阅读说明：本技术 光交联重组胶原蛋白水凝胶、制备方法及其在3d生物打印中的应用 (Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting ) 是由 杨树林 金明杰 王子勋 邓爱鹏 朱帅 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种光交联重组胶原蛋白水凝胶、制备方法及其在3D生物打印中的应用。通过将甲基丙烯酸酐加入到重组胶原蛋白的磷酸盐溶液中,充分搅拌反应得到甲基丙基酸酐改性重组胶原蛋白,再将改性重组胶原蛋白溶解于培养基中,加入光引发剂,混合均匀后光反应交联固化制得光交联重组胶原蛋白水凝胶。本发明的重组胶原蛋白水凝胶前驱液置于模具中或作为3D打印墨水,经光反应交联固化制得组织工程支架或3D打印成品,还可在水凝胶前驱液中加入细胞,制备负载细胞的组织工程支架或3D打印成品。本发明的水凝胶理化性能良好、生物相容性好,且质量稳定,成形性良好,可以制备为任意大小和形状,适于大规模工业化生产。(The invention discloses a photo-crosslinking recombinant collagen hydrogel, a preparation method and application thereof in 3D bioprinting. Methacrylic anhydride is added into phosphate solution of the recombinant collagen, the methyl propyl anhydride modified recombinant collagen is obtained through full stirring reaction, the modified recombinant collagen is dissolved in a culture medium, a photoinitiator is added, and the photo-crosslinking recombinant collagen hydrogel is prepared through photo-reaction crosslinking and solidification after uniform mixing. The recombinant collagen hydrogel precursor liquid is placed in a mould or used as 3D printing ink, and is subjected to photoreaction, crosslinking and curing to prepare a tissue engineering scaffold or a 3D printing finished product, and cells can be added into the hydrogel precursor liquid to prepare a cell-loaded tissue engineering scaffold or a 3D printing finished product. The hydrogel has good physical and chemical properties, good biocompatibility, stable quality and good formability, can be prepared into any size and shape, and is suitable for large-scale industrial production.)

1. The preparation method of the photo-crosslinking recombinant collagen hydrogel is characterized by comprising the following specific steps:

step 1, adding methacrylic anhydride into phosphate solution of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

and 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, uniformly mixing to obtain a hydrogel precursor solution, and carrying out photoreaction, crosslinking and curing to obtain the recombinant collagen hydrogel.

2. The recombinant collagen hydrogel produced by the method according to claim 1.

3. The application of the photocrosslinking recombinant collagen hydrogel in preparing a tissue engineering scaffold is characterized in that the specific application method comprises the following steps:

step 1, adding methacrylic anhydride into phosphate solution of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

step 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, and uniformly mixing to obtain a hydrogel precursor solution;

and 3, placing the hydrogel precursor solution obtained in the step 2 into a mold, and carrying out photoreaction, crosslinking and curing to obtain the tissue engineering scaffold with a specific shape.

4. The application of the photo-crosslinking recombinant collagen hydrogel in 3D bioprinting is characterized in that the specific application method comprises the following steps:

step 1, adding methacrylic anhydride into phosphate solution of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

step 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, and uniformly mixing to obtain a hydrogel precursor solution;

and 3, taking the hydrogel precursor liquid obtained in the step 2 as 3D bioprinting ink, and performing 3D bioprinting photoreaction crosslinking and curing to obtain a 3D bioprinting finished product.

5. The method or use according to claim 1, 3 or 4 wherein the recombinant collagen is of the type I, II, III, IV, V, VI, VII, IX, X, XI or XII repeated with G-X-Y or other small molecular weight collagen; the recombinant collagen is derived from a microbial expression system, such as escherichia coli, streptococcus, hansenula, saccharomyces cerevisiae, or pichia, or a mammalian expression system, such as a transgenic mouse or human embryonic kidney epithelial cell, or an insect expression system, such as an insect cell or a transgenic silkworm, or a plant expression system, such as a transgenic tobacco, barley seed, or corn; the molecular weight of the recombinant collagen is 2-500 Kd; the type of the recombinant collagen is selected from recombinant human collagen, recombinant human collagen or other types of recombinant collagen; the recombinant human collagen is produced by pichia pastoris with the preservation number of CGMCC NO. 5021.

6. The preparation method or the application of claim 1, 3 or 4, wherein in the step 1, the content of the recombinant collagen in the phosphate solution of the recombinant collagen is 5-20% (W/V), and more preferably 10-20% (W/V); in the step 1, the addition amount of the methacrylic anhydride is 3-12% (V)_{Methacrylic anhydride}/V_{Phosphate solution of recombinant collagen}) More preferably 6 to 9% (V)_{Methacrylic anhydride}/V_{Phosphate solution of recombinant collagen}) (ii) a Diluting the supernatant by 3-5 times when adding water; the pH value of the solution before dialysis is 6-8, and more preferably 6.5-7.5.

7. The method or use according to claim 1, 3 or 4, wherein in step 2, the culture medium is a complete medium suitable for the corresponding target cell; the photoinitiator is an ultraviolet photoinitiator or a visible photoinitiator; when the photoinitiator is the ultraviolet photoinitiator 2959, the content of the photoinitiator 2959 in the solution is 0.01-1%, preferably 0.5-1%; when the photoinitiator is a blue light initiator LAP, the content of the photoinitiator LAP in the solution is 0.01-2%, preferably 0.1-0.5%; dissolving the methyl propyl anhydride modified recombinant collagen into a culture medium, wherein the content of the methyl propyl anhydride modified recombinant collagen is 2-30% (W/V); when the ink is used as 3D biological printing ink, the concentration is more preferably 5-15% (W/V); the volume ratio of the photoinitiator aqueous solution to the culture medium containing the methacrylic anhydride modified recombinant collagen is 1: 5-20, and more preferably 1: 10-15; sodium alginate, hyaluronic acid, xanthan gum or gelatin is also added into the culture medium; preferably, the content of the sodium alginate, the hyaluronic acid, the xanthan gum and the gelatin is 0-30% (W/V).

8. The preparation method or application of claim 1, 3 or 4, wherein in the step 3, the specific irradiation wavelength range of the light used for the photoreaction crosslinking curing is 200-800 nm; when the photoinitiator is an ultraviolet initiator 2959, the specific irradiation wavelength range is 365-405 nm, and the most preferable range is 365 nm; when the photoinitiator is a blue light initiator LAP, the specific irradiation wavelength range is 400-450 nm, and the most preferable is 405 nm; the irradiation time is 5-300 seconds.

9. The preparation method or the application of claim 1, 3 or 4, wherein in the step 2, cells are further added into the culture medium to prepare a cell-loaded hydrogel precursor, and the cell-loaded tissue engineering scaffold is prepared by directly irradiating, crosslinking and curing the cell-loaded hydrogel precursor by a light source with a specific wavelength; or taking the cell-loaded hydrogel precursor as 3D bioprinting ink, irradiating by a light source with a specific wavelength, crosslinking and curing to prepare a cell-loaded 3D bioprinting finished product.

10. A tissue engineering scaffold or a 3D bioprinted finished product made by the use of any one of claims 3 to 9.

Technical Field

The invention belongs to the technical field of biological materials, and relates to a photo-crosslinking recombinant collagen hydrogel, a preparation method and application thereof in 3D bioprinting.

Background

Collagen is the most abundant class of structural proteins in animals. The collagen has strong biological activity and biological function, can participate in the migration, differentiation and proliferation of cells, enables connective tissues to have mechanical strength, can promote the growth of the cells, and has good biocompatibility and biodegradability. Based on the characteristics, the collagen has wide application in the medical and health fields of burn and wound repair, eye and cornea disease treatment, wound surface hemostasis, drug delivery and slow release, orthopedics, health care, cosmetology and the like. Common collagen is separated and extracted from animal tissues such as cow bones, cow hides, fish scales, fish skins, pig skins and the like, has the defects and shortcomings of animal virus hidden danger, generation of xenogenous or xenogenous rejection reaction when applied to a human body, uncertain molecular weight, wide distribution range and the like. The inventors' earlier research results overcome these drawbacks well: the human collagen produced by the gene recombination technology replaces the natural collagen. The recombinant collagen takes type III collagen as a template, and a six-tandem sequence is constructed in vitro and is connected with pPIC9K to form a human-like collagen gene six-tandem expression vector pPIC9KG 6. The expression vector is introduced into pichia pastoris to be subjected to high-density fermentation, and the high-purity RHC is obtained through separation and purification (Chinese patent ZL 200610098297.5).

3D biological printing is an important branch of 3D printing, and the biggest characteristic is that cell-carrying biological materials can be accurately distributed and used for constructing complex three-dimensional living tissues or organs. The 3D bioprinting can overcome the defect of insufficient compatibility between artificial tissue construction and natural organism tissues, achieves the refined reproduction of target tissues or organs by integrating the technical advantages of the fields of biological materials science, cell biology, tissue engineering, physics, medicine and the like so as to solve the problem of increasingly serious human organ shortage, and can serve the fields of high-throughput toxicology detection, drug development and the like.

The hydrogel is a soft polymer material which is insoluble in water but has strong hydrophilicity, the molecules of the hydrogel have a unique three-dimensional network-shaped configuration, the hydrophilic group is combined with water molecules to lock the water molecules in the three-dimensional network, and the hydrophobic group expands when meeting water, so that the hydrogel can absorb a large amount of water solution in the water solution and keep a certain shape. The hydrogel is used as a bionic biological functional material with high water content and good biocompatibility and is always applied to the development of tissue engineering scaffold materials for a long time. The hydrogel containing the collagen is used as 3D biological printing ink, and has important application value in the aspects of simulating the in-vivo growth environment of cells, establishing an in-vitro cell culture model, promoting wound repair, filling, regeneration and the like.

The existing 3D biological printing ink uses gelatin as a main raw material, but the intramolecular covalent bond and hydrogen bond of the gelatin are broken, so that the gelatin has no rodlike triple-helix space structure, only three types of triple-helix structures are left, and the triple-helix space structure is maintained by the intramolecular hydrogen bond, so that the bioactivity of the gelatin is lost. Meanwhile, gelatin is insoluble in water but can be dissolved by heating, and is a heat-compatible protein mixture, so that the gelatin-based material has high viscosity, complex processing conditions and high energy consumption, and is not beneficial to industrial popularization (research on physical and chemical properties of silver carp skin gelatin and performance improvement thereof [ D ] 2016. academic papers of the university of Hefei industry; linrong, old shellfish, Wang, et al. collagen properties and research progress of preparation methods thereof [ J ] fishery research, 2018,40(05):73-81 and the like).

Disclosure of Invention

One of the purposes of the invention is to provide a preparation method of photo-crosslinking recombinant collagen hydrogel, which comprises the following specific steps:

step 1, adding methacrylic anhydride into phosphate solution (PBS) of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

and 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, uniformly mixing to obtain a hydrogel precursor solution, and carrying out photoreaction, crosslinking and curing to obtain the recombinant collagen hydrogel.

The second object of the present invention is to provide a recombinant collagen hydrogel prepared by the above preparation method.

The invention also aims to provide the application of the photocrosslinking recombinant collagen hydrogel in preparing a tissue engineering scaffold.

Specifically, the application of the photo-crosslinking recombinant collagen hydrogel in the preparation of a tissue engineering scaffold comprises the following specific application methods:

step 1, adding methacrylic anhydride into phosphate solution (PBS) of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

step 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, and uniformly mixing to obtain a hydrogel precursor solution;

and 3, placing the hydrogel precursor solution obtained in the step 2 into a mold, and carrying out photoreaction, crosslinking and curing to obtain the tissue engineering scaffold with a specific shape.

The fourth purpose of the invention is to provide the application of the photo-crosslinking recombinant collagen hydrogel in 3D bioprinting.

Specifically, the application of the photo-crosslinking recombinant collagen hydrogel in 3D bioprinting comprises the following specific application methods:

step 1, adding methacrylic anhydride into phosphate solution (PBS) of recombinant collagen, fully stirring for reaction, centrifuging to obtain supernatant, adding water to dilute the supernatant, dialyzing, and freeze-drying after dialysis to obtain the methyl propyl anhydride modified recombinant collagen;

step 2, dissolving the methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, and uniformly mixing to obtain a hydrogel precursor solution;

and 3, taking the hydrogel precursor liquid obtained in the step 2 as 3D bioprinting ink, and performing 3D bioprinting photoreaction crosslinking and curing to obtain a 3D bioprinting finished product.

The type of the recombinant collagen can be I, II, III, IV, V, VI, VII, IX, X, XI, XII and G-X-Y repeated sequence type or other low molecular weight collagens. The recombinant collagen can be derived from microbial expression systems (escherichia coli, streptococcus, hansenula, saccharomyces cerevisiae, pichia pastoris, etc.), mammalian expression systems (transgenic mice, human embryonic kidney epithelial cells, etc.), insect expression systems (insect cells, transgenic silkworms, etc.) and plant expression systems (transgenic tobacco, barley seeds, corn, etc.). The molecular weight of the recombinant collagen is 2-500 Kd. The recombinant collagen type is selected from recombinant human collagen, recombinant human collagen or other types of recombinant collagen.

In a specific embodiment of the present invention, the recombinant collagen is recombinant human collagen. In the specific embodiment, the recombinant human collagen of the present invention is a recombinant human collagen obtained by a method disclosed in chinese patent ZL200610098297.5, and more preferably, a recombinant human collagen produced by using pichia pastoris gene engineering bacteria (the strain is deposited in the common microorganism center of the china committee for culture preservation and management of microorganisms, the deposit number is cgmccno.5021) constructed by different numbers of repeated gene recombinant plasmids in series in the same direction through a high-density fermentation and purification method provided in chinese patent ZL 201110327865.5. Compared with animal (especially non-mammalian) collagen, the human collagen has more reliable use safety for human body.

Preferably, in the step 1, the content of the recombinant collagen in the phosphate solution of the recombinant collagen is 5-20% (W/V), and more preferably 10-20% (W/V).

Preferably, in step 1, the addition amount of the methacrylic anhydride is 3-12% (V)_{Methacrylic anhydride}/V_{Phosphate solution of recombinant collagen}) More preferably 6 to 9% (V)_{Methacrylic anhydride}/V_{Phosphate solution of recombinant collagen})。

Preferably, in the step 1, the dilution factor when the supernatant is diluted with water is 3 to 5 times.

Preferably, in step 1, the pH value of the solution before dialysis is 6 to 8, more preferably 6.5 to 7.5.

In the present invention, in step 2, the culture medium is a complete culture medium suitable for the corresponding target cells, and may be a cell or tissue culture medium conventionally used in the art, such as DMEM complete culture medium.

In the present invention, the photoinitiator used in step 2 is a uv initiator or a visible photoinitiator. When the photoinitiator is an ultraviolet photoinitiator 2959, the content of the photoinitiator 2959 in the solution is 0.01-1%, preferably 0.5-1%; when the photoinitiator is a blue light initiator LAP, the content of the photoinitiator LAP in the solution is 0.01-2%, preferably 0.1-0.5%.

Preferably, in the step 2, the methyl propyl anhydride modified recombinant collagen is dissolved in a culture medium, wherein the content of the methyl propyl anhydride modified recombinant collagen is 2-30% (W/V); when the ink is used as 3D biological printing ink, the concentration is more preferably 5-15% (W/V), and when the concentration is too low, the forming control in the 3D biological printing process is not facilitated.

Preferably, in the step 2, the volume ratio of the photoinitiator aqueous solution to the culture medium containing the methacrylic anhydride modified recombinant collagen is 1: 5-20, and more preferably 1: 10-15.

Preferably, in step 2, sodium alginate, hyaluronic acid, xanthan gum or gelatin and the like are further added to the culture medium to increase the mechanical strength of the hydrogel ink after printing or widen the application range. More preferably, the content of the sodium alginate, the hyaluronic acid, the xanthan gum, the gelatin and the like is 0-30% (W/V).

In step 3 of the invention, the specific irradiation wavelength range of the light adopted by the photoreaction crosslinking curing is 200-800 nm. When the photoinitiator is an ultraviolet initiator 2959, the specific irradiation wavelength range is 365-405 nm, and the most preferable range is 365 nm; when the photoinitiator is a blue light initiator LAP, the specific irradiation wavelength range is 400-450 nm, and the most preferable is 405 nm. Irradiating the hydrogel by using a light source with a specific wavelength, wherein the preferable irradiation time is 5-300 seconds.

Preferably, in the step 2, cells are added into the culture medium to prepare a cell-loaded hydrogel precursor, and the cell-loaded hydrogel precursor is directly irradiated by a light source with a specific wavelength, crosslinked and cured to prepare a cell-loaded tissue engineering scaffold; or taking the cell-loaded hydrogel precursor as 3D bioprinting ink, irradiating by a light source with a specific wavelength, crosslinking and curing to prepare a cell-loaded 3D bioprinting finished product.

Compared with the prior art, the invention has the following advantages:

the recombinant collagen provided by the invention has good bioactivity, and meanwhile, the recombinant human collagen has good water solubility, so that the preparation of 3D biological printing ink is facilitated. In addition, the viscosity of the recombinant collagen aqueous solution is low, so that the recombinant collagen aqueous solution can pass through a filter membrane easily, and the sterile effect is achieved. Meanwhile, the photo-crosslinking recombinant collagen hydrogel is irradiated by a light source with a specific wavelength in the crosslinking process, and other chemical crosslinking agents are not required to be additionally introduced, so that the photo-crosslinking recombinant collagen hydrogel is non-toxic and has good biocompatibility. The photo-crosslinking recombinant collagen hydrogel disclosed by the invention is good in physical and chemical properties, free of impurities and good in formability, and can be prepared into any size and shape. The photocrosslinking recombinant collagen hydrogel has wide application fields, can be applied to the fields of 3D bioprinting, cell culture, tissue engineering scaffolds and the like, and is used for skin repair, eye cornea repair, various bone tissue repairs, blood vessel construction, medical cosmetology, repair fillers, drug carriers, drug release carriers, toxicology test models, cosmetic test models, animal model substitutes and the like.

Drawings

FIG. 1: a is a photo-crosslinking collagen hydrogel real object after being irradiated and cured by blue light with the wavelength of 405 nm; and b is a photo-crosslinking hydrogel real object obtained by irradiating and curing the photo-crosslinking collagen hydrogel serving as the 3D biological printing ink by 365nm wavelength ultraviolet light after 3D biological printing.

FIG. 2: a is a 24-hour graph of mouse embryonic fibroblasts (NIH3T3) cultured on the surface of photo-crosslinked collagen hydrogel after irradiation and solidification; b is a 72-hour plot of mouse embryonic fibroblasts (NIH3T3) cultured on the surface of a photocrosslinked collagen hydrogel after irradiation curing. The mouse embryonic fibroblast is proved to be capable of adapting to the environment of photo-crosslinking collagen hydrogel, and has normal proliferation and good cell morphology.

FIG. 3: culturing the photo-crosslinking collagen hydrogel carrying the mouse embryo fibroblast (NIH3T3) after irradiation for 24 hours and 72 hours to obtain an acridine orange/ethidium bromide staining pattern, which proves that the mouse embryo fibroblast can adapt to the environment of the photo-crosslinking collagen hydrogel, can normally proliferate and has no cytotoxicity; a is acridine orange/ethidium bromide staining pattern after 24 hours of culture using NIH3T 3; b is the acridine orange/ethidium bromide staining pattern using NIH3T3 for 72 h of culture.

FIG. 4: the statistical graph of the relative proliferation condition of the Human Umbilical Vein Endothelial Cell (HUVEC) mouse embryo fibroblast (NIH3T3) which is cultured by the recombinant collagen hydrogel leaching liquor after photo-crosslinking and solidification and the corresponding culture medium for 72 hours shows that the photo-crosslinking recombinant collagen hydrogel is qualified and has no cytotoxicity; a is the result of cytotoxicity test using HUVEC for 72 hours; b is the result of cytotoxicity test using NIH3T3 for 72 hours.

Detailed Description

For the purpose of facilitating understanding, the present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. It should be noted that the specific embodiments are only for illustration and do not limit the scope of the invention. It will be apparent to those skilled in the art and to those who work in the relevant art, from this disclosure, that various modifications and adaptations of the invention or areas of applicability thereof may be made within the scope of the invention and these modifications and adaptations or applications in other areas are intended to be within the scope of the invention.

In the following examples, the reagents and raw materials used were conventional products, for example, the collagen was recombinant human collagen prepared according to the inventor's chinese patent ZL201110327865.5, methacrylic anhydride, AO, EB were purchased from Aladdin, mouse embryo fibroblasts, human umbilical vein endothelial cells were purchased from shanghai cell bank of chinese academy of sciences, PBS buffer, DMEM medium, F12 medium, streptomycin mixture was purchased from Hyclone, fetal bovine serum was purchased from sikawa biotechnology limited, photoinitiator 2959 was purchased from basf, blue light initiator LAP was purchased from jiangyinst biotechnology, and MTT cell proliferation and cytotoxicity detection kit was purchased from bi yuntian biology. The recombinant human collagen adopted in the embodiment is the recombinant human collagen obtained by the method disclosed in Chinese patent ZL200610098297.5, and is produced by a pichia pastoris genetic engineering bacterium (the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.5021) constructed by different repetition numbers of gene recombinant plasmids in series in the same direction through a high-density fermentation and purification method provided by Chinese patent ZL 201110327865.5.

In the following examples, the hydrogel preparation process was carried out in a clean bench to ensure aseptic conditions, and the required materials were sterilized by physical or chemical means such as autoclaving, ultraviolet irradiation or gamma irradiation.