradability, structural and mechanical properties, and biomimetic properties. At present, the most widely used type of 3D bioprinting in the field of tissue engineering is extrusion 3D bioprinting. Extrusion 3D bioprinting requires materials that have both sufficient viscoelasticity to maintain the printed shape and shear thinning properties. Pseudoplasticity to mitigate crush damage to cells during printing.

Commonly used 3D bioprinting materials include natural biomaterials (such as sodium alginate, hyaluronic acid, etc.) and synthetic materials (such as polylactic acid, polyethylene glycol, etc.). Synthetic materials may have problems such as foreign body reaction, high infection rate, slow degradation in vivo, and side effects of degradation products, while natural materials are increasingly used for 3D printing due to their superior biocompatibility and natural degradability. raw material. Sodium alginate and hyaluronic acid have been reported as common natural materials for 3D bioprinting, but there are still some defects in the construction of tissue engineered cartilage: the sodium alginate solution is too viscous, and it often blocks the extrusion during the printing process. The 3D printed hyaluronic acid scaffolds often fail to meet the repair requirements due to the rapid degradation of the 3D printed hyaluronic acid scaffold. In summary, there is an urgent need to find natural biomaterials suitable for 3D bioprinting.

Guar gum is a natural polymer polysaccharide extracted from the leguminous plant - guar, with a molecular weight of about 220,000. It is the most effective and water-soluble natural polymer known. Due to its unique molecular structure characteristics and non-Newtonian rheological properties, guar gum has been widely used in various commercial applications. Previous studies have shown that guar gum has good biocompatibility, biodegradability, and good hydrophilicity at room temperature. It can be used as a drug carrier and hydrogel stent, and it has low cost and wide sources. Since guar gum has the above advantages and meets the material requirements of 3D bioprinting, it is expected to become a new type of 3D bioprinting material. The molecular structure of guar gum contains hydroxyl groups, which can undergo esterification reaction, and is expected to be grafted with methacrylic anhydride for UV crosslinking. At present, UV crosslinking (photocuring) is the most commonly used curing method in hydrogel-based 3D bioprinting, which has the advantages of short curing time and low reaction heat. UV-crosslinked (light-cured) hydrogels are increasingly used in tissue engineering due to their porous internal structure, large internal surface area, low diffusion resistance, easy regulation of physicochemical properties, encapsulation of cells and protection of cell growth, etc. .

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a 3D printed guar gum hydrogel scaffold that can be cross-linked by ultraviolet light and can be compounded with stem cells. The scaffold is expected to become an ideal cartilage tissue engineering scaffold and is expected to be applied in clinical practice. Regenerative repair of cartilage defects.

For solving the above-mentioned technical problems, the technical scheme of the present invention is:

A 3D printed guar gum hydrogel scaffold made by the following steps:

S1. Under normal temperature, completely dissolve guar gum in ultrapure water to obtain guar gum aqueous solution, add methacrylic anhydride and fully stir, adjust the pH value of the system to 8-10 with sodium hydroxide solution, and continue the reaction 2-6 hours, the solution obtained by the reaction is dialyzed with ultrapure water for 5-7 days, and guar gum methacrylate is obtained after complete freeze-drying;

S2. In a sterile environment, completely dissolve guar gum methacrylate in a sterile PBS solution containing a photoinitiator to prepare a sol, and uniformly mix the sol with chondrocytes to make a 3D printed guar composite chondrocyte The glue bio-ink is extruded and printed by a 3D bioprinter, and a scaffold is formed by stacking the ejected filaments layer by layer.

Further, in the step S1, the dosage ratio of guar gum, ultrapure water and methacrylic anhydride is 1g:(50-150)mL:(1-3)mL.

Further, in the step S1, the concentration of the sodium hydroxide solution is 5 mol/L.

Further, in the step S2, the dosage ratio of guar gum methacrylate and the sterile PBS solution containing the photoinitiator is 1 g:(20-50) mL, and the photoinitiator is photoinitiator 2959, and the photoinitiator is photoinitiator 2959. The concentration of the initiator in sterile PBS is 0.1-1% w/v.

Further, in the step S2, the dosage ratio of the sol to the chondrocytes is 1 mL: 1×10 ⁷ .

Another technical problem to be solved by the present invention provides the preparation method of the above-mentioned 3D printed guar glue hydrogel scaffold.

In order to solve the above technical problems, the technical solutions are:

A preparation method of 3D printing guar glue hydrogel stent, comprising the following steps:

S1. Under normal temperature, completely dissolve guar gum in ultrapure water to obtain guar gum aqueous solution, add methacrylic anhydride and fully stir, adjust the pH value of the system to 8-10 with sodium hydroxide solution, and continue the reaction 2-6 hours, the solution obtained by the reaction is dialyzed with ultrapure water for 5-7 days, and guar gum methacrylate is obtained after complete freeze-drying;

S2. In a sterile environment, completely dissolve guar gum methacrylate in a sterile PBS solution containing a photoinitiator to prepare a sol, and uniformly mix the sol with chondrocytes to make a 3D printed guar composite chondrocyte The glue bio-ink is extruded and printed by a 3D bioprinter, and a scaffold is formed by stacking the ejected filaments layer by layer.

Further, in the step S1, the dosage ratio of guar gum, ultrapure water and methacrylic anhydride is 1g:(50-150)mL:(1-3)mL.

Further, in the step S1, the concentration of the sodium hydroxide solution is 5 mol/L.

Further, in the step S2, the dosage ratio of guar gum methacrylate and the sterile PBS solution containing the photoinitiator is 1 g:(20-50) mL, and the photoinitiator is photoinitiator 2959, and the photoinitiator is photoinitiator 2959. The concentration of the initiator in sterile PBS is 0.1-1% w/v.

Further, in the step S2, the dosage ratio of the sol to the chondrocytes is 1 mL: 1×10 ⁷ .

Compared with the prior art, the present invention has the following beneficial effects:

1) Using 3D bioprinting technology to print a guar hydrogel scaffold that can composite cells, the shape of the scaffold is controllable, the accuracy is high, the preparation method is simple, the reaction conditions are mild, and the required conditions are not harsh; the hydrogel scaffold occurs Ultraviolet light cross-linking can be completed in a short time, and the fidelity of the printed scaffold can be better achieved. The obtained 3D printed guar gum hydrogel scaffold has good chondrocyte compatibility and biological activity, and can be used for cartilage. Cells provide a good environment for growth and differentiation, and are expected to be used for clinical repair and regeneration of cartilage defects.

2) The reaction requirements are simple, and the pore size, porosity and mechanical strength of the 3D printed guar hydrogel scaffold obtained by the reaction can be adjusted by adjusting the ratio of guar gum and methacrylic anhydride in the reaction system.

Description of drawings

The accompanying drawings described here are used to provide a further understanding of the present invention and constitute a part of this application, and do not constitute an improper limitation of the present invention. In the accompanying drawings:

Fig. 1 is the electron microscope scanning diagram of the embodiment of the present invention 1;

Fig. 2 is the scanning electron microscope diagram of embodiment 2 of the present invention;

Fig. 3 is the compressive modulus of different substitution rate guar gum hydrogels under the same concentration in the present invention;

4 is a schematic structural diagram of Embodiment 1 of the present invention;

5 is a schematic structural diagram of Embodiment 2 of the present invention;

Figure 6 is a cell staining diagram of Example 1 of the present invention cultured in vitro for 7 days;

FIG. 7 is a cell staining diagram of Example 1 of the present invention cultured in vitro for 14 days.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

Example 1

Follow the steps below to prepare 3D printed guar hydrogel scaffolds:

S1. At room temperature, completely dissolve 0.5 g of guar gum in 25 mL of ultrapure water to obtain an aqueous solution of guar gum, add 0.5 mL of methacrylic anhydride and stir well, the molar ratio of methacrylic anhydride to guar gum is approximately equal to 1. Adjust the pH value of the system to 8 with a sodium hydroxide solution with a concentration of 5 mol/L, continue to react for 2 hours, dialyze the solution obtained by the reaction with ultrapure water for 5 days, and obtain guar gum methyl after complete freeze-drying. Acrylate;

S2. In a sterile environment, according to the dosage ratio of 1g:50mL, guar gum methacrylate was completely dissolved in the sterile PBS solution containing photoinitiator 2959 with a concentration of 0.1%w/v to obtain a concentration of 2 %w/v sol, uniformly mix the sol and chondrocytes according to the dosage ratio of 1mL:1×10 ⁷ to make 3D printing guar gum bioink of composite chondrocytes, and use a 3D bioprinter for extrusion printing. The ejected filaments were stacked layer by layer to form a scaffold, and the scaffold was irradiated with ultraviolet light for 3 minutes to make it cross-linked to obtain a 3D printed guar glue hydrogel scaffold.

Example 2

Follow the steps below to prepare 3D printed guar hydrogel scaffolds:

S1. At room temperature, 0.5g of guar gum was completely dissolved in 50mL of ultrapure water to obtain an aqueous solution of guar gum, and 1mL of methacrylic anhydride was added and stirred thoroughly. The molar ratio of methacrylic anhydride and guar gum was approximately equal to 2 , adjust the pH value of the system to 9 with a concentration of 5mol/L sodium hydroxide solution, continue to react for 4 hours, dialyze the solution obtained by the reaction with ultrapure water for 6 days, and obtain guar gum methacrylic acid after complete freeze-drying ester;

S2. In a sterile environment, according to the dosage ratio of 3g:100mL, guar gum methacrylate is completely dissolved in the sterile PBS solution containing photoinitiator 2959 with a concentration of 0.5%w/v to obtain a concentration of 3 %w/v sol, uniformly mix the sol and chondrocytes according to the dosage ratio of 1mL:1×10 ⁷ to make a 3D printing guar gum bioink for composite chondrocytes, and use a 3D bioprinter for extrusion printing. The ejected filaments were stacked layer by layer to form a scaffold, and the scaffold was irradiated with ultraviolet light for 2 minutes to make it cross-linked to obtain a 3D printed guar gum hydrogel scaffold.

Example 3

Follow the steps below to prepare 3D printed guar hydrogel scaffolds:

S1. At room temperature, completely dissolve 0.5 g of guar gum in 50 mL of ultrapure water to obtain an aqueous solution of guar gum, add 1.5 mL of methacrylic anhydride and fully stir, and the molar ratio of methacrylic anhydride to guar gum is approximately equal to 3. Adjust the pH value of the system to 10 with a sodium hydroxide solution with a concentration of 5mol/L, continue the reaction for 6 hours, dialyze the solution obtained by the reaction with ultrapure water for 7 days, and obtain the guar gum methyl group after being completely freeze-dried. Acrylate;

S2. In a sterile environment, according to the dosage ratio of 6g:100mL, guar gum methacrylate is completely dissolved in the sterile PBS solution containing photoinitiator 2959 with a concentration of 1%w/v to obtain a concentration of 6 %w/v sol, uniformly mix the sol and chondrocytes according to the dosage ratio of 1mL:1×10 ⁷ to make a 3D printing guar gum bioink for composite chondrocytes, and use a 3D bioprinter for extrusion printing. The ejected filaments were stacked layer by layer to form a scaffold, and the scaffold was irradiated with ultraviolet light for 1 minute to cross-link to obtain a 3D printed guar glue hydrogel scaffold.

Example 4

Follow the steps below to prepare 3D printed guar hydrogel scaffolds:

S1. At room temperature, completely dissolve 0.5 g of guar gum in 75 mL of ultrapure water to obtain an aqueous solution of guar gum, add 1.5 mL of methacrylic anhydride and stir well, the molar ratio of methacrylic anhydride to guar gum is approximately equal to 3. Adjust the pH value of the system to 8 with a sodium hydroxide solution with a concentration of 5mol/L, continue to react for 6 hours, dialyze the solution obtained by the reaction with ultrapure water for 7 days, and obtain guar gum methyl after complete freeze-drying. Acrylate;

S2. In a sterile environment, according to the dosage ratio of 1g:20mL, guar gum methacrylate was completely dissolved in the sterile PBS solution containing photoinitiator 2959 with a concentration of 0.8%w/v to obtain a concentration of 5 %w/v sol, uniformly mix the sol and chondrocytes according to the dosage ratio of 1mL:1×10 ⁷ to make a 3D printing guar gum bioink for composite chondrocytes, and use a 3D bioprinter for extrusion printing. The ejected filaments were stacked layer by layer to form a scaffold, and the scaffold was irradiated with ultraviolet light for 2 minutes to make it cross-linked to obtain a 3D printed guar gum hydrogel scaffold.

From Figure 1 and Figure 2, it can be seen that the internal pores of the 3D printed guar hydrogel obtained by changing different methacrylic anhydride/guar ratios (molar ratios of 1 and 2, respectively) during the modification reaction For the structure, the molar ratio of methacrylic anhydride/guar gum is 2 (Fig. 2), the porosity is larger and the pore size is smaller.

As can be seen from accompanying drawing 3, when the concentration is 3%w/v, the guar gum hydrogels with different substitution rates (methacrylic anhydride/guar gum molar ratios are respectively 1 and 2 in the modification process) carry out the mechanical test. , the compressive moduli are 79KPa and 151KPa, respectively, and the molar ratio of methacrylic anhydride/guar gum is 2. The guar gum hydrogel obtained by the reaction has a larger compressive modulus.

It can be seen from Figure 4 and Figure 5 that the 3D guar gum scaffold with a sol concentration of 3% w/v (Figure 5) has a higher fidelity than the 3D guar gum scaffold with a sol concentration of 2% w/v (Appendix 4) High.

It can be seen from Fig. 6 and Fig. 7 that chondrocytes can continue to grow and proliferate in the scaffold after being compounded with guar gum hydrogel for 3D printing.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. a 3D printing guar glue hydrogel support is characterized in that: it is made by the following steps: S1. Under normal temperature, completely dissolve guar gum in ultrapure water to obtain guar gum aqueous solution, add methacrylic anhydride and fully stir, adjust the pH value of the system to 8-10 with sodium hydroxide solution, and continue the reaction 2-6 hours, the solution obtained by the reaction is dialyzed with ultrapure water for 5-7 days, and guar gum methacrylate is obtained after complete freeze-drying; S2. In a sterile environment, completely dissolve guar gum methacrylate in a sterile PBS solution containing a photoinitiator to prepare a sol, and uniformly mix the sol with chondrocytes to make a 3D printed guar composite chondrocyte The glue bio-ink is extruded and printed by a 3D bioprinter, and a scaffold is formed by stacking the ejected filaments layer by layer. 2. a kind of 3D printing guar gum hydrogel support according to claim 1, is characterized in that: in described step S1, the consumption ratio of guar gum, ultrapure water, methacrylic anhydride is 1g:(50 -150) mL: (1-3) mL. 3 . The 3D printed guar gum hydrogel support according to claim 1 , wherein: in the step S1 , the concentration of the sodium hydroxide solution is 5 mol/L. 4 . 4. a kind of 3D printing guar gum hydrogel support according to claim 1, is characterized in that: in described step S2, the dosage ratio of guar gum methacrylate and the aseptic PBS solution containing photoinitiator It is 1 g:(20-50) mL, the photoinitiator is photoinitiator 2959, and the concentration of the sterile PBS solution containing photoinitiator is 0.1-1% w/v. 5 . The 3D printed guar gum hydrogel scaffold according to claim 1 , wherein in the step S2, the dosage ratio of sol to chondrocytes is 1 mL: 1×10 ⁷ . 6. The preparation method of a 3D printed guar glue hydrogel scaffold according to any one of claims 1 to 5, characterized in that: comprising the following steps: S1. Under normal temperature, completely dissolve guar gum in ultrapure water to obtain guar gum aqueous solution, add methacrylic anhydride and fully stir, adjust the pH value of the system to 8-10 with sodium hydroxide solution, and continue the reaction 2-6 hours, the solution obtained by the reaction is dialyzed with ultrapure water for 5-7 days, and guar gum methacrylate is obtained after complete freeze-drying; S2. In a sterile environment, completely dissolve guar gum methacrylate in a sterile PBS solution containing a photoinitiator to prepare a sol, and uniformly mix the sol with chondrocytes to make a 3D printed guar composite chondrocyte The glue bio-ink is extruded and printed by a 3D bioprinter, and a scaffold is formed by stacking the ejected filaments layer by layer. 7. The preparation method of a 3D printing guar gum hydrogel support according to claim 6, wherein in the step S1, the dosage ratio of guar gum, ultrapure water and methacrylic anhydride is 1 g : (50-150) mL: (1-3) mL. 8 . The method for preparing a 3D printed guar gum hydrogel scaffold according to claim 6 , wherein: in the step S1 , the concentration of the sodium hydroxide solution is 5 mol/L. 9 . 9. The method for preparing a 3D printed guar hydrogel stent according to claim 6, wherein in the step S2, guar gum methacrylate and a photoinitiator-containing sterile PBS solution The dosage ratio is 1 g:(20-50) mL, the photoinitiator is photoinitiator 2959, and the concentration of the sterile PBS solution containing the photoinitiator is 0.1-1% w/v. 10 . The method for preparing a 3D printed guar glue hydrogel scaffold according to claim 6 , wherein in the step S2 , the dosage ratio of sol to chondrocytes is 1 mL: 1×10 ⁷ .

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