CN116236621B

CN116236621B - 3D printing biological hybrid hydrogel and preparation method and application thereof

Info

Publication number: CN116236621B
Application number: CN202310262444.1A
Authority: CN
Inventors: 汪少芸; 田韩; 陈旭; 蔡茜茜; 程静
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2023-03-17
Filing date: 2023-03-17
Publication date: 2024-08-30
Anticipated expiration: 2043-03-17
Also published as: CN116236621A

Abstract

The invention discloses a 3D printing biological hybridization hydrogel and a preparation method and application thereof. The biological hybrid hydrogel is prepared from gelatin, xanthan gum and an oil gel agent serving as raw materials, has good rheological property, meets the basic requirement of 3D printing, can be used as 3D printing ink to further prepare a porous gel scaffold serving as a tissue scaffold, has good shape retention property, good biocompatibility and no cytotoxicity, and meets the requirement of the tissue engineering scaffold. The invention overcomes the defect of the existing hydrogel ink which is lack of good mechanical property and can be used for biological 3D printing, and is beneficial to the development of tissue engineering and regenerative medicine.

Description

3D printing biological hybrid hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of 3D printing and tissue engineering, and particularly relates to a 3D printing biological hybridization hydrogel and a preparation method and application thereof.

Background

In recent years, research on repair and regeneration of damaged tissues has been increasingly paid attention to. Among the most common damaged tissue repair methods are: tissue-specific cells are isolated from a tissue biopsy of a patient, expanded and seeded into a three-dimensional tissue scaffold. The tissue scaffold mimics the natural extracellular matrix (ECM) of the target tissue, delivers seeded cells to a desired site within the patient, promotes cell-biomaterial interactions, promotes cell adhesion, allows for adequate transport of gases, nutrients and growth factors to ensure proliferation and differentiation of cells, and controls the structure and function of the engineered tissue. The basic requirements for tissue scaffolds are: young's modulus similar to tissue, three-dimensional structure with supported trophic function, ease of ingrowth or infiltration of cells, and negligible degree of inflammation or toxicity in vivo.

Hydrogels are a highly hydrated polymer network, which is considered a good tissue scaffold material due to its cell-friendly aqueous environment, suitable structure and suitable mechanical properties allowing cell interactions and biochemical signaling. In order to promote the attachment, proliferation and differentiation of cells on hydrogel scaffolds, hydrogel tissue scaffolds also typically require pores of a certain size and density to provide clear pathways for cell movement, nutrient penetration and cell metabolite removal. The traditional preparation method of the porous tissue scaffold comprises an emulsion template method, a freeze drying method, a gas foaming method, a photoetching method, electrostatic spinning and the like. However, the hydrogel scaffold produced by the traditional method has the problems of poor aperture accuracy, poor stereo structure accuracy, over-high machine strength, over-high internal acting force and the like. Meanwhile, the cell implantation density of the tissue scaffold manufactured by the traditional process cannot be adjusted, so that the cell implantation density cannot be accurately attached when the cells are manually implanted, and the preparation of the tissue scaffold which needs specific precise cell arrangement, such as blood vessels formed by concentric arrangement of endothelial cells, mineralization arrangement of osteoblasts and the like, cannot be met.

Biological 3D printing is used as a method for additive manufacturing, and can construct in-vitro tissues or organs by accurately positioning biocompatible materials and cells at specified positions, so that the method has great potential in the field of tissue repair and regeneration. The problems of the traditional tissue scaffold preparation method can be well solved by utilizing a 3D printing technology, and a complex and fine biological 3D structure can be manufactured more accurately, so that the regeneration of functional tissues can be promoted more effectively. In order for the printed tissue scaffold to have sufficient mechanical strength and structural fidelity, it is inevitably necessary to form a dense network of biological material for support, but dense structures undoubtedly hinder the spread, migration and proliferation of encapsulated cells. The lack of printable biocompatible, non-cytotoxic bio-inks is considered one of the major obstacles impeding the development of bio-3D printing, particularly the lack of printing inks that have high mechanical strength after curing and shaping. Current 3D printing hydrogel inks fall into three categories: protein hydrogels, polysaccharide hydrogels, and modified hydrogels. The protein hydrogel and the polysaccharide hydrogel have low mechanical strength, and the printed 3D structure collapses, which is not beneficial to molding. Although the modified hydrogel overcomes the problem of mechanical strength, the modification method of the hydrogel is complex and has high cost. Therefore, there is a need to provide a hybrid hydrogel with high mechanical strength suitable for biological 3D printing, so as to solve the current lack of ink suitable for biological 3D printing, and promote development of tissue engineering and regenerative medicine.

Disclosure of Invention

The invention provides a 3D printing biological hybrid hydrogel, a preparation method and application thereof, and aims to overcome the defects and the shortcomings of the prior art.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The invention provides a 3D printing biological hybridization hydrogel, which is prepared from the following raw materials in parts by mass: 5 to 50 parts of oil gel, 5 to 50 parts of xanthan gum solution with the mass concentration of 0.5 to 10 percent and 5 to 20 parts of gelatin solution with the mass concentration of 2 to 50 percent; the oil gel is any one of beeswax, carnauba wax, rice bran wax and candelilla wax.

Preferably, the 3D printing biological hybrid hydrogel is prepared from the following raw materials in parts by mass: 12-20 parts of oil gel, 15-40 parts of xanthan gum solution with the mass concentration of 0.5-5.5% and 9-16 parts of gelatin solution with the mass concentration of 15-40%; the oil gel is beeswax.

The second object of the present invention is to protect the preparation method of the 3D printing biohybrid hydrogel, comprising the steps of:

1) Heating and preserving the temperature of the xanthan gum solution with the mass concentration of 0.5-10% to 50-80 ℃;

2) Adding the melted oil gelling agent into the xanthan gum solution in the step 1), and immediately solidifying the oil gelling agent by ice bath after the oil gelling agent is uniformly dispersed in the xanthan gum solution to obtain a xanthan gum-oil gelling agent mixture;

3) And (3) adding the xanthan gum-oil gel mixture obtained in the step (2) into a gelatin solution with the mass concentration of 2% -50% at the temperature of 35-60 ℃ until the mixture is uniformly dispersed, so as to obtain the 3D printing biological hybrid hydrogel.

A third object of the present invention is to protect a porous hydrogel scaffold prepared using the 3D printed biohybrid hydrogel.

The fourth object of the present invention is to provide a method for preparing a porous hydrogel scaffold, comprising the steps of:

a) 3D printing the 3D printing biological hybridization hydrogel into a required bracket shape;

b) Soaking the printed bracket in a modifying liquid for 16-48 h;

c) And b) heating the modified scaffold in the step b) to 80-120 ℃ to enable the oil gel in the scaffold to be melted and lost, so as to obtain the porous hydrogel scaffold.

Further, the printing temperature in step a) is 25 to 60 ℃.

Further, in the step b), the modifying liquid is glutaraldehyde aqueous solution with the mass concentration of 0.5-3% or genipin aqueous solution with the mass concentration of 0.3-3%.

The porous hydrogel scaffold prepared by the invention is respectively inoculated with Human Skin Fibroblasts (HSF), human liver cancer cells (HepG 2) and mouse mononuclear macrophages (Raw264.7), and the obtained porous hydrogel scaffold is suitable for proliferation and growth of various cells, and has good biocompatibility and no cytotoxicity. It is therefore a fifth object of the present invention to protect the use of the porous hydrogel scaffold in tissue repair or tissue regeneration.

The invention has the following beneficial effects:

The invention provides 3D printing biological hybrid hydrogel prepared from gelatin, xanthan gum and oil gel, which has good rheological property, meets the basic requirement of 3D printing, and can be used as 3D printing ink. Meanwhile, the invention also provides a porous gel scaffold prepared by using the 3D printing biological hybridization hydrogel, the preparation method is simple, and the obtained porous hydrogel scaffold has good shape retention, good biocompatibility and no cytotoxicity, and can meet the requirements of a tissue scaffold. The invention overcomes the defect of the existing hydrogel ink which is lack of good mechanical property and can be used for biological 3D printing, and is beneficial to the development of tissue engineering and regenerative medicine.

Drawings

Fig. 1 is an SEM image of a hydrogel prepared by directly mixing beeswax, xanthan gum solution and gelatin solution.

Fig. 2 is an SEM image of the hydrogel prepared in example 1.

FIG. 3 is a graph of the rheological properties of 3D printed biohybrid hydrogels prepared with varying amounts of beeswax in example 6; wherein the amplitude scanning, shear thinning, shear recovery and temperature recovery are sequentially shown as A-D (I, II, III, IV and V respectively show that the adding amount of beeswax is 12, 15, 17, 19 and 21 g).

FIG. 4 is a three-dimensional model of 3D printed biohybrid hydrogel printing prepared with different amounts of beeswax in example 6.

FIG. 5 is a meniscus and nose scanned, sectioned, and printed using the 3D printed biohybrid hydrogel prepared in application example 2.

FIG. 6 is a graph showing the results of observation of the porous hydrogel scaffold prepared in application example 3 under a confocal laser microscope.

FIG. 7 is a graph showing the results of the air permeability test of the porous hydrogel scaffold obtained in application example 3.

FIG. 8 is a graph showing the results of the porosity and expansion ratio test of the porous hydrogel scaffold obtained in application example 3.

FIG. 9 is a graph showing the results of mechanical strength test of the porous hydrogel scaffold obtained in application example 3.

FIG. 10 is a graph showing proliferation of mouse mononuclear macrophages (Raw264.7) on a porous hydrogel scaffold in application example 3.

FIG. 11 is a graph showing proliferation of human hepatoma cells (HepG 2) on a porous hydrogel scaffold according to application example 3.

Detailed Description

The 3D printing biological hybrid hydrogel is prepared from the following raw materials in parts by mass: 5 to 50 parts of oil gel, 5 to 50 parts of xanthan gum solution with the mass concentration of 0.5 to 10 percent and 5 to 20 parts of gelatin solution with the mass concentration of 2 to 50 percent; the oil gel is any one of beeswax, carnauba wax, rice bran wax and candelilla wax.

The preparation method of the 3D printing biological hybridization hydrogel comprises the following steps:

The method for preparing the porous hydrogel scaffold by using the 3D printing biological hybridization hydrogel comprises the following steps:

b) Soaking the printed bracket in a modifying liquid for 16-48 h;

The printing temperature in step a) is 25-60 ℃.

The modifying liquid in the step b) is glutaraldehyde water solution with the mass concentration of 0.5-3% or genipin water solution with the mass concentration of 0.3-3%.

To investigate how different oleogels have an effect on the printing properties of the resulting 3D printed biohybrid hydrogels. Alternatively, 3 different oleogels, namely carnauba wax, rice bran wax, and candelilla wax, were used to prepare different 3D printing biohybrid hydrogels and used for printing. As a result, it was found that after beeswax is replaced with carnauba wax, rice bran wax or candelilla wax, the prepared 3D printing biological hybrid hydrogel also meets the 3D printing requirement and can be used as 3D ink. But the printing effect is better when beeswax is used than when carnauba wax, rice bran wax and candelilla wax are used. In addition, the rheological results show that the hybrid gel materials prepared by the three materials have certain differences in mechanical strength and temperature recovery time, which are determined by the hardness and heat storage performance of the hybrid gel materials.

In the pre-experiment, an attempt was made to mix 15 g% beeswax directly with 20 g% strength by mass xanthan gum solution and 10 g% strength by mass gelatin solution in a water bath above the melting temperature of beeswax. As a result, beeswax was found to be miscible with gelatin, resulting in poor mechanical properties of the printed hydrogel scaffold (as shown in FIG. 1, there are few beeswax particles in the hydrogel). This is probably due to the fact that the gelatin contains highly hydrophilic polypeptide chains with hydrophobic groups inside, and the hydrophobic groups inside after melting of gelatin interact with lipids in the melted beeswax to be mutually soluble, so that the beeswax cannot form particles after cooling, and thus a porous hydrogel scaffold cannot be prepared.

Another attempt was made to use carrageenan, guar gum, etc. instead of xanthan gum to granulate the beeswax, but this was not effective. When carrageenan and guar gum are used, oil-water two-phase separation is easy in the preparation process, and beeswax is solidified into a whole after the temperature is reduced.

In order to avoid the reaction of molten beeswax and gelatin, an attempt was made to uniformly mix a xanthan gum solution with a mass concentration of 20 g% with a gelatin solution with a mass concentration of 10 g% under a boiling water bath condition, and after the mixture is slightly cooled, the molten beeswax is added to disperse and cool, but as a result, although beeswax particles are formed, but the beeswax particles are not uniform, holes only appear on the surface of the support when the porous hydrogel support is prepared later, and a large amount of beeswax contained in the support cannot be well dissolved out.

Through continuous attempts, it is finally found that after the beeswax and the xanthan gum are mixed, the beeswax in the mixture is solidified into uniform beeswax particles through ice bath, and then the xanthan gum-beeswax mixture is mixed with gelatin at 40 ℃, so that interaction between hydrophobic groups in the gelatin and the beeswax after the beeswax is melted can be avoided, and the porous hydrogel bracket cannot be prepared through melting the beeswax in the follow-up process.

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of 3D printed biohybrid hydrogels

The 3D printing biological hybrid hydrogel is prepared from gelatin, xanthan gum and oil gel, and the preparation method specifically comprises the following steps:

1) Dissolving xanthan gum in water to obtain xanthan gum solution with mass concentration of 5.5%, heating to 60deg.C in water bath, and keeping the temperature for use;

2) Adding 15g of beewax melted by a boiling water bath into the xanthan gum solution prepared in the step 1) of 30g at a stirring speed of 3000 r/min to uniformly disperse beewax in the xanthan gum solution, immediately ice-bathing 10min after dispersing 3 min, and solidifying the beewax to obtain a xanthan gum-beewax mixture;

3) Preparing gelatin solution with mass concentration of 35%, and keeping the temperature at 40 ℃ for standby; adding the xanthan gum-beeswax mixture obtained in the step 2) of 45 g into a 12 g gelatin solution, stirring, dispersing and uniformly dispersing at 3000 r/min to obtain the 3D printing biological hybrid hydrogel.

Fig. 2 is an SEM image of the prepared hydrogel. As can be seen from the figure, the resulting hydrogel contains uniform beeswax particles which not only enhance the mechanical strength of the hydrogel, making it suitable for 3D printing, but also serve as a porogen for the subsequent preparation of porous hydrogel scaffolds.

Example 2 preparation of 3D printed biohybrid hydrogels

The 3D printing biological hybrid hydrogel is prepared from gelatin, xanthan gum and beeswax, and the preparation method specifically comprises the following steps:

1) Dissolving xanthan gum in water to obtain xanthan gum solution with mass concentration of 10%, heating to 70deg.C in water bath, and keeping the temperature for use;

2) Adding the beeswax 5g melted by boiling water bath into the xanthan gum solution prepared in the step 1) of 50 g under the stirring speed of 3000 r/min, uniformly dispersing the beeswax in the xanthan gum solution, immediately ice-bathing 10 min, and solidifying the beeswax to obtain a xanthan gum-beeswax mixture;

3) Preparing gelatin solution with mass concentration of 50%, and keeping the temperature at 40 ℃ for standby; adding the xanthan gum-beeswax mixture obtained in the step 2) of 55 g into 5g gelatin solution, and uniformly stirring at 3000 r/min to obtain the 3D printing biological hybrid hydrogel.

Example 3 preparation of 3D printing biohybrid hydrogels

1) Dissolving xanthan gum in water to obtain xanthan gum solution with mass concentration of 8%, heating to 60deg.C in water bath, and keeping the temperature for use;

2) Adding the beeswax 25 g melted by boiling water bath into the xanthan gum solution prepared in the step 1) of 5g under the stirring speed of 3000 r/min, uniformly dispersing the beeswax in the xanthan gum solution, immediately ice-bathing 10 min, and solidifying the beeswax to obtain a xanthan gum-beeswax mixture;

3) Preparing gelatin solution with mass concentration of 20%, and keeping the temperature at 40deg.C for use; adding the xanthan gum-beeswax mixture obtained in the step 2) of 30 g into a gelatin solution of 20 g, stirring and dispersing uniformly at 3000 r/min to obtain the 3D printing biological hybrid hydrogel.

Example 4 preparation of 3D printed biohybrid hydrogels

1) Dissolving xanthan gum in water to obtain xanthan gum solution with mass concentration of 2%, heating to 60deg.C in water bath, and keeping the temperature for use;

2) Adding 15g of beewax melted by a boiling water bath into the xanthan gum solution prepared in the step 1) of 15g under the stirring speed of 3000 r/min, uniformly dispersing beewax in the xanthan gum solution, immediately ice-bathing 10 min, and solidifying the beewax to obtain a xanthan gum-beewax mixture;

3) Preparing gelatin solution with mass concentration of 40%, and keeping the temperature at 40 ℃ for standby; adding the xanthan gum-beeswax mixture obtained in the step 2) of 30 g into a 9 g gelatin solution, stirring and dispersing uniformly at 3000 r/min to obtain the 3D printing biological hybrid hydrogel.

Example 5 preparation of 3D printed biohybrid hydrogels

1) Dissolving xanthan gum in water to obtain xanthan gum solution with mass concentration of 0.5%, heating to 60deg.C in water bath, and keeping the temperature for use;

2) Adding the beeswax 20g melted by boiling water bath into the xanthan gum solution prepared in the step 1) of 40 g under the stirring speed of 3000 r/min, uniformly dispersing the beeswax in the xanthan gum solution, immediately ice-bathing 10 min, and solidifying the beeswax to obtain a xanthan gum-beeswax mixture;

3) Preparing gelatin solution with mass concentration of 15%, and keeping the temperature at 40 ℃ for standby; adding the xanthan gum-beeswax mixture obtained in the step 2) of 60 g into 16 g gelatin solution, stirring and dispersing uniformly at 3000 r/min to obtain the 3D printing biological hybrid hydrogel.

Application example 1

To verify whether the prepared hydrogel is suitable for 3D printing, the same quality hydrogel is loaded into the barrel of the printer, and the same 3D stereoscopic model is printed.

Specifically, the 3D printing process includes the steps of:

1) The 3D printing biological hybridized hydrogel prepared in the examples 1 to 5 is respectively filled into a charging barrel of a printer for standby;

2) Building a 3D stereoscopic model of a building to be printed by using 3DS MAX software, adjusting the size, opening by using Cura slicing software after a stl file is exported, setting the thickness of the bottom layer of the slice to be 1.0 mm, the thickness of the layer to be 1.2 mm and the filling density to be 80%, slicing, exporting a GCODE file, copying the file to a 3D printer, and identifying and printing; and (3) regulating the printing speed to 25mm/s, wherein the aperture of a printing nozzle is 1.2 mm, and the printing temperature is 45 ℃ for printing.

As a result, it was found that the 3D printing biohybrid hydrogels prepared in examples 1 to 5 can be used for 3D printing, and the 3D printing requirements are satisfied, wherein the 3D printing biohybrid hydrogels prepared in examples 1,4 and 5 have better printing effects, so that the optimal 3D printing biohybrid hydrogel formulation is selected in the dosage range of the 3 examples.

Example 6 Effect of different Cera flava addition amounts on rheological Properties of ink and 3D printing Effect

The effect of different amounts of beeswax addition on the 3D printing performance of 3D printed biohybrid hydrogels was investigated on the basis of examples 1, 4 and 5. The preparation method of the 3D printing biological hybrid hydrogel is the same as that of example 1, the concentration and the dosage of the xanthan gum solution and the gelatin solution are controlled to be unchanged, and the dosage of beeswax is only regulated (wherein, the dosage of the xanthan gum solution with the mass concentration of 5.5% is 30 g, the dosage of the gelatin solution with the mass concentration of 35% is 9 g, and the dosage of beeswax is 12, 15, 17, 19 and 21 g respectively). The 3D printing process is the same as application example 1, the rheological properties of the 3D printed biohybrid hydrogels prepared with different amounts of beeswax are shown in fig. 3 (wherein the amplitude scanning, shear thinning, shear recovery and temperature recovery are shown in a-D in fig. 3 in sequence), and the printed three-dimensional stereoscopic model is shown in fig. 4.

As can be seen from fig. 3, the yield stress (the intersection of the storage modulus G' and the loss modulus G ") of the hybrid gel increased from 36.27 Pa to 78.44 Pa with increasing amounts of beeswax, indicating an increase in the mechanical strength of the hybrid hydrogel. Meanwhile, regardless of the added amount of beeswax, the hybrid hydrogels have shear thinning characteristics (viscosity decreases with increasing shear force and shear stress increases), indicating that they have pseudoplasticity and are suitable for 3D printing. Further by fitting, it was found that after extrusion, as the amount of beeswax added increased, the shear recovery of the hybrid gel 30s increased from 76.3% to 97.21%, and the temperature recovery time decreased from 136.23 s to 86.67 s. This suggests that the addition of beeswax increases the ability of the hybrid gel to recover its original state after being extruded out of the nozzle. The addition of beeswax is advantageous for printing of the hybrid gel material, since the stronger the shear recovery ability and the shorter the recovery time, the more advantageous the shaping of the hybrid gel material after extrusion.

However, too high shear recovery and short gel times may cause ink to clog the nozzles, so that the ink is further evaluated by 3D printing to screen out the optimum amount of addition. As can be seen from FIG. 4, the printing effect of the obtained 3D printing biological hybrid hydrogel is optimal when the beeswax is used in an amount of 17 g.

On the basis, the invention also analyzes the influence of the concentration and the addition amount of the xanthan gum and the gelatin concentration and the addition amount on the rheological property and the 3D printing effect of the obtained 3D printing biological hybrid hydrogel. The result shows that the optimal proportion of each component in the 3D printing biological hybridized hydrogel is as follows: 17 parts of beeswax, 30 parts of xanthan gum solution with the mass concentration of 5.5% and 9 parts of gelatin solution with the mass concentration of 30%.

Application example 2 construction of human meniscus and nose

The 3D model of the prosthetic human meniscus (lxwxh, 47.52×33.24×6.34 mm) and nose (55.14 ×25.73×29.84 mm) were scanned using a commercially available 3D scanner (FreeScan X3) and STL files were output that could be identified by Cura slicing software. Based on the optimal 3D printed biohybrid hydrogel formulation obtained in example 6, a 3D printed biohybrid hydrogel was prepared and used to print human menisci and nose in the manner of example 1, the results of which are shown in fig. 5.

As can be seen from fig. 5, the resulting meniscus and nose printed (bottom panel for photography, 10mm square) did not significantly differ from the scanned meniscus and nose length-width height, and the hybrid hydrogel was able to bear the height of the printed nose, indicating that the resulting biohybrid hydrogel was suitable for accurate 3D printing.

Application example 3 preparation of porous hydrogel scaffold

In order to promote cell attachment, proliferation and differentiation on hydrogel scaffolds, hydrogel tissue scaffolds also typically require pores of a certain size and density to provide clear pathways for cell movement, nutrient penetration and cell metabolite removal. The 3D printing biological hybridized hydrogel can be used for preparing a porous hydrogel bracket.

Based on the optimal 3D printed biohybrid hydrogel formulation obtained in example 6, a 3D printed biohybrid hydrogel was prepared and used to construct a porous hydrogel scaffold according to the method described in example 1, the construction method comprising the steps of:

1) Preparing the 3D printing biological hybridization hydrogel into a required bracket shape through 3D printing according to the method of application example 1;

2) Preparing geni Ping Gaixing liquid with the mass concentration of 1%, and soaking the bracket printed and molded in the step 1) in the modified liquid for 24: 24 h;

3) And cleaning to remove the modifying liquid on the surface of the stent, soaking the stent in deionized water with the pH of 7.4, and heating to 120 ℃ to enable beeswax in the deionized water to be melted and lost, thus obtaining the porous hydrogel stent.

In addition to genipin, pentanediol can be used for modification, and glutaraldehyde aqueous solution with the mass concentration of 0.5-3% or genipin aqueous solution with the mass concentration of 0.3-3% can achieve better effect.

To observe the effect of the prepared porous hydrogel scaffold, the preparation was performed after adding 0.01% nile red dye to melted beeswax; the stents after genipin soaking were divided into two groups (one group was used to melt lost beeswax by heating and one group was not treated as a control); the scaffold was then soaked in 0.01% FITC dye at 1 min and observed with a laser confocal microscope as shown in fig. 6 (where a is the hydrogel scaffold without melting lost beeswax by heating, b is the hydrogel scaffold after melting lost beeswax by heating, green in the figure is FITC staining and red is nile red staining). From the figure, it can be seen that the red beeswax particles in a are wrapped by the green scaffold, and only the green scaffold is shown in b, but the red beeswax particles are not seen, so that the porous hydrogel scaffold can be prepared by using the 3D printing biological hybridization hydrogel.

The air permeability of the porous hydrogel scaffolds prepared by dropping the ink with pigment (the filling density of printing was 100%) was also tested, and the results are shown in fig. 7. As can be seen from FIG. 7, the pigmented ink began to bleed out at 10s, indicating that the air permeability of the resulting porous hydrogel scaffold was good.

The porosity and expansion ratio of the prepared porous hydrogel scaffold were tested, and the results are shown in fig. 8. As can be seen from FIG. 8, the porous hydrogel scaffold has a porosity as high as 58.03.+ -. 7.21% and an expansion ratio of 5.91.+ -. 4.92%, indicating that the porous hydrogel scaffold has a high porosity and a small expansion ratio. The high void fraction is beneficial for the transfer and excretion of information, nutrients and waste between cells. And the small expansion rate is favorable for ensuring the precision of the printing bracket, is favorable for ensuring the precision of 3D printing tissue transplantation, and meets the requirement of accurate medical treatment.

The porous hydrogel scaffold is prepared by printing a cylinder with the diameter of 1 cm and the height of 1 cm by the preparation method, and the mechanical strength is tested, and the result is shown in figure 9, and as can be seen from figure 9, the mechanical strength of the printing structure is as high as 1633.8 +/-187.2 g, and the printing structure can be used for the load growth and propagation of subsequent cells, and meanwhile, the structure with high mechanical strength is favorable for maintaining the shape of tissues.

Further, in order to verify the biocompatibility of the porous hydrogel scaffold material, mouse mononuclear macrophages (raw 264.7) were inoculated with the prepared porous hydrogel scaffold. The inoculation method is that the printed bracket is soaked in 75% ethanol for sterilization, then placed in a culture dish, then the third generation cell is planted on the cell bracket, the cell concentration is 1X 10 ⁶/ml, 30 min and then cell culture medium (DMEM, containing 15% bovine serum and 1% Gibco penicillin-streptomycin) is added. After 3 days of culture, the cells were stained with a staining solution of live dead cells (cell viability assay kit, CALCEIN AM, PI method), and the growth state of the cells was observed by a laser confocal microscope (green for live cells and red for dead cells), and the results are shown in FIG. 10. As can be seen from fig. 10, cells can be grown supported on the scaffold material.

Referring to the above method, after sterilizing the porous scaffold by autoclaving, human hepatoma cells (HepG 2) were inoculated on the scaffold, and the results are shown in fig. 11. As can be seen from FIG. 11, the survival rate of the cells growing on the porous scaffold is more than 80%, further illustrating that the prepared porous hydrogel scaffold is suitable for cell proliferation, indicating that the porous hydrogel scaffold has good biocompatibility and no cytotoxicity, and can be used for tissue repair or regeneration.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The 3D printing biological hybrid hydrogel is characterized by being prepared from the following raw materials in parts by mass: 5 to 50 parts of oil gel, 5 to 50 parts of xanthan gum solution with the mass concentration of 0.5 to 10 percent and 5 to 20 parts of gelatin solution with the mass concentration of 2 to 50 percent;

the oil gel is any one of beeswax, carnauba wax, rice bran wax and candelilla wax;

The preparation method comprises the following steps:

2. A porous hydrogel scaffold prepared using the 3D printed biohybrid hydrogel of claim 1.

3. A method of preparing a porous hydrogel scaffold according to claim 2, comprising the steps of:

b) Soaking the printed bracket in a modifying liquid for 16-48 h;

4. The method for preparing a porous hydrogel scaffold according to claim 3, wherein the printing temperature in step a) is 25-60 ℃.

5. The method for preparing a porous hydrogel scaffold according to claim 3, wherein the modifying liquid in step b) is glutaraldehyde aqueous solution with a mass concentration of 0.5-3% or genipin aqueous solution with a mass concentration of 0.3-3%.