阅读说明：本技术 一种生物3d打印墨水及其制备方法 (Biological 3D printing ink and preparation method thereof ) 是由 宋薇 姚斌 黄沙 付小兵 于 2019-10-30 设计创作，主要内容包括：本发明提供了一种生物3D打印墨水及其制备方法,该制备方法包括以下步骤：S1：制备氢氧化钙饱和溶液,在氢氧化钙饱中加入二氧化硅纳米粒子悬液、离心、取沉淀；即得改性纳米生物玻璃粒子；S2：将明胶和海藻酸钠溶解于去离子水中溶解,再加入步骤S1制得的改性纳米生物玻璃粒子,搅拌均匀制得液态水凝胶,消毒、平衡,即得明胶-海藻酸钠复合水凝胶；S3：将氯化钙固体粉末溶解于去离子水中,制得氯化钙溶液,消毒灭菌,即得交联剂；S4：在所述明胶-海藻酸钠复合水凝胶中加入交联剂进行交联,即得生物3D打印墨水；本发明制备的生物3D打印墨水不仅力学性能显著提高且成胶性能良好、孔隙丰富,具有很好的载细胞潜能。(The invention provides biological 3D printing ink and a preparation method thereof, wherein the preparation method comprises the following steps: s1: preparing a calcium hydroxide saturated solution, adding the silica nanoparticle suspension into the calcium hydroxide saturated solution, centrifuging, and taking a precipitate; obtaining modified nanometer biological glass particles; s2: dissolving gelatin and sodium alginate in deionized water, adding the modified nano bioglass particles prepared in the step S1, uniformly stirring to prepare liquid hydrogel, and sterilizing and balancing to obtain gelatin-sodium alginate composite hydrogel; s3: dissolving calcium chloride solid powder in deionized water to obtain calcium chloride solution, and sterilizing to obtain crosslinking agent; s4: adding a cross-linking agent into the gelatin-sodium alginate composite hydrogel for cross-linking to obtain biological 3D printing ink; the biological 3D printing ink prepared by the invention not only has obviously improved mechanical property, but also has good gelling property, rich pores and good cell-carrying potential.)

1. The modified nanometer bioglass particles applied to biological 3D printing ink are characterized in that the preparation method of the modified nanometer bioglass particles comprises the following steps: preparing a calcium hydroxide saturated solution, adding the silica nanoparticle suspension into the calcium hydroxide saturated solution, centrifuging, and taking a precipitate; thus obtaining the modified nanometer biological glass particles.

2. The biological 3D printing ink prepared from the modified nano bioglass particles, the gelatin-sodium alginate composite hydrogel and the cross-linking agent in the claim 1 is characterized in that the mass ratio of the modified nano bioglass particles to the gelatin-sodium alginate composite hydrogel to the cross-linking agent is 100: 120:1-1: 10-12.

3. A method for preparing the biological 3D printing ink according to claim 2, wherein the method comprises the following steps:

s1: preparing modified nano bioglass particles: preparing a calcium hydroxide saturated solution, adding the silica nanoparticle suspension into the calcium hydroxide saturated solution, centrifuging, and taking a precipitate; obtaining modified nanometer biological glass particles;

s2: preparing the gelatin-sodium alginate composite hydrogel: dissolving gelatin and sodium alginate in deionized water, adding the modified nano bioglass particles prepared in the step S1, uniformly stirring to prepare liquid hydrogel, and sterilizing and balancing to obtain gelatin-sodium alginate composite hydrogel;

s3: preparation of the crosslinking agent: dissolving calcium chloride solid powder in deionized water to obtain calcium chloride solution, and sterilizing to obtain crosslinking agent;

s4: and adding a cross-linking agent into the gelatin-sodium alginate composite hydrogel for cross-linking to obtain the biological 3D printing ink.

4. The method according to claim 2, wherein the step of preparing the saturated solution of calcium hydroxide in step S1 comprises the steps of: weighing calcium hydroxide solid powder, placing the calcium hydroxide solid powder in a beaker, adding deionized water to ensure that the concentration of the calcium hydroxide solid powder in the deionized water is 1.6-1.7g/L, stirring for more than 12 hours by using a constant-temperature magnetic stirrer, sealing the beaker in the stirring process, centrifuging after stirring, removing precipitate, and taking supernatant to obtain a calcium hydroxide saturated solution.

5. The method of claim 3, wherein the step of adding the silica nanoparticle suspension to the calcium hydroxide solution, centrifuging the silica nanoparticle suspension, and taking out the precipitate in step S1 comprises the steps of: placing a saturated calcium hydroxide solution into a beaker, adding 40 wt% of silica nanoparticle suspension, wherein the volume ratio of the saturated calcium hydroxide solution to the 40 wt% of silica nanoparticle suspension is 40:6-7, placing the beaker on a constant-temperature magnetic stirrer for stirring for not less than 72h, keeping the beaker in a sealed state during stirring, centrifuging, taking a precipitate, adding deionized water for washing, centrifuging again, taking the precipitate, adding water for washing, and repeatedly washing for three times to take the precipitate.

6. The preparation method of claim 3, wherein the step S2 of dissolving gelatin and sodium alginate in deionized water, adding the modified nano bioglass particles prepared in the step S1, and uniformly stirring to obtain the liquid hydrogel specifically comprises: weighing gelatin and sodium alginate, placing into a small beaker, adding deionized water, wherein the concentration of the gelatin in the deionized water is 0.01g/ml, the concentration of the sodium alginate in the deionized water is 0.03g/ml, sealing the beaker, dissolving for 25-35min in a water bath kettle at the temperature of 40 +/-5 ℃, adding the modified nano biological glass particles prepared in the step S1, sealing the beaker again, and stirring for 1.5-2.5h at the temperature of 40 +/-5 ℃ by using a constant-temperature magnetic stirrer to obtain the liquid hydrogel.

7. The method of claim 3, wherein the sterilizing and balancing of step S2 includes the following steps: treating liquid hydrogel in 70 deg.C water bath for 30min, placing in 4 deg.C cold bath for 5min, repeating for 3 times, sterilizing, balancing at room temperature for 1 hr, sealing with sealing film, and storing at 4 deg.C.

8. The method according to claim 3, wherein the step S3 of preparing the crosslinking agent comprises the steps of: weighing calcium chloride solid powder, placing in a beaker, adding deionized water, adding 25g of the calcium chloride solid powder into every 1000ml of the deionized water, placing in a constant-temperature magnetic stirrer, stirring for 25-35min to obtain a 2.5% calcium chloride solution, transferring to a high-temperature resistant glass bottle, sealing after high-pressure sterilization of an autoclave, and storing at 4 ℃ to obtain the cross-linking agent.

9. The preparation method according to claim 3, wherein the step S4 of adding a cross-linking agent into the gelatin-sodium alginate composite hydrogel for cross-linking is specifically: and (3) dropwise adding a cross-linking agent to the surface of the 3D printing block until the cross-linking agent completely covers the printing block, removing the cross-linking agent after crosslinking for 10min, and washing once with a DMEM complete culture medium.

10. The use of the modified nano bioglass particles of claim 1 in the preparation of biological 3D printing inks that are mechanically, gel-forming, porous and have good cell-loading potential.

Technical Field

The invention relates to the field of medical model manufacturing and application, in particular to biological 3D printing ink and a preparation method thereof.

Background

The skin is the largest organ of the human body, and has very important function for maintaining the homeostasis of the human body and the normal physiological function of the body, because the damage and the loss of the skin often occur when the skin is exposed to the external environment, for smaller skin defects, the body can completely regenerate to a certain extent through self compensation, but for larger wound surfaces, the skin generally carries out scar repair, and for larger wound surfaces, the defect of the skin often delays healing or can not heal, the wound surface enables the body of the patient to be directly exposed to the external environment, not only can the body fluid balance of the body be maintained, but also enables the patient to be exposed to high risk of infection, so the wound surface is necessary to be sealed in time, in the past, sterile gauze is adopted for wound surface covering in clinical work to play the roles of isolating bacteria and protecting the wound surface, and the final wound surface healing depends on the healing capability of the wound surface of the patient, for diabetic patients and patients with local poor blood circulation, the wound surface is difficult to be closed by the self-repair capability, so that how to solve the problem of wound surface with insufficient repair function becomes a great difficulty in clinical work.

With the increasing change of tissue engineering technology, 3D printing technology and cell-loaded hydrogel are becoming the key points of people's attention, and an ideal wound dressing should have the basic characteristics of adhering to a wound, being soft, permeable/permeable, maintaining homeostasis and the like, and at the same time, should also form a water-retaining layer, and in addition, should have the characteristics of being convenient to carry and use, and the hydrogel can simultaneously meet the above requirements, and thus is widely applied to the research and development of biological dressings.

Different from the microenvironment for transferring seed cells to a scaffold material and assisting in promoting regeneration in the prior tissue engineering, the cell-loaded bio-ink can integrate the bio-material, the seed cells and the regeneration microenvironment into a whole, thereby greatly improving the clinical application possibility of the tissue engineering skin, the selection and the appropriate addition of the bio-material into the regeneration microenvironment are the key points of the current research in the development process of the cell-loaded hydrogel, the gelatin is widely applied to the research of wound dressings due to the excellent biodegradability, biocompatibility, high cell attachment and proliferation, low immunogenicity and the like, in the previous research, the gelatin-sodium alginate cell-loaded bio-ink not only has good biocompatibility, but also can improve the migration, proliferation and differentiation capabilities of cells, however, in the practical application process, the printing gel block hardness of the gelatin-sodium alginate cell-loaded bio-ink is lower, resulting in a softer texture and difficult application to wound models for animals.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a biological 3D printing ink and a method for preparing the same,

the invention provides a modified nano bioglass particle applied to biological 3D printing ink, and a preparation method of the modified nano bioglass particle comprises the following steps: preparing a calcium hydroxide saturated solution, adding the silica nanoparticle suspension into the calcium hydroxide saturated solution, centrifuging, and taking a precipitate; thus obtaining the modified nanometer biological glass particles.

The invention also provides biological 3D printing ink prepared from the modified nano-bioglass particles, the gelatin-sodium alginate composite hydrogel and the cross-linking agent, wherein the mass ratio of the modified nano-bioglass particles to the gelatin-sodium alginate composite hydrogel to the cross-linking agent is 100: 120:1-1:10-12

The invention also provides a preparation method of the biological 3D printing ink, which comprises the following steps:

s1: preparing modified nano bioglass particles: preparing a calcium hydroxide saturated solution, adding the silica nanoparticle suspension into the calcium hydroxide saturated solution, centrifuging, and taking a precipitate; obtaining modified nanometer biological glass particles;

s2: preparing the gelatin-sodium alginate composite hydrogel: dissolving gelatin and sodium alginate in deionized water, adding the modified nano bioglass particles prepared in the step S1, uniformly stirring to prepare liquid hydrogel, and sterilizing and balancing to obtain gelatin-sodium alginate composite hydrogel;

s3: preparation of the crosslinking agent: dissolving calcium chloride solid powder in deionized water to obtain calcium chloride solution, and sterilizing to obtain crosslinking agent;

s4: and adding a cross-linking agent into the gelatin-sodium alginate composite hydrogel for cross-linking to obtain the biological 3D printing ink.

In a further improvement, the preparation of the saturated solution of calcium hydroxide in the step S1 specifically comprises the following steps: weighing calcium hydroxide solid powder, placing the calcium hydroxide solid powder in a beaker, adding deionized water to ensure that the concentration of the calcium hydroxide solid powder in the deionized water is 1.6-1.7g/L, stirring for more than 12 hours by using a constant-temperature magnetic stirrer, sealing the beaker in the stirring process, centrifuging after stirring, removing precipitate, and taking supernatant to obtain a calcium hydroxide saturated solution.

In a further improvement, the step S1 of adding the silica nanoparticle suspension to the calcium hydroxide solution, centrifuging, and taking out the precipitate specifically includes the following steps: placing a saturated calcium hydroxide solution into a beaker, adding 40 wt% of silica nanoparticle suspension, wherein the volume ratio of the saturated calcium hydroxide solution to the 40 wt% of silica nanoparticle suspension is 40:6-7, placing the beaker on a constant-temperature magnetic stirrer for stirring for not less than 72h, keeping the beaker in a sealed state during stirring, centrifuging, taking a precipitate, adding deionized water for washing, centrifuging again, taking the precipitate, adding water for washing, and repeatedly washing for three times to take the precipitate.

Wherein the centrifugation speed is 1500rpm, and the centrifugation time is 5 min.

In a further improvement, the gelatin of step S2 is type a gelatin and is derived from pig skin.

Further improvement, the step S2 of dissolving gelatin and sodium alginate in deionized water, adding the modified nano bioglass particles prepared in the step S1, and uniformly stirring to prepare the liquid hydrogel specifically comprises: weighing gelatin and sodium alginate, placing into a small beaker, adding deionized water, wherein the concentration of the gelatin in the deionized water is 0.01g/ml, the concentration of the sodium alginate in the deionized water is 0.03g/ml, sealing the beaker, dissolving for 25-35min in a water bath kettle at the temperature of 40 +/-5 ℃, adding the modified nano biological glass particles prepared in the step S1, sealing the beaker again, and stirring for 1.5-2.5h at the temperature of 40 +/-5 ℃ by using a constant-temperature magnetic stirrer to obtain the liquid hydrogel.

In a further improvement, the sterilization and balancing of step S2 specifically includes the following steps: treating liquid hydrogel in 70 deg.C water bath for 30min, placing in 4 deg.C cold bath for 5min, repeating for 3 times, sterilizing, balancing at room temperature for 1 hr, sealing with sealing film, and storing at 4 deg.C.

In a further improvement, the preparation of the cross-linking agent of step S3 specifically comprises the following steps: weighing calcium chloride solid powder, placing in a beaker, adding deionized water, adding 25g of the calcium chloride solid powder into every 1000ml of the deionized water, placing in a constant-temperature magnetic stirrer, stirring for 25-35min to obtain a 2.5% calcium chloride solution, transferring to a high-temperature resistant glass bottle, sealing after high-pressure sterilization of an autoclave, and storing at 4 ℃ to obtain the cross-linking agent.

Further improvement, the step S4 of adding a cross-linking agent into the gelatin-sodium alginate composite hydrogel for cross-linking specifically comprises: and (3) dropwise adding a cross-linking agent to the surface of the 3D printing block until the cross-linking agent completely covers the printing block, removing the cross-linking agent after crosslinking for 10min, and washing once with a DMEM complete culture medium.

The invention also provides application of the modified nano-bioglass particles in preparation of biological 3D printing ink with good mechanical property, good gelling property, rich pores and good cell-carrying potential.

The biological 3D printing ink prepared by the preparation method of the biological 3D printing ink provided by the invention has the advantages of remarkably improved mechanical property, good gelling property, rich pores and good cell-carrying potential. In addition, in a cell-loading experiment, the hydrogel can keep an intact shape, and has excellent clinical application potential.

Drawings

FIG. 1 is an appearance picture of the nano bioglass particles before and after modification, wherein A is the appearance picture of the nano bioglass particles before modification, and B is the appearance picture of the modified nano bioglass particles;

FIG. 2 is a state diagram of liquid hydrogel prepared by adding modified nano bioglass particles into gelatin and sodium alginate hydrogel, fully stirring and uniformly mixing the particles and the hydrogel, and respectively standing the liquid hydrogel at room temperature and 37 ℃ for 72 hours; wherein A is a state diagram of the liquid hydrogel prepared from the modified nano bioglass particles after being placed for 72 hours at 37 ℃; b is a state diagram of liquid hydrogel prepared from unmodified nano bioglass particles after being placed for 72 hours at 37 ℃; c is a state diagram of the liquid hydrogel prepared by the modified nano bioglass particles after being placed for 72 hours at room temperature; d is a state diagram of the liquid hydrogel prepared by the unmodified nano bioglass particles after being placed for 72 hours at room temperature;

FIG. 3 is a drawing showing the cross-linking culture and three-day culture of a third-generation mouse fibroblast cell mixed with a gel, wherein A is a state diagram of the hydrogel-loaded cell of the example after cross-linking; b is a state diagram after the hydrogel-loaded cells of the comparative example were crosslinked; c is a state diagram of the hydrogel after 3 days of culture of the hydrogel-carrying cells of the examples; d is a diagram showing the state of the hydrogel after 3 days of culture of the hydrogel-carrying cells of the comparative example;

FIG. 4 is a bar graph showing the cell survival rate and cell inhibitory rate of test example 4; wherein BG is an example, con is a comparative example.

Detailed Description

The present invention will be further illustrated with reference to the following examples; the following examples are illustrative, not limiting, and are not intended to limit the scope of the invention; the equipment used in the invention is the equipment commonly used in the field if no special provisions are made; the methods used in the present invention are those commonly used in the art, unless otherwise specified.