CN117582544A - Composite hydrogel for 3D (three-dimensional) biological printing as well as preparation method and application thereof - Google Patents
Composite hydrogel for 3D (three-dimensional) biological printing as well as preparation method and application thereof Download PDFInfo
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- CN117582544A CN117582544A CN202311292599.6A CN202311292599A CN117582544A CN 117582544 A CN117582544 A CN 117582544A CN 202311292599 A CN202311292599 A CN 202311292599A CN 117582544 A CN117582544 A CN 117582544A
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Abstract
The invention provides a composite hydrogel for 3D biological printing, and a preparation method and application thereof. Composite hydrogels include DAM hydrogels and photocurable hydrogels, including GelMA and HAMA. The preparation method comprises the following steps: obtaining a photo-curing hydrogel raw material and adipose tissue; performing decellularization treatment on adipose tissues to obtain DAM; dissolving the DAM to obtain DAM pregel solution; and dissolving the photo-cured hydrogel raw material, and mixing with the DAM pregelatinized solution to obtain the composite pregelatinized solution for 3D biological printing. According to the invention, the photo-curing hydrogel is added into the DAM to prepare the composite hydrogel, so that the printing adaptability, the printing resolution and the shape fidelity of the DAM are improved, the crosslinking time is shortened, and the printing efficiency and the mechanical strength of the 3D printing support are improved. The biological material can be effectively applied to skin wound surface treatment.
Description
Technical Field
The invention relates to the technical field of regenerative medicine, in particular to a composite hydrogel for 3D biological printing, and a preparation method and application thereof.
Background
Current methods of treating skin defects clinically include mainly autologous skin grafts and skin flap grafts, however, the limited number of donors, secondary damage, and the risk of infection limit the use of the above methods. Wound healing is impaired and scar healing remains a problem to be solved.
Tissue-engineered skin (TES) is a novel method for treating skin defects, and can simulate the physiological structure and biological characteristics of skin tissues, promote wound healing and skin regeneration, and reduce scar formation. TES is currently used mostly as dermal scaffold to induce autologous cells to grow in and produce extracellular matrix (Extracellular matrix, ECM), but due to slow vascularization and delayed epithelialization, secondary skin grafting is often required, prolonging the hospitalization time of patients and reducing the quality of life. Due to the complexity of skin structure and composition, there is still a lack of biological materials for 3D printing that promote wound healing well.
In the traditional tissue engineering, cells are planted on a stent material, and then transplanted to corresponding injury parts after in-vitro culture. Not only does this consume a significant amount of time, but it is also difficult to accurately control the distribution and density of cells within the material.
The 3D biological printing technology has the characteristics of high precision, strong controllability and high efficiency, and can print a three-dimensional biological bracket with accurate size and shape, high resolution and uniform cell distribution by depositing biological ink (composed of cells and biological materials) under the control of a computer, so that the natural tissue microenvironment can be better simulated.
Disclosure of Invention
The invention provides a composite hydrogel for 3D biological printing and a preparation method and application thereof, which are used for solving the problem of wound healing damage in the prior art and effectively promoting wound healing.
A composite hydrogel for 3D bioprinting, comprising: DAM hydrogels and photocurable hydrogels, including GelMA and HAMA.
Specifically, the DAM has a mass-to-volume fraction of 1.125% (w/v), gelMA has a mass-to-volume fraction of 7.5% (w/v), and HAMA has a mass-to-volume fraction of 1% (w/v).
A method of preparing a composite pregel solution for 3D bioprinting, comprising:
obtaining a photo-curing hydrogel raw material and adipose tissue;
performing decellularization treatment on adipose tissues to obtain DAM;
dissolving DAM to obtain DAM pregel solution;
and dissolving the photo-curing hydrogel raw material, and mixing with the DAM pregel solution to obtain the composite pregel solution.
Specifically, adipose tissue decellularization processing to yield a DAM includes:
pretreating adipose tissues to obtain an initial sample;
repeatedly freezing and thawing the initial sample to obtain a first sample;
digesting the first sample by trypsin to obtain a second sample;
carrying out primary extraction on the second sample by using isopropanol to obtain a third sample;
digesting the third sample with trypsin again to obtain a fourth sample;
respectively digesting the fourth sample by using nuclease and lipase to obtain a fifth sample;
and carrying out secondary extraction on the fifth sample to obtain DAM.
Specifically, dissolving the DAM to obtain a DAM pregelatinized solution comprises:
lyophilizing DAM, grinding, and adding digestive enzyme to obtain digestive juice;
and regulating the pH value and the ion concentration of the digestion solution to obtain the DAM pregelatinized solution.
A3D printing hydrogel biological scaffold prepared from the composite pregelatinized solution prepared by the preparation method.
A method for preparing a 3D printed hydrogel bioscaffold, comprising:
obtaining and counting adipose-derived mesenchymal stem cells;
mixing the photoinitiator, the adipose-derived mesenchymal stem cells and the 3D biological printing composite pre-gel solution, and preparing a required bracket shape by a 3D printer to obtain the 3D printing hydrogel biological bracket.
Specifically, each time a layer is printed, the printed product is photocrosslinked.
The invention provides a composite hydrogel for 3D biological printing, a preparation method and application thereof, and biological materials used as a scaffold for cell adhesion and proliferation are required to have good biocompatibility, printing adaptability and mechanical strength. The invention uses DAM as the important component of composite hydrogel, which is obtained by removing cells and immunogenic components in tissues or organs through physical, chemical and/or biological methods, preserving ECM components (mainly comprising collagen, elastin, fibronectin, laminin, matrix cell proteins and the like) and obtaining DAM with good biocompatibility, three-dimensional structure and physical, chemical and biological characteristics very similar to natural adipose tissue, and can better simulate the cell growth microenvironment and regulate and control the adhesion, migration, proliferation and differentiation of cells. The photo-curing hydrogel is GelMA and HAMA, which are respectively products of gelatin and hyaluronic acid after modification, have good biocompatibility, bioactivity and photo-curing crosslinking characteristics, and are suitable biological materials for simulating ECM. Because of low viscosity and low gel speed of DAM, the printing adaptability of DAM is poor, and DAM is difficult to be directly used for 3D printing. Therefore, the invention adds the photo-curing hydrogel into the DAM to prepare the composite hydrogel so as to improve the printing adaptability, the printing resolution and the shape fidelity of the DAM and improve the printing efficiency and the mechanical strength of the 3D printing support.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a process of obtaining DAM powder after decellularization, lyophilization and grinding of adipose tissue in the preparation method of 3D bioprinting hydrogel of the present invention;
FIG. 2 is a HE staining of adipose tissue and DAM in the preparation method of the 3D bioprinting hydrogel of the present invention;
FIG. 3 is a 4', 6-diamidino-2-phenylindole (DAPI) staining of DAM in the preparation method of the 3D bioprinting hydrogel of the present invention;
FIG. 4 is an oil red O staining of DAM in the preparation method of the biological 3D printing hydrogel of the present invention;
fig. 5 shows quantitative detection of deoxyribonucleic acid (DeoxyriboNucleic Acid, DNA) in adipose tissue and DAM in the preparation method of 3D bioprinting hydrogel of the present invention, and the difference therebetween is statistically significant (×p < 0.0001);
FIG. 6 shows a sol-gel phase transition of a pre-gel solution of DAM in a method for preparing a 3D bioprinting hydrogel according to the present invention;
FIG. 7 shows the change of modulus with temperature obtained by performing temperature scanning experiments on DAM-GelMA-HAMA composite hydrogel by using a rheometer in the preparation method of the 3D bioprinting hydrogel of the present invention;
FIG. 8 is a scanning electron microscope (Scanning electronmicroscope, SEM) image of a DAM-GelMA-HAMA composite hydrogel crosslinked in the method for preparing a 3D bioprinting hydrogel of the present invention;
FIG. 9 shows the proliferation of Adipose mesenchymal stem cells (ADSCs) in DAM hydrogel and DAM-GelMA-HAMA composite hydrogel extract, detected by a cell counting kit (Cell Counting Kit-8, CCK-8) method in the preparation method of the 3D bioprinting hydrogel of the present invention;
FIG. 10 is an in vivo biocompatibility test of DAM hydrogels in the method of preparing 3D bioprinted hydrogels of the present invention;
FIG. 11 is a schematic representation of the general appearance of a 3D printed DAM-GelMA-HAMA cell-loaded scaffold in a method of preparing a bioscaffold according to the invention.
Fig. 12 (a) shows the staining of live/dead cells of the das-GelMA-HAMA composite hydrogel-supported ADSCs scaffold 1, 3, 7 days after 3D printing in the preparation method of the biological scaffold of the present invention, the live cells fluoresce green, the dead cells fluoresce red, and the scale bar=500 μm; (B) For the quantitative calculation of the cell viability of ADSCs in scaffolds in the preparation method of the biological scaffold of the present invention, P <0.01 and P <0.0001.
FIG. 13 shows the comparison of the effects of unprinted DAM hydrogel, unprinted DAM-GelMA-HAMA composite hydrogel, 3D bioprinted ADSCs-loaded biological scaffold, conventional dressing change on wound healing and wound healing rate in the preparation method of the biological scaffold of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments that can be obtained by a person of ordinary skill in the art without making any inventive effort are within the scope of the present invention.
Specific details of the invention are described below in connection with fig. 1-13.
A composite hydrogel comprising: adipose tissue acellular matrix (Decellularized adipose tissue extracellular matrix, DAM) hydrogels and photocurable hydrogels. A photocurable hydrogel comprising: methacryloylated gelatin (Methacrylated gelatin, gelMA) and methacryloylated hyaluronic acid (Methacrylatedhyaluronic acid, HAMA).
In order to enable the composite hydrogel to have good printing adaptability, printing resolution, shape fidelity and mechanical strength, the concentration of the composite hydrogel is adjusted, and the optimal concentration of the composite hydrogel in the application is determined as follows: 1.125% (w/v) DAM,7.5% (w/v) GelMA,1% (w/v) HAMA.
The specific preparation process of the composite hydrogel for 3D bioprinting comprises the following steps:
step S10, preparing a photocurable hydrogel material and adipose tissue to be treated.
And step S20, dissolving the photo-cured hydrogel raw material, and filtering and sterilizing to obtain a GelMA and HAMA pregelatinized solution.
Step S30, performing decellularization treatment on the adipose tissues to obtain DAM. As shown in fig. 1, the method comprises the steps of:
step S31, pretreatment is carried out on adipose tissues to obtain an initial sample. Specifically, the swelling liquid and blood components in the sucked adipose tissues are removed by flushing with physiological saline, the adipose tissues are subpackaged into a centrifuge tube in an ultra-clean bench, and the content of the adipose tissues accounts for about half of the capacity of the centrifuge tube.
And S32, repeatedly freezing and thawing the initial sample to break the fat cells, and removing cell contents and liquid through centrifugal delamination to obtain a first sample.
Specifically, the freezing buffer solution with the same volume as the adipose tissue is added into each centrifuge tube, and the centrifuge tubes are respectively placed in a low-temperature and high-temperature environment and repeatedly used for breaking cell membranes, for example, the centrifuge tubes are placed into a deep freezing ice box at the temperature of minus 80 ℃ for 2-3 hours, then placed into a constant-temperature shaking table at the temperature of 37 ℃ for 100 revolutions per minute (Revolutions per minute, rpm) for rewarming, and the freezing buffer solution is replaced and then subjected to freezing and thawing operations again for 3 times.
The cryopreservation buffers used in this example were hypotonic solutions that facilitate the bursting of cells by water, including Tris (Tris hydroxymethyl aminomethane, tris) and ethylenediamine tetraacetic acid (Ethylene diamine tetraacetic acid, EDTA). Taking 1L volume of cryopreservation buffer as an example, 1.21g Tris and 1.46g EDTA powder are dissolved in 800mL pure water, the solution is fully stirred to be dissolved, the volume is fixed to 1L, and the pH is adjusted to 8.0,0.22 mu m, and the filtration is carried out for sterilization.
After repeated freeze thawing for 3 times, most of fat cells in the adipose tissue are broken, the upper layer of grease and the lower layer of liquid are discarded after centrifugation for 3min at 7000rpm and room temperature, and the middle adipose tissue is reserved as a first sample.
In step S33, the first sample is digested with trypsin-EDTA to obtain a second sample.
To separate cells from ECM, the first sample needs to be treated with trypsin-EDTA to obtain a second sample. In this example, an equal volume of 0.25% trypsin-EDTA digest of the original adipose tissue was added to the centrifuge tube, digested on a constant temperature shaker at 37℃for 16h at 7000rpm, centrifuged at room temperature for 3min, and the lower precipitated tissue was collected. The digested tissue was transferred to a new centrifuge tube, added with an equal volume of phosphate buffer (Phosphate Balanced Solution, PBS), washed 3 times at 100rpm on a constant temperature shaker at 37℃for 30 min/time.
And step S34, carrying out primary extraction on the second sample by using isopropanol to obtain a third sample.
Specifically, the fat tissue contains a large amount of lipid components, and the lipid components in the fat tissue are removed by an organic solvent extraction method in this example. And adding isopropanol with the concentration of 99% which is equal to that of the initial adipose tissue into the second sample by taking the isopropanol as an organic solvent for extraction, carrying out extraction operation on a constant temperature shaking table at the speed of 100rpm and the temperature of 37 ℃, and replacing the isopropanol every 12 hours for 4 times and 48 hours in total to obtain a third sample.
And step S35, digesting the third sample again by trypsin-EDTA to obtain a fourth sample.
Specifically, after removal of the lipid component, digestion with 0.25% trypsin-EDTA was again performed to isolate cells that were not previously isolated from the ECM. The third sample was transferred to a new centrifuge tube, added with an equal volume of PBS solution, washed 3 times at 100rpm on a constant temperature shaking table at 37℃for 30 min/time to wash the organic solvent. Again, 0.25% trypsin-EDTA digest was added and digested on a shaking table at 37℃for 6h at 100 rpm. The digested tissue was transferred to a new centrifuge tube, added with PBS solution, and washed 3 times at 37℃for 30 min/time on a constant temperature shaker at 100 rpm.
S36, respectively digesting the fourth sample by nuclease and lipase to obtain a fifth sample.
In addition to pancreatin digestion, to remove nucleic acid components and triglycerides from adipose tissue, digestion with a digestion solution containing benzonase nuclease and type II lipase, respectively, was required overnight at 100rpm on a constant temperature shaker at 37 ℃. Wherein the working concentration of nuclease is 1000U/ml, and the working concentration of lipase is 80U/ml.
And S37, extracting the fifth sample again by using isopropanol to obtain DAM.
Specifically, the present example employs an organic solvent extraction method to further remove lipid components from adipose tissue. Adding 99% isopropanol with the same volume as the initial adipose tissue into the centrifuge tube, extracting at 100rpm on a constant temperature shaking table at 37 ℃ for 6-8h, washing with PBS solution for 3 times, adding pure water with the same volume as the initial adipose tissue, washing at 100rpm on a constant temperature shaking table at 37 ℃ for 30min, and repeating for 3 times.
As shown in FIG. 1, a large amount of fat can be seen to float on the upper layer of the solution after 3 times of freeze thawing, the fat component is removed after centrifugation, the lower layer of the solution can be seen to be a yellow-white fiber flocculent tissue after 1 st time of trypsin-EDTA digestion of cells, a small amount of incompletely decellularized yellow fat tissue is doped between the lower layer of the solution and the upper layer of the solution can be seen to be a small amount of fat. After 4 times of isopropanol treatment with organic solvent, the lipid component is removed, the color of the flocculent fiber tissue gradually lightens, the flocculent fiber tissue changes from yellow-white to white, the DAM is in a primary form, and the DAM is dried and flocculent, and the DAM is washed by PBS solution and then rehydrated to form gel. Subsequent trypsin-EDTA digestion, nuclease and lipase digestion, isopropanol extraction and washing processes, the appearance of the DAM was not significantly changed. After decellularization, a DAM in the form of a white fiber gel was obtained.
To examine the decellularization effect of adipose tissue, the DAM and adipose tissue were subjected to histopathological examination and DNA was extracted for quantitative analysis. Histopathological examination was accomplished by paraffin sections and frozen sections after tissue fixation, followed by microscopic observation. As shown in fig. 2, the fat cells in normal adipose tissue are intact in morphology, the cell body is composed of one huge lipid droplet, a thin layer of cytoplasm is visible outside the lipid droplet, and the cell nucleus is extruded to the cell edge by the lipid droplet. DAM loses normal adipocyte structure, has no nuclear component, and a large number of ECM components are seen. The results suggest that adipose tissue is treated by the above-described decellularization method, the overall structure of adipose tissue and adipocytes is destroyed, and ECM components are retained.
As shown in FIG. 3, DAPI staining of DAM did not see strong blue fluorescent spots, suggesting that the DNA components had been substantially removed after treatment of adipose tissue by the decellularization method described above. As shown in FIG. 4, the oil red O staining of DAM did not see red-stained lipid droplets, suggesting that the triglycerides have been substantially removed after the treatment of adipose tissue by the decellularization method described above. DNA was extracted from adipose tissue and DAM, respectively, and quantitatively analyzed. As shown in FIG. 5, the DNA content in the dry-weight adipose tissue is 643.6 + -69.7 ng/mg, the DNA content in the dry-weight DAM is 24.5+ -7.1 ng/mg, and less than 50ng/mg, which meet the currently accepted decellularization standard, and the difference between the two is statistically significant (P < 0.0001), which indicates that most of DNA components are removed after the adipose tissue is treated by the above-mentioned decellularization method, and the decellularization standard is met.
And S40, dissolving the DAM to obtain a DAM pregel solution.
Specifically, in one implementation, the DAM is digested with pepsin to obtain a DAM pregelatinized solution.
The preparation of the DAM pregel solution in this example comprises the following steps:
and (3) freeze-drying and grinding the DAM, and adding digestive enzymes to obtain digestive juice.
In particular, pepsin digests proteins by hydrolyzing peptide bonds, which can hydrolyze proteins in the adipose tissue ECM into soluble peptide chains or protein monomers. An acetic acid solution with an optimum pH of 2-4,0.5mol/L provides an appropriate pH for pepsin hydrolysis of peptide bonds.
Firstly, preparing pepsin digestive juice. 0.3ml of anhydrous acetic acid solution was taken, 9.7ml of distilled water was added to obtain 10ml of acetic acid solution having a concentration of 0.5mol/L, 33mg of pepsin powder was weighed and added to the acetic acid solution to have a concentration of 0.33% mass volume fraction (w/v), and the mixture was sufficiently shaken to be completely dissolved, and filtered and sterilized by a 0.22 μm filter, and then used for digestion and dissolution of DAM powder.
DAM powder was added to the pepsin digest, and 330mg of DAM powder was dissolved in 10ml of digest (DAM powder mass: pepsin mass=10:1), and digested on a biological shaking table at 200rpm for 72 hours at room temperature to completely dissolve the DAM powder, to obtain 10ml of DAM digest before alkali neutralization.
The pH and ion concentration of the DAM digest prior to alkali neutralization are then adjusted to physiological levels. The needed solution and the gun head of the liquid-transferring gun are sterilized in advance, so that DAM digestive juice is spontaneously assembled into gel state in the processes of acid-base neutralization and ion concentration adjustment, and the solution and the gun head are placed in a refrigerator at 4 ℃ to be precooled in advance before use and are operated at 0-4 ℃ (on ice). Specifically, acetic acid in the DAM digest was neutralized to ph=7.4 with 0.5ml of 10mol/L sodium hydroxide solution. The ion concentration was then adjusted by adding 10 XMEM medium to give a light pink DAM pregel solution.
In order to concentrate the DAM, the DAM pregelatinized solution is lyophilized and ground again to obtain DAM powder after pepsin digestion, the main component of which is collagen monomer. Dissolving the mixture by using a culture medium to obtain the DAM pregel solution with the required mass and volume fraction.
As shown in FIG. 6, a light pink DAM pregel solution with fluidity was obtained. When the temperature is gradually increased to 37 ℃ from low temperature, the DAM pre-gel solution undergoes sol-gel phase transition, and the DAM hydrogel can resist self gravity and maintain a specific appearance form.
And S50, dissolving the photo-curing hydrogel raw material, and mixing with the DAM pre-gel solution to obtain the 3D biological printing pre-gel solution.
Specifically, 450mg of GelMA freeze-dried powder is weighed and dissolved in 2.7ml of culture medium, the culture medium is placed in a water bath box at 40 ℃ for 30min to completely dissolve the freeze-dried powder, the GelMA solution is obtained, the GelMA solution is filtered and sterilized by a 0.22 mu m filter membrane, and the GelMA solution is placed in a refrigerator at 4 ℃ for light shielding for standby. 60mg of HAMA freeze-dried powder is weighed and dissolved in 0.6ml of culture medium to obtain HAMA solution, and the HAMA solution is subjected to pasteurization and placed in a refrigerator at 4 ℃ for light shielding for standby. 67.5mg of DAM powder was weighed and dissolved in 2.7ml of medium at low temperature to obtain DAM pregelatinized solution. The solution was removed by syringe, sterilized by filtration through a 0.22 μm filter, and placed in a refrigerator at 4℃in the absence of light (both dissolution of DAM and post-dissolution operations were performed on ice to prevent gel at room temperature). The three were mixed to give a composite pregelatinized solution of 1.125% (w/v) DAM,7.5% (w/v) GelMA and 1% (w/v) HAMA.
In this example, the complex pregelatinized solution was subjected to rheological testing to observe the change in properties. Before use, the parallel plates of the rheometer and DAM-GelMA-HAMA pregelatinized solution are pre-cooled to 0 ℃, the pregelatinized solution is in gel state at the temperature, and the gel-like composite pregelatinized solution is placed on the parallel plates with the diameter of 40mm and the interval of 1 mm. The sample was then observed for changes in properties by temperature scanning. The independent variable is temperature, the variation range is 0-35 ℃, the linear temperature rise is carried out, and the speed is 3 ℃/min. The sample was measured using the dynamic shear mode of the rheometer, and fixed shear conditions were applied to the sample: the shear frequency was 1Hz and the strain was 2%. The resulting data is plotted as a curve. As shown in FIG. 7, when the temperature was gradually increased from 0℃to 35℃both the storage modulus (G ') and the loss modulus (G') were gradually decreased, and when the temperature was about 14℃both were suddenly decreased, the slope was significantly increased. The falling rate of G ' is faster than G ", and the temperature at which the G ' and G" curves intersect is about 17.5 ℃, which is the cross-linking temperature of the composite pregel solution, where G ' =g "=8pa. When the temperature is lower than the crosslinking temperature, G 'is larger than G', the material is mainly elastically deformed, and the composite hydrogel is in a gel state. When the temperature is higher than the crosslinking temperature, G 'is higher than G', the material mainly generates viscous deformation, and the composite pregelatinized solution has fluidity and is in a sol state. The DAM-GelMA-HAMA composite pregel solution undergoes gel-sol phase transition with the temperature rise, and the temperature at which the phase transition occurs is 17.5 ℃.
If the 3D bioprinting pregel solution is to be crosslinked, a photoinitiator is also added. Mixing DAM-GelMA-HAMA composite pre-gel solution with photoinitiator LAP to make the working concentration of LAP be 0.25%, obtaining 1.125% (w/v) DAM-7.5% (w/v) GelMA-1% (w/v) HAMA composite pre-gel solution to be crosslinked, and using SEM to observe internal structure after crosslinking.
200 μl of the above compound pregelatinized solution to be crosslinked is sucked and placed into a 96-well plate, gelMA and HAMA are photo-crosslinked by irradiation with 405 nm ultraviolet light for 10 seconds, and then the 96-well plate is placed into a cell incubator at 37deg.C for 60min to self-assemble DAM into hydrogel. And (3) freeze-drying the sample in a vacuum freeze dryer, cutting the freeze-dried sample by a scalpel, exposing the internal structure, placing the cross section of the freeze-dried sample on the conductive double-sided adhesive tape upwards, and sputtering and spraying iridium for 30s. SEM images were collected at 15kV acceleration voltage at 10mm working distance. As shown in FIG. 8, the inside of the composite hydrogel is in a three-dimensional net-like honeycomb, the pores are uniformly distributed and have unequal sizes, the porosity is high, and the pores are closely adjacent. The average pore size was calculated by software to be 73.+ -.18. Mu.m, and the porosity 65%.
This example is a method for detecting biological activity of the biological material of the present invention by cell proliferation. 200 μl of the composite pregel solution to be crosslinked was pipetted into a 24-well plate, irradiated with 405 nm ultraviolet light for 10 seconds to photocrosslink GelMA and HAMA, and then the 24-well plate was placed into a 37℃cell incubator for 60min to assemble the DAM into a hydrogel. 200 μl of 1.125% (w ∈)v) DAM pre-gel solution was placed in a new 24-well plate and placed in a 37℃cell incubator for 60min to allow DAM self-assembly into hydrogel. 2ml of culture medium is added into the crosslinked DAM-GelMA-HAMA composite hydrogel and DAM gel respectively, the mixture is placed into a 37 ℃ and 5% CO2 cell incubator for soaking, and the leaching solution is collected after 3 days. ADSCs cell suspension was collected, counted and inoculated into 96-well cell culture plates so that the number of cells in each well was about 5X 10 3 And each. DAM-GelMA-HAMA composite hydrogel and DAM gel are used as test groups, 100 μl of the two gel extracts and conventional culture medium are added into the cell culture well, and the culture medium is replaced every 3 days. 3 replicates were set at different time points for each group. The proliferation of cells was examined by CCK-8 method, and after 1, 3 and 5 days of culture, the culture solution was discarded, and washed with PBS solution, 100. Mu.l of a medium containing CCK-8 solution (10% volume fraction) was added to each well, and the wells were incubated in a cell incubator for 2 hours. The optical density value of the solution at 450 nm is measured by an enzyme-labeled instrument, and a cell proliferation curve is drawn. As shown in FIG. 9, the increasing optical density values of ADSCs in the two gel extracts over time indicate that ADSCs can maintain good cell activity and proliferate in both DAM-GelMA-HAMA composite hydrogels and DAM gels, and both gels have good biological activity.
The biological material applied to tissue engineering needs to have good biocompatibility and safety, and the embodiment is verified by in-vivo experiments of animals. 6 BALB/c nude mice were selected, and 200. Mu.l of DAM-GelMA-HAMA gel after crosslinking was transplanted under the skin of 3 nude mice as a test group, and 3 untreated healthy nude mice were used as a control group. The nude mice are subjected to abdominal anesthesia after 3 weeks of transplantation, abdominal aorta is used for blood sampling, and 0.5ml of blood sample is collected and is filled in an anticoagulation tube for blood routine detection; in addition, blood samples are collected and filled in an anticoagulation tube, and are centrifuged for 15min at 3000rpm by a centrifuge, and upper serum is collected for biochemical detection, wherein the presence or absence of obvious differences between liver function indexes (such as glutamic pyruvic transaminase and glutamic oxaloacetic transaminase) and kidney function indexes (blood urea nitrogen and creatinine) are mainly concerned. And finally, killing nude mice, taking out heart, liver, spleen, lung and kidney organs of two groups of experimental animals, fixing tissues, and performing HE staining after paraffin section preparation. As shown in fig. 10, the blood biochemical and blood routine indexes of the experimental group and the control group have no statistical difference (P > 0.05) with that of the healthy nude mice, HE staining shows no pathological changes such as cell necrosis, congestion and hemorrhage in heart, liver, spleen, lung and kidney organs, and the DAM-GelMA-HAMA gel has good biocompatibility and safety in vivo.
The 3D biological printing hydrogel can be used for preparing a cell-carrying biological scaffold by 3D printing. The preparation process of the biological scaffold comprises the following steps:
a10, obtaining stem cells.
Specifically, in this embodiment, ADSCs are taken as an example, and the process will be described. ADSCs are cells that have been identified as conforming to the International cytotherapeutic Association definition of mesenchymal stem cells and can be resuscitated for expansion by passaging for use.
A20, mixing the photoinitiator, the stem cell suspension collected after passage and the 3D biological printing composite pregel solution, and 3D printing into a required bracket shape to obtain the cell-carrying biological bracket.
Specifically, the photoinitiator, the cell suspension and the 3D biological printing composite pregelatinized solution are mixed to be used as biological ink, and an extrusion type 3D printer is used for printing out the required shape of the bracket, so that the cell-carrying biological bracket is obtained. To maintain the stability of the scaffold structure, the printed product was photocrosslinked after each layer was printed.
Specific parameters for 3D printing are shown in the following table:
TABLE 2.1 printing parameters
As shown in FIG. 11, a cell-loaded biological stent prepared by an extrusion type 3D printer, which is a cylinder with a diameter of 8mm and a layer height of 720 μm, has a shape and a structure substantially conforming to those of the stent model originally designed. The bracket is almost transparent, is in a crisscross grid shape, has uniform line width and line spacing, and has a regular pore shape, and pores are visible in the middle.
As shown in fig. 12, to examine the survival of cells in 3D bioprinted biological scaffolds, ADSCs in scaffolds were stained with calcein-AM/propidium iodide double staining kit. Live cells can be detected under a fluorescence microscope for green fluorescence, dead cells can be detected for red fluorescence, a fluorescence staining image is obtained and the cell viability is calculated, cell viability = live cell number/(live cell + dead cell number). Cell viability in the bioscaffold was 66.64±1.04%, 86.78 ±3.15%, 77.17 ±1.90% on days 1, 3, 7 after 3D bioprinting and photocrosslinking were completed, respectively. At different time points after printing, the survival rates of ADSCs in the ADSCs-carrying bracket of the DAM-GelMA-HAMA composite hydrogel are different and have statistical differences and are in a trend of ascending and descending (day 1vs day 3,P<0.0001;day 1vs day 7,P<0.01;day 3vs day 7,P<0.01). The scaffold structure formed by the DAM-GelMA-HAMA composite hydrogel for 3D biological printing can be maintained for at least 7 days, has strong structural stability, and has good cell survival in the scaffold within 7 days after printing.
To verify the role of cell-bearing bioscaffold in tissue repair, the invention randomly divided 8 nude mice into 4 groups of 2, a group: DAM gel group, group b: DAM-GelMA-HAMA composite hydrogel group, group c: 3D biological printing carries ADSCs biological scaffold group, D group: conventional dressing change sets. In the embodiment, a full-layer skin defect wound surface model is established on the back of a nude mouse. Immediately after operation, the visible semitransparent gel of the group a and the group b is covered on the wound surface, the visible biological bracket of the group c is covered on the wound surface, and the wound surface of the skin is seen by the group d; on the 7 th day after operation, the wound healing rates of a, b, c, d groups are 38.88 +/-2.12%, 39.68+/-1.99%, 47.99 +/-8.04% and 27.37+/-1.71% (a vs d, P <0.001;b vs d,P<0.001;c vs d,P<0.05), and the wound healing rates of a, b and c groups are all larger than that of d groups and have statistical differences; on the 10 th day after operation, the wound healing rates of the four groups are 56.79 +/-1.73%, 59.44 +/-1.43%, 78.23 +/-5.04% and 51.33+/-1.23%, the wound healing rate of the c group is larger than that of the a group, the b group and the d group and has statistical differences (a vs c, P <0.01;b vs c,P<0.05;c vs d,P<0.01), and the wound healing rate of the a group and the b group is larger than that of the d group and has statistical differences (a vs d, P <0.05;b vs d,P<0.001); on day 14 after surgery, the wound healing rates of the four groups are 73.92+/-1.88%, 76.45+/-1.75%, 87.27 +/-3.13% and 71.49 +/-1.12%, respectively, the wound healing rate of the c group is larger than that of the a group, the b group and the d group and has statistical differences (a vs c, P <0.01;b vs c,P<0.01;c vs d,P<0.01), and the wound healing rate of the b group is larger than that of the d group and has statistical differences (b vs d, P < 0.05). The 3D biological printing cell-carrying biological scaffold group can better promote wound healing compared with DAM gel and DAM-GelMA-HAMA composite hydrogel.
According to the invention, DAM, gelMA and HAMA with photo-curing characteristics are innovatively mixed to prepare the composite hydrogel, so that the printing adaptability, printing resolution and shape fidelity of the DAM hydrogel are improved, the gel time is shortened, the printing efficiency is improved, and the mechanical strength of the 3D printing support is improved. The shear thinning characteristics of the GelMA hydrogels can maintain good continuous filament morphology of the composite hydrogels during and after printing, enabling the DAM hydrogels to be printed out by an extrusion 3D printer. Subsequent uv light irradiation can crosslink the photocurable material to form a gel state, which enables the DAM to maintain a complex multi-layered porous network morphology without the use of external support structures. The DAM-GelMA-HAMA composite hydrogel is found to have good in vivo biocompatibility and safety through being transplanted under the skin of a nude mouse. Extrusion type 3D printing can better control the porosity, shape and cell distribution of a printing structure, compared with a liquid drop type 3D printing technology, the extrusion type 3D printing efficiency is higher, the number of the adaptive biological ink types is more (including cell aggregates, high-viscosity hydrogel, microcarriers and extracellular matrix component), and the mechanical strength of a printed matter is higher. And the extrusion type 3D printing can print out a porous grid structure, promote the exchange of nutrient substances and metabolites, and realize a better bionic effect. Therefore, the invention selects to use extrusion type 3D printing to prepare the composite hydrogel ADSCs biological scaffold, and cells can survive well on the biological scaffold.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A composite hydrogel for 3D bioprinting comprising: DAM hydrogels and photocurable hydrogels, including GelMA and HAMA.
2. The composite hydrogel for 3D bioprinting of claim 1, wherein the DAM has a mass-to-volume fraction of 1.125% (w/v), gelMA has a mass-to-volume fraction of 7.5% (w/v), and HAMA has a mass-to-volume fraction of 1% (w/v).
3. A method for preparing a composite pregel solution for 3D bioprinting, comprising:
obtaining a photo-curing hydrogel raw material and adipose tissue;
the adipose tissue is subjected to decellularization treatment to obtain DAM;
dissolving the DAM to obtain DAM pregel solution;
and dissolving the photo-curing hydrogel raw material, and mixing with the DAM pregel solution to obtain the composite pregel solution.
4. The method for preparing a composite pregel solution for 3D bioprinting according to claim 3, wherein the adipose tissue decellularization process to obtain DAM comprises:
preprocessing the adipose tissue to obtain an initial sample;
repeatedly freezing and thawing the initial sample to obtain a first sample;
digesting the first sample by trypsin to obtain a second sample;
performing primary extraction on the second sample by using isopropanol to obtain a third sample;
digesting the third sample again by trypsin to obtain a fourth sample;
digesting the fourth sample with nuclease and lipase respectively to obtain a fifth sample;
and performing secondary extraction on the fifth sample to obtain DAM.
5. A method of preparing a composite pregel solution for 3D bioprinting according to claim 3, wherein dissolving the DAM to obtain the DAM pregel solution comprises:
lyophilizing and grinding DAM, and adding digestive enzyme to obtain digestive juice;
and regulating the pH value and the ion concentration of the digestion liquid to obtain the DAM pregelatinized solution.
6. A 3D printed hydrogel bioscaffold prepared using a composite pre-gel solution prepared by the method of any one of claims 3-5.
7. A method of preparing a 3D printed hydrogel bioscaffold according to claim 6, comprising:
obtaining and counting adipose-derived mesenchymal stem cells;
and mixing the photoinitiator, the adipose-derived mesenchymal stem cells and the 3D biological printing composite pre-gel solution, and preparing a required bracket shape by a 3D printer to obtain the 3D printing hydrogel biological bracket.
8. The method of preparing a 3D printed hydrogel bioscaffold according to claim 7, wherein each layer is printed, the printed product is photocrosslinked.
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