Patent Application
20240108784
This application claims priority benefits to Chinese Patent Application No. 202211199865.6, filed 29 Sep. 2022, the contents of which are incorporated herein by reference.
The present invention relates to the technical field of biomedical polymer hydrogels, and in particular to hydrogel for cell-laden bioprinting, bioink, and a preparation method and an application thereof.
Information of the related art part is merely disclosed to increase the understanding of the overall background of the present invention, but is not necessarily regarded as acknowledging or suggesting, in any form, that the information constitutes the prior art known to a person of ordinary skill in the art.
In the prior arts, the treatment solutions for large-area skin trauma and organ tissue injury generally rely on the patient's own repair capacity and organ transplantation. However, for the large-area skin trauma and organ injury that exceeds the repair capacity of the human body, an autologous transplantation or an allogeneic transplantation is usually adopted. However, the autologous transplantation will cause greater secondary damage to the patient, and the allogeneic transplantation is difficult to match a suitable donor. Therefore, an effective treatment of the large-area skin trauma and organ tissue injury has been a major problem in the medical field.
With the progress of science and technology, a 3D bioprinting technology provides a new clinical medical strategy for tissue and organ repair, and has been applied in the medical field, such as tissue and organ substitute construction, organ transplantation and regeneration, drug screening models and anatomical models. The 3D bioprinting has many advantages for tissue and organ repair, allowing complex tissue structure printing, cell-laden printing, controllable precise deposition for different cell numbers and cell densities, and easy access to the gradient of physicochemical property biomaterials. Bioink capable of loading living cells is particularly important for the 3D bioprinting, and proper physical and chemical properties of hydrogel materials are the premise for cell adhesion, migration, and proliferation. Therefore, the hydrogel material needs to have good biocompatibility, which can provide both a specific microenvironment for cell growth and a basis for the induction of cell functionalized behavior; at the same time, it must also have good printability, a degradation rate that matches the regeneration rate of tissue cells, a swelling rate suitable for cell growth, and good mechanical properties, etc.
In a three-dimensional culture of cells, in order to ensure printability, biological scaffold materials need to have a high elastic modulus. During in vitro culture, the mechanical properties of biological scaffolds affect the activity, proliferation and differentiation of cells, and different cells have different requirements for the mechanical properties of the biological scaffolds. Therefore, in order to ensure the good biocompatibility of the biological scaffolds, it is necessary to make the mechanical properties of biological scaffold as close as possible to the mechanical properties required by cultured cells in vivo environment. In order to support cell proliferation and migration as well as nutrient transport, a certain porosity of the biological scaffold is required. At present, the preparation of hydrogel materials with good comprehensive properties is still the difficulty of 3D bioprinting.
For overcoming the problems in the prior art, it is an object of the present invention to provide hydrogel for cell-laden bioprinting, capable of advantages such as adjustable mechanical properties, adjustable porosity, high biocompatibility, high printing accuracy, and high customizability, which can widely support the printing of human tissues such as spinal cord, cartilage, and heart. According to the present invention, by adding different cell-specific materials to a matrix material, bioactive tissues can be printed directly, which has good prospects for application in tissue repair, organ transplantation and so on.
To achieve the above-mentioned objectives, the present invention provides the following technical solutions.
The first objective of the present invention is to provide hydrogel for cell-laden bioprinting, being polymer gel formed by adding a cell-specific material into a matrix of alginate and gelatin and crosslinking and curing, wherein the cell-specific material is polypeptide.
The second objective of the present invention is to provide a method for preparing cell-laden bioink, comprising the steps of: a preparation for cell-laden bioink precursor: adding sodium alginate to phosphate buffered saline (PBS) and dissolving by stirring with warming to obtain alginate solution, adding and dissolving gelatin and cell-specific materials to the PBS to obtain gelatin composite solution; mixing the alginate solution and the gelatin composite solution evenly to obtain bioink solution; centrifuging cell suspension, and then adding the bioink solution and blowing the cells being dispersed and even to obtain the cell-laden bioink precursor.
In one preferred embodiment of the present invention, a concentration range of the alginate is 2-5% w/v and a concentration range of the gelatin is 2-8% w/v.
In another preferred embodiment of the present invention, for L929 cells, the cell-specific material is polypeptide CMP27, the concentration of the alginate is 2.5% w/v and the concentration of the gelatin is 6% w/v.
The third objective of the present invention is to provide a method for preparing a cell-laden scaffold, comprising the steps of: loading the cell-laden bioink prepared by the above-described preparation method into a printer syringe, extruding the cell-laden bioink onto a cryogenic platform through a pneumatic nozzle on the 3D printer to prepare a grid-like cell-laden scaffold, then soaking the printed cell-laden scaffold in 4.5% w/v calcium chloride solution for 5 min, and then obtaining a final structure of the cell-laden scaffold after being washed with PBS (phosphate buffered saline).
The fourth objective of the present invention is to provide a cell-laden scaffold prepared by using a method for preparing the cell-laden scaffold as described above.
The fifth objective of the present invention is to provide a Band-aid, comprising the cell-laden scaffold as described above.
The sixth objective of the present invention is to provide a method of preparing tissues and organs using a freeform reversible embedding of suspended hydrogels (FRESH) extrusion printing, comprising the steps of: fixing a gelatin support bath on a cryogenic platform, loading cell-laden bioink prepared using the method for preparing the cell-laden bioink as described above into a syringe of a 3D bioprinter, extruding and printing the bioink into the support bath through a pneumatic nozzle, raising the temperature of the cryogenic platform after the printing is completed, releasing a structure being printed and washing the structure with the PBS.
In another preferred embodiment of the present invention, comprising: incubating prepared solution containing 5% (w/v) gelatin and 1% (w/v) CaCl2 at 4° C. for 24 h, then crushing the incubated solution using a crusher after adding CaCl2 solution with 3 times the volume; centrifuging gelatin granules obtained from the crushing, then removing supernatant, then centrifuging the gelatin granules again to obtain gelatin slurry after being washed three times with 1% (w/v) CaCl2 solution, and then transferring the gelatin slurry to a petri dish.
The seventh objective of the present invention is to provide an application of hydrogel for cell-laden bioprinting as described above or a cell-laden scaffold as described above in the biomedical field.
One or More Technical Solutions Provided in the Present Invention at Least have the Following Technical Effects or Advantages:
1. According to the present invention, the bioink for cell-laden 3D bioprinting can independently regulate the mechanical properties and biological properties of the printed structure. By adjusting the composition ratio of the matrix materials, the quantitative adjustment of the porosity and mechanical properties of the hydrogel can be achieved simultaneously. By adding different components of additive materials, the biocompatibility requirements of different cells can be met to induce the migration and differentiation of cells.
2. According to the present invention, designing two ways of cryogenic printing and FRESH printing by combining the bioink to construct the printing structure, which can simultaneously meet the requirements of rapid printing of simple cell scaffolds and high-precision printing of complex tissues and organs, and the whole process uses natural biological materials, which will not harm cells.
3. According to the present invention, optimizing the cryogenic printing process for the bioink, making the survival rate of the printed cells up to 96%; and the printing process is simple and rapid, which can be completed within 5 min, realizing an application of the rapid custom printing of cell-laden dressings; optimizing the FRESH printing process, making the temperature of the whole printing process is kept within the suitable range for cells, which can meet the printing application of a long time and large scale complex tissues and organs.
The advantages of additional aspects of the present invention will be given in the following description, and partially will become apparent from the following description, or will be learned from the practice of the present invention.
In order to make the above objectives, features, and advantages of the present invention more obvious and understandable, the following is a detailed description of the preferred embodiments in combination with the attached drawings.
The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary examples of the present invention and descriptions thereof are used to explain the present invention, and do not constitute an improper limitation of the present invention.
The spacing or size between each other is exaggerated to show the position of each part, and the schematic diagram is only used for illustration.
It should be pointed out that the following detailed descriptions are all illustrative and are intended to provide further descriptions of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those usually understood by a person of ordinary skill in the art to which the present invention belongs.
The present invention proposed multifunctional hydrogel bioink with adjustable mechanical properties for cell-laden 3D bioprinting, comprising polymer gel formed by using alginate, gelatin, and cell-specific additive materials as monomers and cross-linking and curing. Wherein, the cell-specific materials are polypeptides, and different polypeptides are selected according to different laden cells. For examples, for L929 fibroblasts, osteoblasts, and neural stem cells, the cell adhesion polypeptides can be added, including, and not limited to RGD polypeptides, CMP27 and other collagen-mimetic polypeptides; for skin tissue cells, antimicrobial peptides can be added, including and not limited to ε-poly-L-lysine, PMAP-class polypeptides, and Protegrin-class polypeptides, etc. The hydrogel can be molded by both temperature-sensitive cross-linking and ionic cross-linking. The elastic modulus of the scaffold can be adjusted by adjusting the concentration of alginate, and the porosity during long-term incubation can be adjusted by adjusting the concentration of gelatin. Specifically:
The alginate, isolated from brown algae, has high biocompatibility. The guluronic acid on the alginate molecule can bind to calcium ions thus forming a three-dimensional network molecule in the form of ionic cross-linking to form a hydrogel network, which can be stable in the medium for a long time. By changing the concentration of alginate in the gel, the mechanical behavior of the gel can be changed.
The gelatin, containing many bioactive sites, has both good biodegradability and modulation of cell adhesion, and gelatin solutions have good temperature sensitivity and can form a hydrogel network by physical cross-linking.
According to the present invention, a method for preparing cell-laden bioink comprises the steps of:
Using the cell-laden bioink for 3D bioprinting obtained using the preparation method as described above to construct a cell-laden scaffold with adjustable mechanical properties and porosity.
According to the mechanical properties of the specific tissue, the mechanical behavior of the printed tissue may be regulated by adjusting the concentration of alginate matrix in the bioink. According to the rate of cell proliferation and migration, the porosity of the tissue scaffold in long-term culture may be regulated by adjusting the concentration of gelatin in the bioink.
The present invention provides a method for rapid preparation of cell-laden scaffolds by cryogenic extrusion printing, comprising:
storing cell-laden bioink of the present invention in a cryogenic printer syringe, and setting a temperature of the syringe at 26° C. and a temperature of a printer platform at 5° C.; constructing a grid-like cell-laden scaffold by printing layer by layer, and soaking and incubating the scaffold with 4.5% (w/v) CaCl2 solution for 5 min after the print is completed, then transferring the scaffold to complete medium for culture after being washed twice with 1×PBS.
The present invention further provides a cell-laden hydrogel trauma dressing, i.e., a Band-aid prepared using a cell-laden scaffold described above.
The present invention further provides a method for high-precision printing of vitro tissues, comprising:
Accordingly, the structure may be printed and shaped by both a cryogenic extrusion printing and a FRESH extrusion printing. Wherein, the cryogenic extrusion printing can quickly construct simple cell-laden three-dimensional structures that can be directly used for applications such as trauma repair; the FRESH extrusion printing can construct complex structures with high precision that can be used to construct tissues and organs. The printed structures have the advantages of adjustable mechanical properties, adjustable porosity, high biocompatibility, high printing accuracy, and high customizability, etc., which have good prospects for application in tissue repair, organ transplantation and so on.
A Method for Preparing Cell-Laden Bioink
1. Preparation of Alginate Solution:
weighing and dissolving 0.5 g of sodium alginate powder in 10 ml of 1×PBS (phosphate buffered salt solution), stirring the solution at 920 r/min for 2 h using a magnetic stirrer, while heating the solution to 40° C. and keeping it until the alginate is completely dissolved to obtain 5% (w/v) alginate solution; then, degassing the alginate solution using an ultrasonic cleaner for 2 min and then transferring the degassed alginate solution to a clear glass vial and sterilizing it using 260 nm UV light for 24 h; after sterilization, transferring the alginate solution to 4° C. for storage.
2. Preparation of Gelatin/Polypeptide CMP27 Composite Solution:
weighing and dissolving 1.2 g of gelatin powder and 4 mg of polypeptide CMP27 (a specific additive material for L929 cells, which is existing biomaterial-collagen mimetic polypeptide, and its role is to promote L929 cell adhesion, reference: collagen mimetic peptide and its effect on fibroblast adhesion) in 10 ml of 1×PBS, stirring the solution at 700 r/min for 1 h using the magnetic stirrer, while heating the solution to 37° C. and keeping it until the dissolvent is completely dissolved to obtain gelatin/polypeptide composite solution; after stirring, degassing the composite solution using an ultrasonic cleaner for 2 min and then sterilizing it by filtering with 0.22 um filter.
3. Preparation of Cell-Laden Bioink Precursor:
heating the prepared alginate solution and gelatin/polypeptide CMP27 composite solution to 40° C. and mixing them in a ratio of 1:1, mixing the mixture evenly using a vortex oscillator to obtain bioink precursor, which is then kept at 40° C. for use.
4. Cell Culture and Preparation of the Cell-Laden Bioink:
selecting L929 mouse fibroblasts and culturing them in medium containing 10% fetal bovine serum and 1% penicillin/streptomycin; keeping the cultures in a humidified incubator with 37° C. and 5% CO2, and changing the medium every other day; before preparing the bioink, digesting the cells, with a count cell density of 3×106 ml−1; aspirating 2 ml of cell suspension according to the cell density of 1×10 6 ml 1 in bioink and centrifuging it; aspirating 6 ml of the bioink precursor solution and blowing the cells evenly to obtain L929-specific cell-laden bioink.
A Construction of Cell-Laden 3D Grid Scaffolds by Cryogenic Extrusion Printing
1. Preparation of Bioink:
Same as Steps 1, 2 and 3 in Example 1.
2. Preparation of L929 Cell-Laden Bioink:
Same as Step 4 in Example 1.
3. Printing of the Cell-Laden 3D Grid Scaffold:
transferring cell-laden bioink into a syringe of a 3D bio-printer, setting a temperature of the syringe to 26° C., and incubating the bioink for 10 min before printing; selecting the printing mode to print one by one, and conducting the printing in a petri dish with 41035 mm; setting a temperature of the printing platform at 5° C., a needle size to 22 G, a layer height to 0.3 mm, a printing speed to 100 mm/min, a movement speed to 900 mm/min, a return axis speed to 2000 mm/min, a brim width (edge width) to 1 mm, and a brim speed (edge printing speed) to 100 mm/min; setting a printing pressure to 16 kpa, to print a three-layer grid structure, a printed size to 12.0 mm×12.0 mm, a layer height to 0.3 mm, and a spacing to 1 mm×1 mm.
4. Culture of the Cell-Laden Scaffold:
further cross-linking the scaffolds using 5% (w/v) CaCl2 solution soaked for 5 min after printing, then transferring the scaffolds to culture medium containing 10% (w/v) fetal bovine serum, 1% (w/v) penicillin/streptomycin after washing twice with 1×PBS; placing the cultures in an incubator at 37° C., 5% CO2.
Constructing a High-Precision Complex Structure with Low Elastic Modulus by a FRESH Extrusion Bioprinting
1. Preparation of Bioink:
Same as Steps 1, 2 and 3 in Example 1.
2. Preparation of a Support Bath:
measuring out 50 ml of ultrapure water, weighing 2.5 g of gelatin granules and 0.5 g of CaCl2) granules, stirring the solution at 700 r/min for 1 h using a magnetic stirrer, heating the solution to 37° C. while the stirring, degassing the solution using an ultrasonic cleaner for 2 min after the stirring is completed, to obtain 5% (w/v) gelatin solution, then transferring the gelatin solution to a 50 ml centrifuge tube and incubating the gelatin solution at 4° C. for 24 h to wait for the gelatin to crosslink; weighing and dissolving 3 g of CaCl2 granules in 300 ml of ultrapure water to prepare 1% (w/v) CaCl2 solution, then putting the cross-linked gelatin gel into a crusher after being cut into squares of 10 mm 3, and adding the 1% (w/v) CaCl2 solution with 3 times the volume, then crushing them at 10000 r/min for 60 s to obtain the solution of gelatin granules, then centrifuging the solution of gelatin granules under the centrifugal force of 2000 g with a centrifuge, removing supernatant after centrifugation, and then centrifuging and washing the solution of gelatin granules after adding the 1% (w/v) CaCl2 solution with 3 times the volume again to obtain gelatin slurry.
3. Setting of the FRESH Bioprinting Process:
slicing a designed 3D model of tissue using the Slic3r (3D slicing software), wherein setting a layer height to 0.2 mm, a fill pattern to Rectilinear (straight line), a fill density to 30%, and sending a G code generated after the slice is completed to the bioprinter;
transferring the prepared gelatin support bath to a petri dish of Φ35 mm and placing the petri dish on a cryogenic platform of the bioprinter, setting a temperature of the cryogenic platform to 15° C.; transferring the cell-laden bioink into a syringe of the bioprinter, setting a temperature of the syringe to 26° C. and carrying out the printing after the bioink being incubated for 10 min, wherein setting a needle size to 25 G, a layer height to 0.2 mm, a printing speed to 100 mm/min, a movement speed to 900 mm/min, a return axis speed to 2000 mm/min, a Brim Width (edge width) to 1 mm, a Brim speed (edge printing speed) to 100 mm/min, and a printing pressure to 30 kpa; performing the printing for a series of complex spatial structures, as shown in
In the following, the advantages of the hydrogel material in the examples in the application of tissue engineering scaffold or cell-laden printed tissue will be explained through the following tests.
1. Rheological Tests
A rheology evaluation for the cell-laden hydrogel of the present invention was carried out. As shown in
In addition, the thixotropy properties of the bioink were also tested, as shown in
For the gel kinetics of the cells, a test for the bioink was carried out in a shaking mode at a frequency of 1 Hz and in the range of 4-37° C. As shown in
2. Mechanical Properties Test
A test for mechanical properties of the bioink with different concentrations was carried out, and a printable range of the bioink is investigated from a view of elastic modulus. The test results are shown in
3. Observation of Hydrogel Morphology
Morphologies of the hydrogel material of the present invention after curing, freeze drying and gold spraying under a SEM are shown in
4. Analysis of Cell Activity
For evaluating the biocompatibility of the bioink, observing the activity and proliferation of the cells in the grid structure printed by using the bioink loaded with L929 cells. The cell viability was analyzed using a live/dead staining kit on the 1st, 3rd, 5th, and 7th day of culture respectively. The cells were observed by using an inverted fluorescence microscope, as shown in
The proliferation status of the cells was tested on the 1st, 3rd, 5th, and 7th day of culture using the CCK8 kit. Wherein, the tests on the first and third day after printing showed that the absorbance did not change much and the number of cells remained approximately the same, which mainly due to changes in the extracellular environment during printing; the tests on the fifth and seventh day showed a large increase in absorbance and a large proliferation of cells. It indicated that the printing process did not cause irreversible damage to the state of the cells and that the biomaterials used have good biocompatibility and can support cell proliferation and migration.
Although the specific embodiments of the present invention are described above in combination with the accompanying drawings, it is not a limitation on the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical scheme of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.
Finally, it should be noted that, if there is no conflict, the examples of the present invention and various features in the examples can be combined with each other, and all are within the protection scope of the present invention. Additionally, all or a portion of the steps of the above-described methods may be performed in a computer system, such as a set of computer-executable instructions, and although the steps are listed in the order of 1, 2, 3, and so on, in some cases, the steps shown or described may be performed in an order other than that shown herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211199865.6 | Sep 2022 | CN | national |
