CN112755248A

CN112755248A - Preparation method and application of 3D printing composite biological ink based on ovary or vagina acellular matrix

Info

Publication number: CN112755248A
Application number: CN202011601206.1A
Authority: CN
Inventors: 黄向华; 张敬坤; 郑嘉华; 侯晨晓
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-05-07

Abstract

The invention discloses a preparation method and application of 3D printing composite biological ink based on an ovary or vagina acellular matrix. The preparation method comprises the following steps: a. adding prepared ovarian or vaginal acellular matrix powder into pepsin solution for digestion; b. dropping alkali liquor into the obtained acellular matrix solution to ensure that the pH value is 7.0-7.3; c. preparing a mixed gel solution of gelatin and sodium alginate; d. and c, adding the mixed gel solution obtained in the step c into the acellular matrix solution treated in the step b, and uniformly mixing to obtain the composite biological ink. The composite biological ink with smooth and stable silk output, stable printed matter forming and better biocompatibility is obtained by mixing the acellular matrix solution and the mixed glue solution according to a specific proportion. Experiments prove that the tissue material printed by the composite biological ink disclosed by the invention is closer to a natural tissue matrix, and is more beneficial to cell growth and tissue repair.

Description

Preparation method and application of 3D printing composite biological ink based on ovary or vagina acellular matrix

Technical Field

The invention relates to the technical field of biological ink, in particular to a preparation method and application of 3D printing composite biological ink based on ovary or vagina acellular matrix.

Background

Ovarian cancer, premature ovarian failure and polycystic ovarian syndrome can cause infertility caused by ovum development and ovulation disorder of women, and seriously affect the reproductive health of women. Artificial ovaries are one of the promising fertility preservation technologies in the future. Artificial ovaries are intended to mimic two representative functions of the ovary: egg production and steroid hormone release. Anatomically or ideally, the artificial ovary should contain follicles isolated from cryopreserved ovarian tissue or other ovarian tissue, and also require a suitable delivery scaffold that is biocompatible, minimally inflammatory, angiogenesis-competent, and degradable after implantation so as not to interfere with follicle growth and migration. According to the source of the scaffold material, the tissue engineering scaffold material mainly comprises natural polymer material and synthetic polymer material. Natural polymer materials are high molecular weight compounds having a basic structure of linear long chains formed by connecting repeating units, and are high molecular substances present in animals, plants, and living bodies. The material has wide source, low price, good biocompatibility, biodegradability and hydrophilicity, and is nontoxic and harmless. However, some materials have poor mechanical strength and processability, are difficult to process into a stent and have the problem of high degradation speed. At present, the most used natural polymer materials include collagen, alginate, gelatin, agarose, chitosan, chitin, cellulose, hyaluronic acid, and the like. Synthetic high molecular materials such as polylactic acid, polyvinyl alcohol, and polybutanoic acid refer to polymers obtained by a certain polymerization reaction using monomers with known structures and relative molecular masses as raw materials. One of the greatest advantages of the material is that the mechanical properties can be tailored to the specific requirements of clinical application, and it can also be manufactured in large quantities and uniformly and has a long shelf life. However, the degradation products may cause some immunological rejection reactions, adversely affect the organism, and do not contain molecules necessary for cell adhesion, and bioactive factors are added to stimulate this function. The choice of which matrix to use for ex vivo transplantation of follicles and cells is one of the most challenging and critical factors in the development of bioengineered artificial ovaries.

At present, both natural and synthetic polymeric materials have been used to create artificial ovarian prototypes. However, the research shows that the natural polymer material is the first substrate for forming the artificial ovary. The decellularized extracellular matrix has been primarily studied for the construction of artificial ovaries due to its many advantages, such as retention of physiological components of the natural extracellular matrix, low immunogenicity, etc. In 2015, Laronda et al succeeded for the first time in constructing scaffolds from decellularized bovine and human ovarian tissue that support follicular growth and restoration of ovarian function in ovariectomized mice. In 2017, another study decellularized the ovaries of Bama miniature pigs, followed by assessment of ultrastructure and biocompatibility of the extracellular matrix of decellularized ovaries in combination with in vitro and in vivo studies. In 2019, Pors et al optimized the decellularization scheme of human ovarian tissue, and tested the biological function of decellularized scaffolds in vitro and in vivo by reseeding mouse and human pre-antral follicles and other ovarian cells, and studies showed that decellularized human ovarian tissue can support human follicle survival and mouse follicle growth. Despite these promising results achieved by decellularized ovarian tissue matrices, finding an exact match in the natural pores of the matrix that fits different sized follicles is very challenging, severely impacting the number of follicles planted and the survival rate. An alternative is to convert the matrix into a temperature sensitive hydrogel. This method allows for perfect encapsulation of isolated follicles and other ovarian cells while retaining the decellularized ovarian tissue component. Chiti et al have characterized and converted bovine decellularized ovarian extracellular matrix to a hydrogel, demonstrating that isolated pre-antral follicles in mice can successfully survive in this matrix.

Vaginal tissue loss can be caused by various congenital and acquired factors, such as congenital vaginal-free syndrome (MRKH syndrome), amphoteric malformation, trauma, tumor operation, and the like. The patient suffers physical pain and great psychomental stress. The traditional vagina reconstruction method using the non-vagina tissue to reconstruct has certain functional limitations, is greatly different from a normal vagina in morphology and histology, and can cause contracture, necrosis, prolapse, intestinal obstruction, catastrophe and the like of a new vagina to need secondary operations. The subject group independently develops a porcine vagina acellular matrix material, obtains a national invention patent, and the tissue engineering vagina constructed by applying the composite mesenchymal stem cells is superior to the tissue engineering vagina constructed by the traditional matrix material under the small intestine mucosa, and is similar to a normal vagina in morphology and functional science. The traditional tissue engineering technology achieves certain results in vaginal reconstruction, but has the defects of low survival rate of seed cells, rough construction, lack of individuation and the like. The 3D bioprinting can accurately and quickly realize the integrated construction of the complicated macroscopic appearance and the internal fine structure of the stent, can realize the personalized production aiming at specific patients and specific tissues and organs, and is expected to realize the perfect form repair and the real functional reconstruction of the vagina.

The hydrogel made of the pure acellular matrix material has poor mechanical properties, has the defects of too fast dripping and no forming of printed matters in 3D biological printing, and is not suitable for being directly used for 3D printing. Therefore, the research on the high-activity biological ink which is smooth and stable in filament discharging from the nozzle, stable in printed matter forming and good in biocompatibility has important significance for solving the technical problems in construction of tissue engineering vagina and ovary.

Disclosure of Invention

The invention aims to provide a preparation method and application of 3D printing composite biological ink based on ovary or vagina acellular matrix, and aims to solve the problems of low seed cell survival rate, rough construction, lack of individuation and the like of the conventional tissue engineering vagina and ovary.

The technical scheme adopted by the invention is as follows: a preparation method of 3D printing composite bio-ink based on ovarian or vaginal acellular matrix comprises the following steps:

a. adding pre-prepared ovarian or vaginal acellular matrix powder into a pepsin solution for digestion to obtain an acellular matrix solution with the mass fraction of 3.0-3.5%; wherein the concentration of the pepsin solution is 18-22 mg/mL;

b. dropwise adding alkali liquor into the obtained acellular matrix solution to make the pH value between 7.0 and 7.3 so as to obtain a flowing acellular matrix solution;

c. preparing a mixed gelatin solution of gelatin and sodium alginate, wherein the mass fraction of the gelatin is 13-17%, and the mass fraction of the sodium alginate is 2-4%;

d. and c, adding the mixed gelatin solution of gelatin and sodium alginate obtained in the step c into the acellular matrix solution treated in the step b, and uniformly mixing to obtain the composite biological ink, wherein the volume ratio of the mixed gelatin solution to the acellular matrix solution is 1-3: 3.

In step a, the ovarian acellular matrix powder is prepared by the following method:

(1) mechanical treatment: taking fresh pig/cattle/human ovarian tissues, removing tissues around the ovaries, shearing the ovarian tissues into tissue blocks, and washing with cold normal saline;

(2) and (3) treating a hypotonic solution: placing the ovarian tissue in deionized water containing phenylmethylsulfonyl fluoride at the temperature of 3-5 ℃, carrying out oscillation treatment for 36-60 h, and changing the liquid once every 12 h;

(3) ionic detergent treatment: placing the ovarian tissue treated in the step (2) in a Tris-HCl solution containing phenylmethylsulfonyl fluoride and sodium dodecyl sulfate, wherein the pH value of the Tris-HCl solution is 7.8-8.2, the temperature of the Tris-HCl solution is 3-5 ℃, performing oscillation treatment for 10-14 h, and then sequentially washing with a PBS solution and deionized water;

(4) non-ionic detergent treatment: placing the ovarian tissue treated in the step (3) in a Tris-HCl solution containing phenylmethylsulfonyl fluoride and Triton X-100 with the pH of 7.8-8.2 and the temperature of 3-5 ℃, shaking for 6-8 days, and changing the solution once every 12 hours; then sequentially washing the substrate by using a PBS solution and deionized water;

(5) enzyme treatment: placing the ovary tissue treated in the step (4) in a Tris-HCl solution containing deoxyribonuclease and ribonuclease, wherein the pH value of the Tris-HCl solution is 7.3-7.6, the temperature of the Tris-HCl solution is 35-38 ℃, performing oscillation treatment for 12-13 h, and then sequentially cleaning the ovary tissue with a PBS solution and deionized water;

(6) punching: placing the ovarian tissue treated in the step (5) in a peroxyacetic acid solution with the temperature of 3-5 ℃ and the mass fraction of 4-6%, oscillating for 1.5-2.5 h, and then sequentially washing with a PBS (phosphate buffer solution) and deionized water;

(7) and (4) sterilizing the ovarian tissues treated in the step (6), freeze-drying, and grinding the freeze-dried blocks to obtain the ovarian acellular matrix powder.

In the step a, the pepsin solution is prepared by 0.01mol/L hydrochloric acid solution, and the digestion treatment comprises the following steps: shaking at 37 ℃ for 24-26 h.

In the step b, the alkali liquor is 0.1mM NaOH solution.

In the step c, the preparation process of the mixed glue solution comprises the following steps: and putting the weighed gelatin and sodium alginate into a sterile deionized water solution, and melting and mixing for 20-40 min at 50-55 ℃.

In the step d, the volume ratio of the mixed gelatin solution of gelatin and sodium alginate to the acellular matrix solution is 2: 3.

The composite biological ink prepared by the method is applied to 3D bioprinting of an artificial ovary or an artificial vagina.

The composite biological ink is pasteurized and then wraps seed cells, the seed cells are placed in an aseptic printing cylinder, the temperature is controlled at 3-5 ℃ for 10-20 min, the seed cells are then placed in a 3D printing nozzle, the temperature of the nozzle is set to be 18-22 ℃, the temperature of a platform is controlled at 3-5 ℃ for 5-20min, a model is led in, parameters are set, 3D printing is started, and after printing is finished, 5% CaCl2 is used for crosslinking for 5-10 min, so that printed matter is formed stably.

According to the invention, sodium alginate and gelatin are mixed according to a certain proportion, and the temperature control crosslinking characteristic of the gelatin and the crosslinking characteristic of alginate and calcium ions are utilized to modify the mechanical properties of the acellular matrix hydrogel. The acellular matrix solution and the mixed gel solution are mixed according to a specific proportion to obtain the composite biological ink which is smooth and stable in silk output, stable in printed matter forming and good in biocompatibility. Experiments prove that the tissue material printed by the composite biological ink disclosed by the invention is closer to a natural tissue matrix, and is more beneficial to cell growth and tissue repair.

Drawings

FIG. 1 is a graphical representation of the overall and local morphology of an ovarian decellularized matrix. The left panel is fresh ovarian tissue (native ovary); the middle panel is ovary acellular matrix (Unfreeze-dried dcems) before freezing; the right panel shows the Freeze-dried ovarian acellular matrix (Freeze-dried dECMs) which is tough and tough, and the pores in which the growing follicles were located prior to acellular growth (as indicated by the arrows).

FIG. 2 is a graph of histological characteristics of ovarian acellular matrix (HE staining and DAPI staining).

FIG. 3 is a diagram of analysis of DNA content of ovarian acellular matrix.

FIG. 4 is an SDS-PAGE image of ovarian acellular matrix proteins or peptides.

FIG. 5 is a photograph of the ovarian acellular matrix collagen and proteoglycan assays.

FIG. 6 is a scanning electron micrograph of decellularized ovarian stroma.

FIG. 7 is a diagram of the raw gelatin preparation process.

FIG. 8 is a scanning electron micrograph of virgin rubber.

Fig. 9 is a native gel rheology detection map.

FIG. 10 is a crude gel circular dichroism chart. Wherein, A is normal temperature circular dichroism spectrum, B is variable temperature circular dichroism spectrum, and C is secondary fitting structure.

Fig. 11 is a photograph of a virgin rubber test print.

Fig. 12 is a photograph of a test print of composite bio-ink.

FIG. 13 is a scanning electron micrograph of composite bio-inks of different volume ratios.

Fig. 14 is a rheological map of composite bio-inks of different volume ratios.

FIG. 15 is a diagram of the results of normal temperature circular dichroism spectroscopy tests on composite bio-inks of different volume ratios.

FIG. 16 is a graph of temperature-variable circular dichroism spectroscopy results of composite bio-inks of different volume ratios.

FIG. 17 is a graph of the results of cell death experiments with different volume ratios of composite bio-ink.

FIG. 18 is a graph showing the results of MTT method for detecting cell proliferation activity of composite bio-ink with different volume ratios.

FIG. 19 is a graph showing the results of biocompatibility measurements after the composite bio-ink xenotransplantation.

Fig. 20 shows growth of rat bone marrow mesenchymal stem cells encapsulated with vaginal acellular matrix-based bio-ink.

Detailed Description

The present invention is described in detail below with reference to specific examples, wherein reagents and procedures not mentioned in the examples are all performed according to the routine procedures in the art. The vaginal acellular matrix material of the present invention can be prepared by a method disclosed in patent document (application No. 201510584734.3).

Example 1 preparation of ovarian acellular matrix

Fresh ovarian tissues (as shown in figure 1) are taken within 10min after the death of the sow, and after the sow is fully washed by cold normal saline, the following treatment is carried out:

(1) mechanical treatment: periovarian tissue was removed and the ovarian tissue was cut into 5mm by 5mm tissue pieces and washed again with cold saline.

(2) And (3) treating a hypotonic solution: ovarian tissue was placed in deionized water containing 0.1mM PMSF at 4 ℃ for 130 rotations, 48 h, with changes every 12 h.

(3) Ionic detergent treatment: the mixture was placed in 10mM Tris-HCl solution containing 0.1mM PMSF and 0.1% SDS at 4 ℃ at pH 8.0, 130 rpm, 12 h. Then, PBS was changed every 15min at 4 ℃ for 30min at 130 rpm. Deionized water was changed every 15min at 4 deg.C for 30min at 130 rpm.

(4) Non-ionic detergent treatment: the mixture was placed in 10mM Tris-HCl solution containing 0.1mM PMSF and 1% Triton X-100 at 4 ℃ at pH 8.0 for 130 cycles for 7 days with changes every 12 h. Then, PBS was changed every 15min at 4 ℃ for 30min at 130 rpm. Deionized water was changed every 15min at 4 deg.C for 30min at 130 rpm.

(5) Enzyme treatment: nuclease solution (containing 50U/ml DNase I and 1U/ml RNase A), pH 7.5, 37 ℃, 80 rpm, 12 h. Then, PBS was changed every 15min at 4 ℃ for 30min at 130 rpm. Deionized water was changed every 15min at 4 deg.C for 30min at 130 rpm.

(6) Punching: 5% peroxyacetic acid, 4 ℃, 80 revolutions, 2 hours. PBS 4 ℃, 130 rotation, 30min, liquid change every 15 min. Deionized water was changed every 15min at 4 deg.C for 30min at 130 rpm.

(7) And (3) disinfection: 0.1% peracetic acid and 20% ethanol solution at 25 deg.C for 2h at 130 rpm. Sterile PBS was changed at 4 ℃ for 30min every 15min at 130 rpm. Sterile deionized water is changed every 15min at 4 ℃, 130 r.p.30 min.

(8) Freeze-drying for later use: ovarian tissue (excess water on tissue surface was discarded) was spread on a UV-irradiated plastic plate and freeze-dried in a-80 ℃ lyophilizer and stored at-20 ℃ until use (photographs before and after lyophilization are shown in FIG. 1).

And (3) performing characterization detection on the prepared acellular matrix material:

(1) HE staining: and (3) taking ovary tissues with the size of about 1cm multiplied by 0.3cm after freeze-drying, fixing by 4% paraformaldehyde, directionally embedding paraffin, and continuously slicing by 5 mu m. Slicing paraffin, conventional dewaxing and hydrating, separating with 0.5% hydrochloric acid and ethanol for 10s, performing running water bluing for 10s, washing with water, separating eosin solution for 10s, dehydrating, sealing with neutral gum, and observing with light microscope to obtain cell component residue (shown in FIG. 2).

(2) DAPI staining: and (3) taking paraffin sections, performing conventional dewaxing and hydration on the DAPI 10 mu L, directly sealing the sections, and observing the sections by using a light microscope, wherein no cell component is left (as shown in figure 2).

(3) DNA content detection

Fresh ovarian tissues and decellularized and freeze-dried ovarian tissues are taken and weighed, an animal tissue genome DNA extraction kit (Tiangen) is used for extracting DNA, and the DNA content is measured by a spectrophotometer NanoDrop 2000 according to the operation of the specification. The DNA content of acellular matrix is 48.4 +/-1.88 ng/m, and compared with the content of fresh ovarian tissue (861.83 +/-18.06 ng/mg), the DNA content is statistically different (P < 0.0001) (as shown in the right graph of figure 3).

(4) DNA size detection

Agarose gel electrophoresis was used to detect the size of DNA fragments contained in the ovarian tissue after cell removal treatment, and fresh ovarian tissue was used as a control. The results show that the gel electrophoresis image of the acellular matrix material running out of 1.5% agarose shows only the migration of the DNA band in fresh pig ovarian tissue, and no significant DNA band after the acellular treatment (the results are shown in the left panel of FIG. 3).

(5) Detection of protein or polypeptide: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed until the protein bands were clear, and photographed. The results indicated the presence of various proteins in the ovarian acellular matrix (as shown in figure 4).

(6) Detection of collagen and proteoglycan: masson staining and Toluidine Blue (TB) staining. Ovarian acellular matrix collagen fibers are similar to the pre-acellular ovarian tissue and are continuously present throughout the ovary (Masson staining); staining with Toluidine Blue (Toluidine Blue, TB) also revealed that proteoglycans remained unchanged after decellularization (as shown in FIG. 5).

(7) And observing the ultrastructure of the ovarian acellular matrix by a scanning electron microscope. After decellularization, each section had no cell component residue (fig. 6A-6E), and the three-dimensional structure and integrity were well preserved. The photomicrographs of the ovarian decellularized matrix (FIGS. 6A-6B) show that once filled with different types of cells, a complex fiber network is formed with a porous structure. Under high power microscopy (FIGS. 6C-6E), the follicle extracellular matrix structure was intact, collagen fibers were visible in the walls of the extracellular matrix pore (white arrows, FIGS. 6E-6F), and flexible fibronectin fibers were also contained in the walls (black arrows, FIGS. 6E), with little effect on the orientation and structure of these fibers.

Example 2 preparation of acellular matrix solution (raw gelatin) (the procedure is shown in FIG. 7)

(1) Grinding: adding a proper amount of liquid nitrogen into the freeze-dried pig ovary acellular matrix in a grinding machine, grinding into powder, and preserving at the temperature of-20 ℃ for later use.

(2) Digestion: 0.1g of the above powder was put into 3mL of 0.01mol/L hydrochloric acid solutions containing 15mg (5 mg/mL), 30mg (10 mg/mL), 40mg (15 mg/mL) and 60mg (20 mg/mL) of pepsin, respectively, to prepare a 3.33% acellular matrix solution, i.e., virgin rubber, and the shaking table was set at 37 ℃ for 80 revolutions for 24 hours.

(3) Neutralizing: about 0.7mL of 0.1mM NaOH solution was added dropwise to the solution so that the pH of the solution became neutral (pH 7.0-7.3) from acidic (pH 3.2-3.5) and the gel became mobile and irreversible.

The raw rubber prepared by detecting the concentrations of 4 different proteases by a bottle inversion method accords with the characteristics of hydrogel, but the hydrogel prepared by only 20mg/mL pepsin does not contain any blocky indigestion, so that the condition that the pinholes are blocked by the indigestion during 3D printing is avoided, and the raw rubber is selected as the hydrogel for subsequent 3D printing.

Identifying the primary rubber prepared by the following steps:

(1) and observing the primary colloid super-microstructure by a scanning electron microscope. The raw rubber exhibits a porous network-like structure (as shown in fig. 8).

(2) Raw rubber rheology test

Placing the prepared crude rubber on a sample table of an MAL1160979 advanced rotary rheometer, performing rheological characterization, and measuring changes of G '(storage modulus) and G' (loss modulus) along with amplitude, frequency and time. Testing accessories: PTD200, DG26.7 test system. An amplitude scan is performed. The results show that G' is much higher than G ", the material shows solid properties (as shown in FIG. 9).

(3) Circular dichroism detection of crude rubber

And (3) opening nitrogen, adjusting the flow meter to 3L/min, purging for 20min, then starting a xenon lamp, and opening Chirascan software to set experiment parameters. The crude rubber is diluted into 5mg/mL solution by ultrapure water, and the solution is tested on a machine, and the ultrapure water is used as a background solvent to subtract a baseline. And carrying out normal-temperature circular dichroism spectrum and variable-temperature circular dichroism spectrum detection. The result shows that the maximum positive absorption peak is positioned at 222.5nm, the maximum negative absorption peak is positioned at 197.5nm, and the maximum negative absorption peak is in an inverted S shape and is in a typical collagen triple-helix conformation. It was shown from the thermal denaturation curves that unwinding of the triple helix structure occurred mainly between 45-70 ℃ with Tm =55.61 ℃ (as shown in fig. 10).

Example 3 virgin rubber test printing

And starting the Jieno flying second-generation biological printing system.

The neutral crude rubber 1mL is loaded into a sterile cylinder (specification: 5 mL), the diameter of a needle is 0.34mm, the cylinder is embedded into a low-temperature nozzle and fixed on a biological printer, the temperature of the nozzle is set to be 20 ℃, and the temperature of a printing table is kept at 4 ℃. Setting air pressure → starting to discharge the filament. By adjusting different parameters such as filament-discharging pressure, temperature control time and the like, the primary rubber can not normally discharge filament, and is in a liquid water drop shape (as shown in figure 11).

Example 4 preparation of composite bio-ink

Respectively adding 3mL, 2mL, 1mL and 0.75mL of mixed liquid of 15% gelatin and 3% seaweed (melted and uniformly mixed at 55 ℃ for 30 min) into the 3mL neutral raw rubber to form 4 composite biological inks with volume ratios (the volume ratios of the raw rubber to the mixed gel solution are respectively 3: 3, 3: 2, 3: 1 and 4: 1), pasteurizing, and placing in a refrigerator at 4 ℃ for 3D printing. The results show that 4 inks with different volume ratios (3: 3, 3: 2, 3: 1 and 4: 1) are all the temperature (more than 37 ℃) when the gel is denatured and melted, and the temperature (4 ℃) when the gel is renaturated and solidified, thus being the thermosensitive hydrogel.

Example 5 composite bio-ink test printing

And drawing a 3D structure with a grid geometric structure by using CAD software, converting and storing the 3D structure into an STL format, and printing by using a Sinuofei second-generation biological printing system. 1mL of the above ink with each volume ratio was put into a sterile cartridge (specification: 5 mL), and a needle with a diameter of 0.34mm was inserted into a low-temperature head and fixed to a bioprinter, the head temperature was maintained at 20 ℃ and the print table temperature was controlled at 4 ℃. Model introduction → parameter setting (layer number, layer thickness, pinhole diameter, air pressure, filament discharge speed, etc.) → device calibration (stage height measurement, head height measurement) → start of printing. The results show that 3 inks with volume ratios (3: 3, 3: 2, 3: 1) can form uniform lines and normally produce filaments. The 4: 1 volume ratio ink jet was shorter and had a dripping phenomenon (as shown in FIG. 12). The different volume ratio printing parameter settings are as follows:

the above 3 volume ratios of inks were tested:

(1) and (3) carrying out vacuum freeze drying on the prepared ink with the volume ratio for 24 hours in a freeze dryer with the temperature of-80 ℃, and observing by using a scanning electron microscope. The results show that the porosity of the 3: 1 volume ratio bio-ink is not uniformly dense, the porosity of the 3: 2 volume ratio bio-ink is relatively uniformly dense, and the porosity of the 3: 3 volume ratio bio-ink is more uniformly dense (as shown in fig. 13).

(2) The results of the rheological tests showed that the mechanical strength of the 3: 3 bio-ink was the best, the strength of the 3: 2 bio-ink was the second best, and the strength of the 3: 1 bio-ink was the weakest (as shown in FIG. 14).

(3) Circular dichroism chromatogram detection shows that 3 kinds of ink have typical collagen triple helix conformation (inverted S shape), the unwinding of the triple helix structure of the 3: 3 and 3: 2 biological ink mainly occurs between 45 ℃ and 70 ℃, and the Tm is 59.2 and 59.0 ℃ respectively. Unwinding of the 3: 1 bio-ink triple helix structure occurs mainly between 40-80 ℃ with Tm =49.2 ℃ (as shown in fig. 15 and 16). The secondary fitting structure is shown in the following table:

。

(4) cell death experiment: 3 kinds of biological ink are respectively wrapped on Kunming mouse ovary granular cells for 3D printing (each milliliter of ink is wrapped by 1x 10)⁶Cells), 3 samples were set for each ink, LIVE/DEAD cell viability/toxicity assays were performed in D1 and D14, respectively, confocal slides. Observing that the ovary cells in 3D printing ovaries with the biological ink with the volume ratios are uniformly distributed, and the ovary cells are regular in shape, spherical, free of swelling and shrinkage; live cells fluoresce green and dead cells fluoresce red. The number of cells in the 3: 1 volume ratio bio-ink at 1d and 14d was greater than that in the other two volume ratio bio-inks, and the 3: 3 volume ratio bio-ink at 1d was similar to that in the 3: 2 volume ratio bio-ink, but the number of cells at 14d was the least (as shown in fig. 17).

(5) MTT method for detecting cell proliferation activity: 3 kinds of biological ink are respectively wrapped on Kunming mouse ovary granular cells for 3D printing (each milliliter of ink is wrapped by 2x 10)⁶Individual cell), placing the printed ovarian scaffold in a 96-well plate, culturing the ovarian scaffold in 150 muL culture medium per well, setting 5 multiple wells with the culture medium not added in the ovarian scaffold as a blank control, and changing the culture medium every 2d for 1 time, wherein the culture medium is obtained by performing the stepsAdding 20 muL MTT every 24h, and placing at 37 ℃ and 5% CO₂Incubating the incubator for 4h, carefully absorbing culture solution in the holes, adding 150 mu L dimethyl sulfoxide, oscillating the culture solution by using an enzyme linked immunosorbent assay detector for 10min to dissolve purple crystal A, and measuring absorbance values of the holes at 490 nm. It can be seen that the cell proliferation effect of the 3D printing ovary of different volume ratios of biological ink is more than 3: 1, more than 3: 2, more than 3: 3 (as shown in FIG. 18).

In summary, the greater the proportion of virgin gelatin, the better the cell viability, while the greater the proportion of gelatin and alginate, the greater the mechanical strength of the bio-ink, and the 3: 2 bio-ink is certainly the best choice.

Example 6 biocompatibility testing

The experimental mouse is anesthetized by 0.04mg/kg of 0.5% sodium pentobarbital, skin preparation is carried out on the back after corneal reflex and righting reflex disappear, 75% alcohol is disinfected, and biological ink with the volume ratio of 3: 2 is injected into the two sides of the back of the mouse subcutaneously.

Mice were euthanized at

weeks

1, 2, 4, and 9, respectively, and the samples were taken, 6 tissue blocks were collected at each time period, fixed by immersing in 4% paraformaldehyde, embedded in wax blocks for tissue section preparation, and subjected to HE staining, CD45 antibody immunohistochemical staining, and light microscopic observation.

All rats survived within 9 weeks after implantation with no complications. After one week of implantation, angiogenesis reaction can be seen on the surface of the composite biological ink. HE and immunohistochemical results showed a gradual increase in inflammatory cells at 1-2 weeks post-implantation and a gradual decrease in inflammatory cells at 2-9 weeks. The volume of the bio-ink gradually decreases with time. These data indicate that the bio-ink is good in biocompatibility in xenogenic subcutaneous implants (as shown in figure 19).

Example 7 detection of vaginal acellular matrix composite bio-ink

The composite biological ink based on the porcine vaginal acellular matrix is prepared according to the same method, and similar detection is carried out, the composite biological ink has similar properties with the composite biological ink based on the ovarian acellular matrix, and the composite biological ink wraps mesenchymal stem cells and is detected, and the result shows that the cell growth state is good (as shown in figure 20).

Claims

1. A preparation method of 3D printing composite biological ink based on ovary or vagina acellular matrix is characterized by comprising the following steps:

2. The method according to claim 1, wherein in step a, the ovarian acellular matrix powder is prepared by the following method:

3. The preparation method according to claim 1, wherein in the step a, the pepsin solution is prepared by 0.01mol/L hydrochloric acid solution, and the digestion treatment comprises the following steps: shaking at 37 ℃ for 24-26 h.

4. The method according to claim 1, wherein in step b, the alkali solution is 0.1mM NaOH solution.

5. The method according to claim 1, wherein the mixed gum solution is prepared by the following steps: and putting the weighed gelatin and sodium alginate into a sterile deionized water solution, and melting and mixing for 20-40 min at 50-55 ℃.

6. The method according to claim 1, wherein the volume ratio of the mixed gelatin solution of gelatin and sodium alginate to the acellular matrix solution in step d is 2: 3.

7. The application of the composite biological ink prepared according to any one of claims 1 to 6 in 3D bioprinting of an artificial ovary or an artificial vagina.

8. The use of claim 7, wherein the composite bio-ink is pasteurized and coated on seed cells, then placed in an aseptic printing cylinder, controlled at 3-5 ℃ for 10-20 min, and then placed in a 3D printing nozzle, set at 18-22 ℃ and 3-5 ℃ on a platform for 5-20min, model introduction, and parameter setting, and 3D printing is started, and after printing is completed, 5% CaCl is used₂And crosslinking for 5-10 min to make the printed matter stably formed.