WO2020206799A1

WO2020206799A1 - Method for preparing three-dimensional bioprinting ink and application thereof

Info

Publication number: WO2020206799A1
Application number: PCT/CN2019/086571
Authority: WO
Inventors: 吕培军; 李箐
Original assignee: 北京大学口腔医学院
Priority date: 2019-04-11
Filing date: 2019-05-13
Publication date: 2020-10-15
Also published as: CN109876190A

Abstract

A three-dimensional biological printing ink, a method for preparing the three-dimensional printing ink, and an application thereof. Gelatin, sodium alginate, and type I collagen are used to prepare a collagen hydrogel, to which calcined calf bone meal is loaded, to prepare a three-dimensional bioprinting ink, which has good biocompatibility, certain mechanical strength, and a three-dimensional porous structure. A bone graft material produced with the three-dimensional bioprinting ink improves biocompatibility compared with a bone graft material produced with porous apatite ceramic as a printing ink, and improves the plasticity and clinical operability of bone graft material products compared with a bone graft material produced with calcined calf bone meal.

Description

Preparation method and application of three-dimensional bioprinting ink

Technical field

The invention relates to the field of biologically active composite bone graft materials, in particular to a three-dimensional bioprinting ink, a preparation method and application of the three-dimensional bioprinting ink.

Background technique

Among the members of the hydroxyapatite family, the crystal structure of nano-hydroxyapatite (Hydroxyapatite, abbreviated as HA) is basically the same as that of mineral particles in bone tissue, and calcium phosphate (Tricalcium Phosphate, abbreviated as TCP) is in bone tissue. It also occupies a certain proportion, and its degradation rate is much higher than that of HA, which is easy to degrade in the body. In addition to the chemical composition, the degradation rate is also related to the contact area with body fluids. The best design principle of porous apatite ceramics is to take into account the degradation rate and the growth of new bone tissue, that is, the large pores of 300um-500um and the small pore structure of micrometers. Although the forming technology of calcium-phosphorus ceramics has been greatly developed, such as computer-controlled three-dimensional ceramic printing technology. However, when reading the structural information of natural bone tissue, it is still limited by measurement and characterization technology and processing technology. It is still a long-term goal to shorten the gap with natural bone tissue.

Deprotein Bovine Bone (DBB for short) is one of the existing bone repair materials. It not only retains inorganic calcium and phosphorus minerals, but also basically retains the porous structure of bone tissue. It has achieved good results in clinical applications. Evaluation. However, calf calcined bone has poor strength. Due to high temperature firing, collagen is completely lost, and the nanocrystals have changed and are not easily degraded; it is not easy to shape when directly used in 3D printing.

With the development of bone tissue three-dimensional bioprinting technology, the use of cells and bioactive matrix materials to print bio-ink composed of hydrogel and gelatin can construct a preset microstructure and have a high cell survival rate (>90 %). In order to ensure that the hydrogel can be extruded smoothly, the matrix material needs to have a certain degree of fluidity. The filaments falling into the printing platform will be deformed due to their own gravity and the superstructure accumulation, making it difficult to construct high-precision and complex models. Although methods such as chemical cross-linking can be used in current research to promote rapid formation of hydrogels, excessive cross-linking has been proven to affect cell migration, proliferation and differentiation. In addition, due to the viscosity of the printing material, calcium-phosphorus (Ca-P) materials are usually nano-sized, and therefore lack micro-sized pores.

In order to build a three-dimensional structure with good biocompatibility, certain mechanical strength, and three-dimensional porous structure, and a proper degradation rate to maintain the growth of new tissues, research on calf calcined bone bioprinting ink that can be rapidly shaped and mixed with cells It is the most important content in the current biological 3D printing.

Summary of the invention

The present invention provides a three-dimensional bioprinting ink, a preparation method and application of the three-dimensional bioprinting ink, so as to at least solve the problem of poor biocompatibility or plasticity of bone graft material products in related technologies.

In the first aspect, an embodiment of the present invention provides a method for preparing a three-dimensional bioprinting ink, including:

Step 1. Prepare an aqueous solution of sodium alginate;

Step 2: Add gelatin to the sodium alginate aqueous solution to form a sodium alginate-gelatin uniform hydrogel;

Step 3. Use beef tendon as raw material, and after extracting collagen, remove telopeptide to obtain type I collagen;

Step 4. Select calf bone joint head or tibia epiphysis as bone raw material, modify the bone raw material with a modifier, and obtain calcined bone powder after calcination;

Step 5: Add the type I collagen and the calcined bone meal to the hydrogel, stir evenly, and prepare a three-dimensional bioprinting ink.

Optionally, the step 3 includes:

Choose beef tendon, clean it, and use a grinder to cut large pieces of beef tendon into smaller sizes;

Use flash extraction technology to process beef tendon, add acetic acid to the extract to obtain crude collagen, and then go through centrifugation and dialysis steps to purify and separate to obtain crude collagen type I;

The type I collagen crude material is treated with pepsin to obtain atelopeptide-free immunogenic type I collagen, which is the type I collagen.

Optionally, the step 4 includes:

Choose calf bone joint head or tibia epiphysis as bone material, wash it with deionized water, and basically remove collagen by physical or biochemical methods;

Use diammonium phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifiers to modify the bone salt of the bone raw material from which collagen is basically removed, so that the calcium-phosphorus atom ratio Ca/P of the bone salt is reduced to 1.5 to 1.67;

After high temperature calcination at 800°C-1100°C to remove immunogenicity, the calcined bone powder is obtained.

Optionally, the step 5 includes:

Adding the type I collagen to the hydrogel at a mass ratio of hydrogel/type I collagen of 0.2-0.5, and mixing uniformly to obtain a collagen hydrogel;

Select calcined bone powder with porosity of 70%-80% and particle size of 60-100 mesh. According to the ratio of calcined bone powder/collagen hydrogel relative mass ratio of 0.10-0.25, add calcined bone powder to collagen hydrogel, mix evenly, and adjust The viscosity range is 30-107 mPa/s, and the pH is 6.5-7.5 to obtain the three-dimensional bioprinting ink.

In a second aspect, an embodiment of the present invention provides a three-dimensional bioprinting ink prepared by the method described in the first aspect.

In a third aspect, embodiments of the present invention provide an application of the three-dimensional bioprinting ink described in the second aspect in three-dimensional printing of bone graft materials, including:

In a sterile environment, the three-dimensional bioprinting ink is used for three-dimensional bioprinting and shaping, and freeze-dried to obtain a bone graft material product.

Optionally, using the three-dimensional bioprinting ink to perform three-dimensional bioprinting and shaping includes:

After sterilizing the three-dimensional bioprinting ink, load cells and biological factors;

Use 3D bioprinting ink loaded with cells and biological factors to shape 3D bioprinting;

Among them, the ambient temperature for 3D bioprinting is around 37°C.

Optionally, the crosslinking agent used in the three-dimensional bioprinting and shaping is a 100 mmol/L CaCl ₂ solution.

According to the three-dimensional bioprinting ink and the preparation method and application of the three-dimensional bioprinting ink provided by the embodiments of the present invention, a collagen hydrogel is prepared with gelatin, sodium alginate and type I collagen, and calf calcined bone meal is loaded to prepare the obtained three-dimensional Bio-printing ink has good biocompatibility, certain mechanical strength and a three-dimensional porous structure. In the three-dimensional bioprinting ink, the collagen composite calcined bone is conducive to the adhesion and proliferation of osteoblasts. The collagen components will be slowly absorbed within a few weeks without barrier effect. Using CaCl ₂ solution as a crosslinking agent can transform the ink into a homogeneous solid phase within 3-5 minutes, the curing time is appropriate, and the product has certain strength and elasticity. Through the present invention, the problem of poor biocompatibility or plasticity of the bone graft material products in the related art is solved. The bone graft material made of the three-dimensional bioprinting ink according to the embodiment of the present invention is compared with porous apatite ceramics. As the bone graft material made by printing ink, the biocompatibility is improved, and the plasticity and clinical operability of the bone graft material product are improved compared with the bone graft material made by calcining calf bone.

Description of the drawings

The drawings described here are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 is an infrared characteristic spectrum of collagen according to an embodiment of the present invention;

2 is a schematic structural diagram of a collagen gel calcined calf bone three-dimensional bioprinting bone graft material according to an embodiment of the present invention;

Fig. 3 is an X-ray diffraction pattern confirming that the hydroxyapatite of the scaffold includes HA and βTCP according to an embodiment of the present invention, where DBB/Col is calf calcined bone and collagen group, and DBB is calf calcined bone group;

4 is a schematic diagram of the porous characteristics of the material according to an embodiment of the present invention;

5 is a schematic diagram of the growth of cells closely attached to apatite particles in a three-dimensional bioprinting body structure according to an embodiment of the present invention;

Fig. 6 is a schematic diagram of cells uniformly distributed and grown in a three-dimensional bioprinting scaffold network according to an embodiment of the present invention;

Fig. 7 is a schematic diagram of osteogenic differentiation of cells in a three-dimensional bioprinting scaffold network according to an embodiment of the present invention.

detailed description

The features and exemplary embodiments of various aspects of the present invention will be described in detail below. In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not used to limit the present invention. For those skilled in the art, the present invention can be implemented without some of these specific details. The following description of the embodiments is only to provide a better understanding of the present invention by showing examples of the present invention.

It should be noted that in this article, the terms "including", "including" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, It also includes other elements not explicitly listed, or elements inherent to the process, method, article, or equipment. If there are no more restrictions, the element defined by the sentence "including..." does not exclude the existence of other same elements in the process, method, article, or equipment that includes the element.

Example 1

In this embodiment, a method for preparing 3D bioprinting ink for 3D bioprinting bone graft material is provided, and the method includes the following steps:

Step 1. Prepare an aqueous solution of sodium alginate;

Choose sodium alginate with a molecular weight of about 5000-50000 to dissolve it in distilled water, while mechanically stirring and ultrasonically mixing to prepare an aqueous solution with a concentration of 10-30mg/ml.

Step 2. Use the aqueous solution of step 1 to make a homogenous sodium alginate-gelatin hydrogel;

A certain amount of gelatin is used to adjust the viscosity of the aqueous solution obtained in step 1, and then the mixed solution is stirred under heating to form a hydrogel, and cooled to room temperature. Changing the ratio of gelatin/sodium alginate can adjust the amount of deformation of the colloid during storage.

Step 3. Choose fresh beef tendon as raw material, and after extracting collagen, remove telopeptide to obtain type I collagen;

Choose fresh beef tendons, clean them, use a grinder to cut large pieces of beef tendons into small-size raw materials; use flash extraction technology to process the beef tendons, and then use a certain amount of acetic acid to initially obtain crude collagen. After centrifugation and purification and separation through dialysis steps, a crude type I collagen is obtained; the prepared crude type I collagen is then treated with pepsin to obtain an atelo-immunogenic type I collagen. Type I collagen is composed of three peptide chains, including α(Ⅰ), α(Ⅱ) chains and one β chain. The final obtained collagen is generally a white powder with relative molecular weight ranging from about 2kD to 300kD. It is insoluble in cold water, dilute acid, dilute alkali solution, and has good water retention and emulsification. Its infrared characteristic spectrum is shown in the figure. 1. The curve a represents the collagen group, and the curve b represents the atelocollagen group.

Step 4. Select the new calf bone joint head or the epiphysis of the tibia as the bone raw material, modify the bone raw material with a modifier, and completely remove the immunogenicity of the bone material after calcining and modification to obtain calcined bone meal;

Choose calf bone joint head or tibia epiphysis as bone material, wash it with deionized water, and basically remove collagen by physical or biochemical methods. Using diammonium phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifiers, the bone matrix (bone salt) is modified to reduce the Ca/P atomic ratio of the bone matrix to 1.5-1.67. After calcination at 800℃-1100℃, the immunogenicity is completely removed; at the same time, the X-ray diffraction (XRD) patterns before and after the calcination of the bone block show that the bone matrix crystal phase changes from HA to HA/β-TCP dual-phase structure. Scanning electron microscopy (SEM) observations show that the calcined bone powder contains not only macropores ranging from 300μm to 500μm, but also microporous structures at the micron level.

Step 5: Add type I collagen and calcined bone meal to the hydrogel prepared in Step 2, and stir uniformly to prepare a three-dimensional bioprinting ink.

The hydrogel is mixed with type I collagen in a mass ratio of 0.2-0.5 to obtain a collagen hydrogel. Select calcined calf bone powder with porosity (volume ratio) of 70%-80%, particle size of 60-100 mesh, and add calcined bone powder to collagen hydrogel according to the relative mass ratio of calcined bone powder/collagen hydrogel to 0.10-0.25 , Mixed uniformly to prepare a three-dimensional bioprinting ink, by adjusting the mixing ratio and ambient temperature, adjust the viscosity range to 30-107mPa/s; adjust the pH to 6.5-7.5. In addition, changing the adding ratio of calcined bone powder can adjust the deformation and strength of the colloid during storage.

Example 2

This embodiment also provides a three-dimensional bio-printing ink prepared by the method for preparing three-dimensional bio-printing bone graft material provided in Example 1.

Wherein, the cross-linking agent used when the above-mentioned three-dimensional bio-printing ink is used for three-dimensional bio-printing shaping is a 100 mmol/L CaCl ₂ solution.

The collagen gel system of the three-dimensional bioprinting ink of this embodiment uses the interpenetrating network structure formed by gelatin/sodium and type I collagen as the scaffold, and the porous Ca-P bone meal is filled in this network structure. Biological factors use protein gel as a carrier to greatly improve the biological activity and mechanical properties of bone graft materials, providing a new type of repair material for bone defect repair treatment.

Example 3

In this embodiment, an application of a three-dimensional bioprinting ink in a three-dimensional bioprinting bone graft material is also provided, including:

In a sterile environment, the three-dimensional bioprinting ink prepared in Example 1 was used for three-dimensional bioprinting and shaping, and freeze-dried at -20°C to obtain a series of personalized three-dimensional collagen materials made of bone graft materials.

Wherein, the crosslinking agent used in the three-dimensional bioprinting and shaping is a 100 mmol/L CaCl ₂ solution.

Example 4

In this embodiment, another application of 3D bioprinting ink in 3D bioprinting bone graft material is also provided, including:

The three-dimensional bioprinting ink prepared in Example 1 is sterilized and mixed with biological factors or cells to prepare a three-dimensional bioprinting ink with biologically active factors or cells; the ink can be used to print various biologically active bone grafts material.

Wherein, the crosslinking agent used in the three-dimensional bioprinting and shaping is a 100 mmol/L CaCl ₂ solution. It can be transformed into a homogeneous solid phase by cross-linking with 100mmol/L CaCl2 solution for 3-5 minutes at a physiological body temperature of 37°C, with certain strength and flexibility.

The curing time of the material has a great impact on 3D bioprinting, and it should have a suitable curing time. Too short a time will cause premature curing and block the print nozzle; too long a curing time will lead to unsatisfactory shaping. The ideal curing time should be in the interval of 7-10 minutes. This is because bone powder has a lot of pores and absorbs moisture. Experiments show that when 10%-25% calf calcined bone powder is added, the compressive strength is about 0.1-0.3Mpa, and it has two phases of hydroxyapatite/β-tricalcium phosphate (Figure 3), the porosity is 60%-80%, and the pore size is about 150μm-500um (Figure 4). In vitro biocompatibility tests show that in the three-dimensional bioprinted scaffold network, osteoblasts are evenly distributed in the scaffold structure (Figure 5), and the cells are closely attached to the apatite particles to grow (Figure 6), and differentiate towards osteogenic (Figure 7).

The bioactive bone graft material prepared in this example has a certain morphology and mechanical strength, and at the same time, it can slowly release the bioactive factors that promote bone formation, and it can also meet the personalized design requirements for clinical bone defect repair. It is for maxillofacial bone and other defects. The repair treatment provides a new type of repair material.

In Example 3 and Example 4, gelatin and sodium alginate were selected to make an aqueous gel, and CaCl ₂ with good biocompatibility was used as a cross-linking agent to form a sodium alginate penetrating network structure (gelatin and sodium alginate penetrating the network In addition to the cross-linking effect of CaCl2, the formation of the structure includes the complexation of Mg2+ and colloid to form a hydrogel), and this gel network structure is used to load type I collagen and porous calf calcined bone meal. A series of 3D bioprinting bone graft materials can be formed by adjusting the colloid composition and concentration, adjusting the operating temperature and cross-linking time of 3D bioprinting, adding cells and bioactive factors, and adjusting the proportion of type I collagen and calcined calf bone meal. , And has excellent biological activity, biocompatibility, mechanical tolerance and sustained release properties. It can be widely used in periodontal and implant bone repair and restoration and reconstruction of alveolar ridge atrophy and absorption.

To sum up, the above-mentioned embodiments of the present invention aim at the disadvantages of the existing three-dimensional bioprinting bone graft materials, such as poor strength, difficult to decompose, poor biological activity, and lack of natural microporous structure. Therefore, modified calcined bone particles are selected as the matrix. Sodium alginate-gelatin is mixed with type I collagen to obtain collagen hydrogel. The collagen hydrogel is cross-linked with CaCl ₂ with good biocompatibility, and cells and biological factors can be loaded in the collagen hydrogel. The printed bone graft material has high strength, is easy to degrade and has osteoinductive properties.

It should be clear that the present invention is not limited to the specific configuration and processing described above. For brevity, a detailed description of the known method is omitted here. In the above embodiment, several specific steps are described and shown as examples. However, the method process of the present invention is not limited to the specific steps described and shown, and those skilled in the art can make various changes, modifications and additions, or change the order between the steps after understanding the spirit of the present invention.

The foregoing descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc., made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

A method for preparing three-dimensional bioprinting ink, which is characterized in that it comprises:

Step 1. Prepare an aqueous solution of sodium alginate;

Step 2: Add gelatin to the sodium alginate aqueous solution to form a sodium alginate-gelatin uniform hydrogel;

Step 3. Use beef tendon as raw material, and after extracting collagen, remove telopeptide to obtain type I collagen;

Step 4. Select calf bone joint head or tibia epiphysis as bone raw material, modify the bone raw material with a modifier, and obtain calcined bone powder after calcination;

Step 5: Add the type I collagen and the calcined bone meal to the hydrogel, stir evenly, and prepare a three-dimensional bioprinting ink.
The method according to claim 1, wherein the step 3 comprises:

Choose beef tendon, clean it, and use a grinder to cut large pieces of beef tendon into small-size beef tendons;

Use flash extraction technology to process beef tendon, add acetic acid to the extract to obtain crude collagen, and then go through centrifugation and dialysis steps to purify and separate to obtain crude collagen type I;

The type I collagen crude material is treated with pepsin to obtain atelopeptide-free immunogenic type I collagen, which is the type I collagen.
The method according to claim 1, wherein the step 4 comprises:

Choose calf bone joint head or tibia epiphysis as bone material, wash it with deionized water, and basically remove collagen by physical or biochemical methods;

Use diammonium phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifiers to modify the bone salt of the bone raw material from which collagen is basically removed, so that the calcium-phosphorus atom ratio Ca/P of the bone salt is reduced to 1.5 to 1.67;

After high temperature calcination at 800°C-1100°C to remove immunogenicity, the calcined bone powder is obtained.
The method according to claim 1, wherein the step 5 comprises:

Adding the type I collagen to the hydrogel at a mass ratio of hydrogel/type I collagen of 0.2-0.5, and mixing uniformly to obtain a collagen hydrogel;

Select calcined bone powder with porosity of 70%-80% and particle size of 60-100 mesh. According to the ratio of calcined bone powder/collagen hydrogel relative mass ratio of 0.10-0.25, add calcined bone powder to collagen hydrogel, mix evenly, and adjust The viscosity range is 30-107 mPa/s, and the pH is 6.5-7.5 to obtain the three-dimensional bioprinting ink.
A three-dimensional biological printing ink, characterized in that the three-dimensional biological printing ink is prepared by the method according to any one of claims 1 to 4.
An application of the three-dimensional bioprinting ink according to claim 5 in three-dimensional printing bone graft material, characterized in that it comprises:

In a sterile environment, the three-dimensional bioprinting ink is used for three-dimensional bioprinting and shaping, and freeze-dried to obtain a bone graft material product.
The application according to claim 6, wherein using the three-dimensional bioprinting ink to perform three-dimensional bioprinting and shaping comprises:

After sterilizing the three-dimensional bioprinting ink, load cells and biological factors;

Use 3D bioprinting ink loaded with cells and biological factors to shape 3D bioprinting;

Among them, the ambient temperature for 3D bioprinting is around 37°C.
The application according to claim 6 or 7, characterized in that the cross-linking agent used in the three-dimensional bioprinting and shaping is a 100 mmol/L CaCl 2 solution.