CN111375091B

CN111375091B - Photosensitive composite biological ink for 3D printing and preparation method thereof

Info

Publication number: CN111375091B
Application number: CN201811645797.5A
Authority: CN
Inventors: 郑雄飞; 郭凯; 王赫然; 李松; 朱慧轩
Original assignee: Shenyang Institute of Automation of CAS
Current assignee: Shenyang Institute of Automation of CAS
Priority date: 2018-12-30
Filing date: 2018-12-30
Publication date: 2021-09-10
Anticipated expiration: 2038-12-30
Also published as: CN111375091A

Abstract

The invention discloses photosensitive composite biological ink for 3D printing and a preparation method thereof, and belongs to the technical field of biological materials and cell-containing hydrogel. The ink has temperature-sensitive and photosensitive characteristics, and the characteristics of the double gel are very beneficial to the forming of 3D printing. The ink has two components, one of which is collagen fiber. Research shows that the mesenchymal stem cells can be more effectively differentiated into osteoblasts on the surface of the collagen fibers. The component II is photosensitive gelatin which retains the temperature-sensitive characteristic of gelatin, has the rapid photocuring performance and is particularly suitable for precise 3D printing and stacking. Particularly, the combination of the two can produce the additive effect, the collagen fiber changes the original gel structure of the photosensitive gelatin, and the cells have stronger proliferation and extension capability in the composite gel with the phase separation structure. In addition, the ink was demonstrated to be able to promote differentiation of mesenchymal stem cells into osteoblasts.

Description

Photosensitive composite biological ink for 3D printing and preparation method thereof

Technical Field

The invention relates to the technical field of biological materials and cell-containing hydrogel, in particular to photosensitive composite biological ink for 3D printing and a preparation method thereof.

Background

3D printing is a new manufacturing technology, and the technology is to build a three-dimensional model of a structure by means of three-dimensional scanning and the like, and then quickly print various structures by accurate accumulation of materials. At present, printing technologies for traditional materials such as metal materials and plastics are widely applied in production and life. The bioprinting technology refers in particular to the construction of a printing structure by using biocompatible materials, and the structure is used for replacing tissues or organs of a human body, so that the aim of accurate and rapid personalized repair is fulfilled.

At present, products such as meninges, bone defect substitutes and the like are applied to clinic. At present, biological printing materials mainly comprise two types, namely synthetic macromolecules such as PCL and PLGA, and although the materials are well formed, the materials are slowly degraded, inflammatory reaction is aggravated in a report, and cells cannot be loaded; natural macromolecules such as sodium alginate, chitosan, hyaluronic acid, gelatin and the like can load cells, but the solidification and the molding are poor, and a good printing structure is often difficult to obtain. Transplantation of printed tissue containing cells to promote tissue regeneration has been widely recognized. Part of the biological materials have good cell loading capacity and show high survival rate and high extensibility of cells, such as collagen and fibrinogen. But both have no printable properties due to low viscosity.

Photosensitive gelatin is a printing material widely used in recent years, and although its printability is good, its gel network is too tight and cell proliferation is limited. In addition, the traditional synthetic method has the advantages of large modifier consumption, large smell in the preparation process, low yield and slow dialysis. Therefore, it is very urgent to develop a printable material having a certain viscosity and printable property in which cells have a good proliferation ability and stretching property.

Disclosure of Invention

The invention aims to provide photosensitive composite biological ink for 3D printing and a preparation method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of photosensitive composite bio-ink for 3D printing comprises the following steps:

(1) preparation of collagen solution:

taking I type collagen as a raw material, dissolving the I type collagen in an acid solution with the concentration of 0.05-0.5mol/L to obtain a collagen solution with the I type collagen content of 1-10 mg/mL;

(2) preparing collagen fibers:

adding 0.1mol/L PBS solution, 1mg/mL phenol red solution and 1mol/L sodium hydroxide solution into the collagen solution obtained in the step (1), and neutralizing the solution to red by adjusting the adding amount to obtain a neutral collagen solution; pouring the obtained neutral collagen solution into a PBS solution, and magnetically stirring in a water bath at the temperature of 20-37 ℃ for 1-4 hours; centrifuging the obtained material, removing supernatant, and collecting precipitate as collagen fiber;

(3) preparation of modified photosensitive gelatin:

weighing a certain amount of gelatin, adding the gelatin into PBS buffer solution, heating to 60 ℃, stirring until the gelatin is completely dissolved, and cooling to 50 ℃ to obtain gelatin solution; dropwise adding an acetone solution of methacrylic anhydride into a gelatin solution, adjusting the pH value to be 8-9, and continuing to react for 1-5 hours; dialyzing the reaction product with large volume of distilled water, intercepting the molecular weight of the dialysis bag to be 8000Da, dialyzing for 3 days, and freeze-drying to obtain modified photosensitive gelatin;

(4) adding the modified photosensitive gelatin obtained in the step (3) into 0.01mol/L PBS solution to prepare modified photosensitive gelatin solution with the modified photosensitive gelatin content of 0.05-0.4g/mL, adding a photoinitiator, adjusting the pH value of the solution to 7, filtering the obtained mixed material by using a microporous filter membrane with the aperture of 0.22 mu m to obtain modified photosensitive gelatin solution containing the photoinitiator for later use;

(5) and (3) mixing the collagen fiber prepared in the step (2) with the modified photosensitive gelatin solution containing the photoinitiator prepared in the step (4) according to the weight ratio of 10:1-1:10, and adding a cell suspension into the obtained mixed material to obtain the photosensitive composite biological ink for 3D printing.

In the step (1), the type I collagen is bovine collagen, rat tail collagen or pig collagen; the acid solution is acetic acid solution, hydrochloric acid solution or malonic acid solution.

In the step (2), when the neutral collagen solution is poured into the PBS solution, the concentration of the PBS solution is 0.001-0.05mol/L, and the volume of the PBS solution is 5-20 times of that of the neutral collagen solution; the magnetic stirring speed is 200-.

In the step (3), the acetone solution of methacrylic anhydride is prepared by mixing the modifier methacrylic anhydride: acetone ═ 3: 10 by volume in acetone.

In the step (4), the photoinitiator is phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate (LAP) or 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (I2959); adding an initiator into the modified photosensitive gelatin solution, wherein the content of the modifier in the solution is 0.5-5 mg/mL.

In the steps (3) and (4), the pH value of the solution is adjusted by adopting 1mol/L NaOH solution.

In the step (5), the volume of the cell suspension added is 10% of the volume of the mixture.

The photosensitive composite biological ink for 3D printing is prepared by the method, and when the biological ink is used for 3D printing, 365-²Irradiating with the light for 5-200 seconds to obtain the composite gel for promoting differentiation.

The design mechanism of the invention is as follows:

the new method is used for preparing the propenized photosensitive gelatin, the modifier takes acetone as a solvent, and the pH value of a reaction system is adjusted, so that the yield of the propenized gelatin is improved, the dosage of a cross-linking agent is reduced, the reaction time and the dialysis time are reduced, and the synthesis cost is saved. The patent also discloses a novel method for preparing collagen fibers, which can control the size of the collagen fibers and show good differentiation promoting effect in the composite hydrogel. The traditional propylene photosensitive gelatin has certain defects, and the crosslinked molecular network is too compact, so that the cell expansion is limited. Studies have reported that the network structure is modified by adding PEO or collagenase, etc. to improve the expansion ability of cells. According to the invention, collagen fibers with different proportions are added into the allene photosensitive gelatin, so that the gel structure of the photosensitive gelatin is changed, and the expansion and proliferation capacities of cells are improved. Meanwhile, research shows that the collagen fibers have the capacity of promoting the differentiation of stem cells into osteoblasts. Experiments show that the collagen fiber-photosensitive gelatin material disclosed by the invention can obviously promote osteogenic differentiation of stem cells and has considerable printable performance. Compared with the traditional hydrogel, the printing ink disclosed by the invention has the advantages that all components are derived from mammals, the bioactivity is good, the gel has adhesion sites, the combination of a wound surface and the outside of the gel is facilitated, and the expansion and proliferation of cells in the gel are also facilitated.

The invention has the following advantages and beneficial effects:

1. the preparation method of the collagen fiber can obtain the collagen fibers with different structure scales, and is beneficial to the design of hydrogel systems with different functional strengths. Collagen is a material with extremely high biocompatibility, and after collagen fibers are introduced into a photosensitive gelatin system, the biocompatibility of the gel is improved. In addition, the fibers are distributed in the gel in an intricate way, so that an excessively compact network structure of the gelatin is damaged, and the expansion and proliferation of cells are facilitated.

2. In the improved method for preparing the photosensitive gelatin, methacrylic anhydride is dissolved in acetone, and the pH is adjusted to 9. Wherein, the acetone, the water and the methacrylic anhydride are mixed and dissolved, the methacrylic anhydride is directly added by the traditional method, and the existence of the anhydride which is not mixed and dissolved with the water can be always seen in the reaction process. The improved method has the advantages of improved reaction efficiency and reduced consumption of acid anhydride under the same degree of substitution. The reaction efficiency of the acid anhydride and the amino is high when the pH value is 8-9, and the reaction efficiency of the acid anhydride is improved by the improved method. In conclusion, methacrylic anhydride is currently expensive, and the improved process reduces the amount of anhydride and reduces the cost. In addition, less acid anhydride consumption can greatly shorten the dialysis time, prolong the service life of the dialysis bag and save a large amount of distilled water.

3. The biological ink component is derived from mammals, has good biocompatibility and degradability, is provided with an adhesion site, and is very suitable for growth, adhesion and proliferation of cells.

4. The biological ink can obtain printing materials with different mechanical properties by adjusting the proportion of the collagen fibers and the photosensitive gelatin and controlling the printing temperature, is suitable for 3D printing forming, and can be quickly cured by illumination.

5. The induction medium is used for inducing the mesenchymal stem cells to osteoblasts in a two-dimensional environment, and the time is generally 21 days. In the ink of the invention, the mesenchymal stem cells can be differentiated into osteoblasts only in 10 days. The ink provided by the invention has the capacity of promoting the differentiation of stem cells into osteoblasts.

Drawings

FIG. 1 is a diagram of a photosensitive composite bio-ink and 3D printing structure according to the present invention; wherein: (a) the growth state of the bone marrow mesenchymal stem cells in the gel added with the collagen fibers within 7 days, wherein the dye is calcein; (b) the growth state of the bone marrow mesenchymal stem cells in the gel without the collagen fibers for 7 days, wherein the dye is calcein; (c) alkaline phosphatase staining 10 days after induction of the printed bone scaffolds; (d) alizarin staining was performed 10 days after induction of the printed bone scaffolds.

Detailed Description

The present invention is described in detail below with reference to examples.

In the following examples, collagen solutions were prepared from bovine achilles tendon collagen obtained by the following extraction procedure:

(1) adding pepsin into an acetic acid solution with the concentration of 0.1-0.5mol/L, and uniformly mixing to obtain the acetic acid solution of the pepsin; the content of pepsin in the acetic acid solution of the pepsin is 1-5 mg/mL.

(2) Dissolving the bovine achilles tendon in an acetic acid solution containing pepsin, stirring for 72h, and then sequentially performing salting-out and acetic acid dialysis, wherein the cut-off molecular weight of a dialysis bag is 3000-10000Da, so as to obtain the bovine achilles tendon collagen. The salting-out process adopts 4mol/L NaCl solution, and the dialysis process adopts 0.1-0.5mol/L acetic acid. The process is aseptic.

Example 1:

the raw material of the collagen solution is self-extracted bovine achilles tendon collagen, and the collagen solution is obtained by degreasing, enzymolysis, salting out and other steps, and the bovine achilles tendon collagen is dissolved in acetic acid solution with the concentration of 0.1mol/L to prepare 6mg/mL acidic collagen solution.

8.09mL of the above collagen solution, 1mL of 10-fold concentration PBS, 0.81mL of 1mol/L sodium hydroxide, and 0.1mL of 1mg/mL phenol red were mixed. At this time, the solution was poured into a 10-fold volume of 0.01mol/L PBS solution, and magnetically stirred in a water bath at 37 ℃ for 1 hour at a stirring speed of 1000 rpm. Centrifuging the solution at 10000g for 1h, removing supernatant, and obtaining the precipitate as collagen fiber. The steps are all aseptic operation.

Adopting an improved photosensitive gelatin synthesis method: weighing 10g of gelatin, dissolving in 100mL of PBS, heating to 60 ℃, stirring to completely dissolve, and cooling to 40 ℃ to obtain a gelatin solution. 3mL of methacrylic anhydride was added to 10mL of acetone to obtain an acetone solution of methacrylic anhydride, the acetone solution was added dropwise to the gelatin solution, and the pH was maintained to 9 using 1mol/L sodium hydroxide solution. After the pH stabilized, the reaction was continued for 1 hour. Subsequently, dialyzing with distilled water, cutting off molecular weight of 8000Da in dialysis bag, dialyzing for 3d, and lyophilizing.

A gelatin solution prepared from the above gelatin using 0.01mol/L PBS was added with 3mg/mL of a photoinitiator lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate. Adjusting the pH value to 7, and filtering with a filter membrane with the pore diameter of 0.22 mu m for later use.

Mixing the gelatin solution and collagen fiber at a mass ratio of 1:1, adding 10 volume tenth of the mixture⁷/mL of mesenchymal stem cell suspension. 5mW/cm²Irradiating at 365nm for 30 seconds to obtain the composite gel containing cells. A control group without collagen fiber is arranged, and the rest proportion is the same as that of the experimental group. After 10 days of culture in DMEM low-sugar medium, cells in two gel groups were stained with calcein, wherein the gel with collagen fibers was shown in fig. 1(a), and the gel without collagen fibers was shown in fig. 1 (b). As can be seen, the mesenchymal stem cells in the gel added with the collagen fibers have stronger expansion capability.

Example 2:

the raw material for preparing the collagen solution is self-extracted bovine achilles tendon collagen, and the acidic collagen solution with the concentration of 0.1mol/L is obtained by degreasing, enzymolysis, salting out and the like, wherein the acidic collagen solution with the concentration of 8mg/mL is obtained by using acetic acid solution as a solvent. 8.09mL of the above collagen solution, 1mL of 10-fold concentration PBS, 0.81mL of 1mol/L sodium hydroxide, and 0.1mL of 1mg/mL phenol red were mixed. At this time, the solution was poured into 5 volumes of 2-fold concentrated PBS solution, and magnetically stirred in a water bath at 25 ℃ for 2 hours at 1000 rpm. Centrifuging the solution at 10000g for 1h, removing supernatant, and obtaining the precipitate as collagen fiber. The steps are all aseptic operation. An improved photosensitive gelatin synthesis process is employed.

Weighing 10g of gelatin, dissolving in 100mL of PBS, heating to 60 ℃, stirring to completely dissolve, and cooling to 40 ℃ to obtain a gelatin solution. 2mL of methacrylic anhydride was added to 10mL of acetone to obtain an acetone solution of methacrylic anhydride. The acetone solution was added dropwise to the gelatin solution and the pH was maintained to 9 using 1mol/L sodium hydroxide solution. After the pH value was stabilized, the reaction was continued for 3 hours. Subsequently, dialyzing with distilled water, cutting off molecular weight of 8000Da in dialysis bag, dialyzing for 3d, and lyophilizing. A gelatin solution prepared from the above gelatin using 0.01mol/L PBS was added with 2mg/mL of a photoinitiator lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate. Adjusting pH to 7, filtering with 0.22 μm filter

Mixing the gelatin solution and collagen fiber at a mass ratio of 2:1, and adding 10 volume tenth of the mixture⁷/mL of mesenchymal stem cell suspension. Can be used for preparing biological ink containing cells. Placing the ink in a 3D printer nozzle, wherein the nozzle temperature is 22 ℃, the soleplate temperature is 4 ℃, executing a hydrogel-PCL composite printing program, and after printing is finished, using 5mW/cm²The printed product was irradiated with light at 365nm for 30 seconds and placed in a medium. After culturing for 10 days in the osteogenic induction medium, the results of alkaline phosphatase staining are shown in FIG. 1(c), wherein the opaque portion is PCL-supported, and the cells were induced for 10 days in the osteogenic medium, and the cells were stained with alkaline phosphatase using the kit, whereby it was revealed that the stem cells had differentiated into osteoblasts; alizarin staining results are shown in fig. 1(d), where the opaque part is PCL-supported, and induced for 10 days using osteogenic medium, and where alizarin staining was used, it was seen that stem cells had differentiated into osteoblasts. As a result, it was found that the stem cells were partially differentiated into osteoblasts.

Claims

1. A preparation method of photosensitive composite biological ink for 3D printing is characterized by comprising the following steps: the method comprises the following steps:

(1) preparation of collagen solution:

(2) preparing collagen fibers:

adding 0.1mol/L PBS solution, 1mg/mL phenol red solution and 1mol/L sodium hydroxide solution into the collagen solution obtained in the step (1), and neutralizing the solution to red by adjusting the adding amount to obtain a neutral collagen solution; pouring the obtained neutral collagen solution into a PBS solution, and magnetically stirring in a water bath at the temperature of 20-37 ℃ for 1-4 hours; centrifuging the obtained material, removing supernatant, and collecting precipitate as collagen fiber; wherein, when the neutral collagen solution is poured into the PBS solution, the concentration of the PBS solution is 0.001-0.05mol/L, and the volume of the PBS solution is 5-20 times of the volume of the neutral collagen solution; the magnetic stirring speed is 200-5000 r/min;

(3) preparation of modified photosensitive gelatin:

weighing a certain amount of gelatin, adding the gelatin into PBS buffer solution, heating to 60 ℃, stirring until the gelatin is completely dissolved, and cooling to 50 ℃ to obtain gelatin solution; dropwise adding an acetone solution of methacrylic anhydride into a gelatin solution, adjusting the pH value of the solution to be 8-9, and continuing to react for 1-5 hours; dialyzing the reaction product with distilled water, intercepting the molecular weight of the dialysis bag to be 8000Da, dialyzing for 3 days, and freeze-drying to obtain modified photosensitive gelatin;

2. The method of preparing the photosensitive composite bio-ink for 3D printing according to claim 1, wherein: in the step (1), the type I collagen is bovine collagen, rat tail collagen or pig collagen; the acid solution is acetic acid solution, hydrochloric acid solution or malonic acid solution.

3. The method of preparing the photosensitive composite bio-ink for 3D printing according to claim 1, wherein: in the step (3), the acetone solution of methacrylic anhydride is prepared by mixing a modifier of methacrylic anhydride according to the following ratio of methacrylic anhydride: acetone ═ 3: 10 by volume in acetone.

4. The method of preparing the photosensitive composite bio-ink for 3D printing according to claim 1, wherein: in the step (4), the photoinitiator is phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate or 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone; the proportion of the added photoinitiator to the modified photosensitive gelatin solution is (0.05-5) mg: 1 mL.

5. The method of preparing the photosensitive composite bio-ink for 3D printing according to claim 1, wherein: in the step (3) and the step (4), the pH value of the solution is adjusted by adopting 1mol/L NaOH solution.

6. The method of preparing the photosensitive composite bio-ink for 3D printing according to claim 1, wherein: in the step (5), the adding volume of the cell suspension is 10% of the volume of the mixed material.

7. A photosensitive composite bio-ink for 3D printing prepared using the method of claim 1.

8. The photosensitive composite bio-ink for 3D printing according to claim 7, wherein: when the biological ink is used for 3D printing, 365-405nm wavelength light is used for irradiating for 5-200 seconds, and the composite gel for promoting differentiation can be obtained.

9. The photosensitive composite bio-ink for 3D printing according to claim 8, wherein: the light intensity is 1-100mW/cm²。