Construction method and application of in-vitro callus-like organ

Abstract

The invention discloses a construction method and application of an in-vitro callus-like organ, the survival rate of the organ is improved by improving freeze-formed bio-ink, and a stable directional channel structure is further prepared, so that the internal mass transfer problem of the callus-like organ is improved, and the material exchange efficiency is improved. The forming mode of the callus-like organ is simple and effective, the stent material is easy to prepare, and the callus-like organ has mature cross-linking and printing characteristics, is expected to be applied to drug screening research or bone defect repair, is clear in process and simple in composition, and has relatively high operability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology and relates to a method for constructing and applying in vitro callus-like organs. Background Technology

[0002] Human bone formation relies on two pathways: intramembranous ossification and endochondrial ossification. Endochondrial ossification refers to the process by which cartilage is gradually replaced by bone after mesenchymal coagulation and chondrogenic differentiation, building upon a pre-formed cartilage rudiment. Some studies have shown that, compared to intramembranous ossification, where coagulated mesenchyme directly calcifies into bone, bone defect repair via endochondrial ossification typically results in higher bone volume and density in the newly formed bone tissue. However, in vitro models of endochondrial ossification are relatively scarce in this field. Furthermore, past tissue-engineered osteogenic processes often rely on complex physiological environments and rarely use materials alone to induce osteogenic formation, generally exhibiting dependence on exogenous growth factors and low implant survival rates (such as cells and organoids).

[0003] In the current context of bone tissue engineering research, researchers often combine stem cells (pluripotent stem cells, embryonic stem cells, or adult stem cells) and their derived osteoblast cell lines with tissue engineering scaffolds. GelMA is a stable and widely used tissue engineering scaffold material. However, due to its poor mass transfer properties, when GelMA is used as a carrier material for cells in tissue engineering preparation, it often leads to reduced cell activity or even apoptosis.

[0004] To address this issue, researchers have employed methods to construct directional channels within materials to improve cell survival. However, the fabrication of directional scaffolds typically utilizes 3D printing, a method that suffers from time-consuming processes, complex designs, and high instrument precision requirements when fabricating large-volume, precision-structured scaffolds (such as micrometer-scale channel structures). Cryo-directed prototyping is an emerging approach to overcome these challenges. It involves the cryo-directed fabrication of highly aligned, directional biomaterial templates, forming microchannels within the material to improve cell survival and nutrient exchange. However, the freezing process itself can significantly damage the viability of seed cells loaded within the scaffold, substantially hindering the subsequent tissue repair mediated by the scaffold material in vivo. Therefore, the bio-ink used in the cryo-directed prototyping process needs to be specially designed to provide excellent cryoprotection.

[0005] Therefore, a significant challenge at present is to develop a model that can reproduce the endochondral osteogenic process in vitro, transform cartilage organoids into callus organoids in an in vitro culture environment, and ultimately construct an in vitro callus organoid model that can be used for bone defect repair or drug screening. This model should also have the characteristics of low preparation cost, low preparation difficulty, and good in vitro osteogenic effect. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing and applying in vitro callus organoids. The materials used in this method have a solid foundation of prior research and clinical validation, making it suitable for establishing in vitro endochondral ossification models in multiple species. The process is clear, the structure is simple, and it is highly operable. It provides unique insights into the physiological mechanisms of endochondral ossification in vitro and is suitable for widespread application.

[0007] To solve the above-mentioned technical problems, the method for constructing in vitro callus organoids of the present invention comprises the following steps:

[0008] Step 1. Collagen hydrogel presolution

[0009] (1) Dissolve the freeze-dried collagen in hydrochloric acid to prepare a stock solution with a certain collagen concentration.

[0010] (2) Under ice bath conditions, mix the cartilage induction culture medium and collagen solution by blowing and mixing, then adjust the solution to pH=7, and finally dilute the collagen solution to a certain concentration.

[0011] (3) The obtained collagen solution was placed in an ice bath for half an hour for prepolymerization.

[0012] Step 2. Construction of cartilage organoids

[0013] (1) Proliferate P2 generation mesenchymal stem cells to 60%-80% of the culture dish area, digest the cells with trypsin, and repeatedly rinse the culture dish with growth medium to collect the cells. Take a small amount of cell suspension and drop it onto a cell counting plate, count the number of cells under a microscope, and calculate the total number of collected cells. Discard the excess cell suspension according to the target number of organoid cells, and centrifuge to obtain pure mesenchymal stem cells.

[0014] (2) Mix pure mesenchymal stem cells with the collagen hydrogel precursor solution after incubation in step 1, and resuspend them into a uniform cell-collagen hydrogel precursor solution mixture suspension. Then inject the mixture into a cylindrical mold and transfer it to a 37°C carbon dioxide constant temperature incubator for incubation until complete gelation. After that, transfer it to cartilage induction medium for culture.

[0015] Step 3. Preparation of freeze-molding ink

[0016] (1) Weigh out the gelatin and dissolve it in deionized water under a 50-degree water bath. After the gelatin is completely dissolved, add methacrylic anhydride (MA) in several portions to adjust the pH of the reaction system to around 9.

[0017] (2) The GelMA aqueous solution after the reaction was freeze-dried into a white sponge. The freeze-dried sponge was soaked and washed, then redissolved and transferred to a dialysis bag for dialysis. The dialyzed solution was freeze-dried again to obtain the finished GelMA sponge.

[0018] (3) The GelMA sponge was reconstituted in PBS phosphate buffer containing LAP photoinitiator to prepare a GelMA hydrogel precursor solution.

[0019] (4) Add the inducible biomaterial to the GelMA hydrogel precursor solution according to a certain solid content ratio, and disperse it evenly by ultrasonication to obtain a GelMA / HAp precursor solution that can be used for photocrosslinking; then add 10% DMSO and a certain amount of polysaccharide to the precursor solution to prepare a cryo-molded ink with cryoprotection effect.

[0020] Step 4. Preparation of callus organoid precursors

[0021] The aforementioned cartilaginous organoids were loaded with a certain proportion of organoids and cryo-forming ink onto each callus organoid mold, and then transferred to a mold placed on a cryoplate for cryo-forming. During the process, ultraviolet light was used to irradiate the material on the cryoplate to completely cross-link it, thus obtaining the callus organoid precursor.

[0022] In one or more embodiments of the present invention, the collagen in step 1 is type I collagen.

[0023] In one or more embodiments of the present invention, the hydrogel presol in step 1 may also be hyaluronic acid or decellularized extracellular matrix.

[0024] In one or more embodiments of the present invention, the cartilage induction culture medium in step 2 mainly contains VC, dexamethasone, ITS liquid culture medium supplement, and TGF-β.

[0025] In one or more embodiments of the present invention, the inducible biomaterial in step 3 may be hydroxyapatite powder (HAp), tricalcium phosphate powder, or biaxial calcium phosphate powder.

[0026] In one or more embodiments of the present invention, the polysaccharide in step 3 is mainly menotriose.

[0027] In one or more embodiments of the present invention, the cartilaginous organoids in step 4 are obtained after 14 days of chondrogenic induction.

[0028] In one or more embodiments of the present invention, the mold dimensions in step 4 are approximately 3-8 mm inner diameter and approximately 5-10 mm height.

[0029] In one or more embodiments of the present invention, the number of cartilaginous organoids in step 4 is 10-25.

[0030] In one or more embodiments of the present invention, the freezing temperature in step 4 is -20°C.

[0031] Compared to existing technologies, this invention, through the design of the scaffold material components, endows the scaffold material with the ability to induce the development of cartilage organoids into callus organoids in vitro. By cryo-directing, channels of specific sizes are formed to improve the mass transfer performance within the callus organoids. Furthermore, the directional structure is simple, effective, easy to prepare, and possesses mature cross-linking properties. Simultaneously, this invention has researched and designed the cryo-forming ink components to ensure that the activity of the frozen organoids is not significantly affected, allowing them to exert their intended biological characteristics during subsequent osteogenic development. Attached Figure Description

[0032] Figure 1 This is an experimental flowchart illustrating the method for constructing in vitro callus organoids in Example 1.

[0033] Figure 2 Matrix phenotype of the cell-collagen hydrogel prepared in Example 1 after cartilage induction for different number of days.

[0034] Figure 3 Cross-sectional view of the oriented GelMA / HAp hydrogel prepared in Example 1 on a -20°C frozen plate.

[0035] Figure 4 This is a distribution diagram of HAp powder uniformly deposited on the hydrogel in Example 1.

[0036] Figure 5 Example 1: Cell viability assay of cartilage organoids after cryopreservation.

[0037] Figure 6 This is a cross-section of the callus organoid prepared in Example 1.

[0038] Figure 7 This is a schematic cross-sectional view of the callus-like organoid prepared in Example 1, cut along the diameter of the bottom surface of the material.

[0039] Figure 8 This is an alkaline phosphatase staining image of the callus-like organoid prepared in Example 1.

[0040] Figure 9 Alizarin Red staining image of the callus organoid prepared in Example 1. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the following embodiments are only further illustrations of the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0042] Example 1

[0043] This embodiment relates to a method for constructing in vitro callus organoids, the specific method being as follows:

[0044] 1. Preparation method of collagen hydrogel presol:

[0045] (1) The freeze-dried collagen was dissolved in a certain volume of 2M hydrochloric acid to prepare a stock solution with a collagen concentration of 10mg / mL.

[0046] (2) Under ice bath conditions, the cartilage induction culture medium was mixed with a certain amount of 10 mg / mL collagen solution by blowing and mixing. Then, an appropriate amount of 0.2 M sodium hydroxide was added to the solution to adjust the pH to 7. Finally, the collagen solution was diluted to 3 mg / mL.

[0047] (3) The obtained 3 mg / ml collagen solution was placed in an ice bath for half an hour for prepolymerization.

[0048] 2. The method for constructing the cartilaginous organoids:

[0049] (1) When the P2 generation mesenchymal stem cells proliferate to about 80% of the culture dish area, the cells are digested with trypsin and the culture dish is repeatedly rinsed with growth medium to collect the cells. A small amount of cell suspension is dropped onto a cell counting plate, the number of cells is counted under a microscope, and the total number of collected cells is calculated.

[0050] (2) Discard excess cell suspension according to the target cell quantity of organoids, and centrifuge at 1100 r / min for min to obtain pure mesenchymal stem cells. Mix the cells with the collagen hydrogel precursor solution after incubation, and resuspend to form a homogeneous cell-collagen hydrogel precursor solution mixture suspension. Inject 100 μl of cell-collagen hydrogel solution into a cylindrical PDMS mold (Φ4×6 mm), and transfer to a 37℃ carbon dioxide constant temperature incubator for incubation until complete gelation.

[0051] (4) The collagen hydrogel loaded with mesenchymal stem cells was transferred to chondrogenic medium for culture.

[0052] (5) After 14 days of culture, histological and immunohistochemical experiments were performed on the cartilaginous organoids.

[0053] 3. Preparation of freeze-curing ink:

[0054] (1) Weigh 10g of gelatin and dissolve it in 200ml of deionized water under a 50°C water bath (gelatin concentration is 50mg / ml). After the gelatin is completely dissolved, add 10ml of MA in 6 portions, one portion every 30 minutes, while continuously adding NaOH to adjust the pH of the reaction system to around 9.

[0055] (2) The GelMA aqueous solution after the reaction was freeze-dried into a white sponge. The freeze-dried sponge was soaked and washed with anhydrous ethanol to remove unreacted MA. Then it was redissolved in RO water and transferred to a dialysis bag for dialysis for 4-5 days. The dialyzed solution was freeze-dried to obtain the finished GelMA sponge.

[0056] (3) The GelMA sponge was reconstituted in PBS phosphate buffer containing LAP photoinitiator to prepare a GelMA hydrogel precursor solution.

[0057] (4) Add hydroxyapatite powder (HAp) to the GelMA hydrogel precursor solution at a solid content ratio of 30%, and disperse it evenly by ultrasonication to obtain a GelMA / HAp precursor solution that can be used for photocrosslinking.

[0058] (5) Dissolve 10% v / v DMSO and 8% w / v mesotriose in GelMA / HAp precursor solution to prepare cryogenic ink.

[0059] 4. Preparation of callus organoid precursors

[0060] The aforementioned cartilaginous organoids, with each callus organoid containing 22 cartilaginous organoids, were mixed with cryoforming ink and transferred to a mold placed on a cryoplate for cryoforming. The mold dimensions were approximately 4 mm in inner diameter and 6 mm in height. During extrusion, the material on the cryoplate was simultaneously cross-linked under 405 nm ultraviolet light to obtain a cryoform organoid precursor containing cartilaginous organoids, prepared through cryo-directed processing.

[0061] Example 2: Characterization of the induction effect of cartilage organoids

[0062] Mesenchymal stem cells were mixed with a prepolymerized collagen hydrogel solution and incubated in a 37°C CO2 incubator to form a gel. The gel was then cultured in chondrogenic medium. The spheroids formed by the cell-collagen mixed hydrogel were collected on days 7, 14, and 21 after culture, respectively, and stained with safranin to observe the phenotype of chondrogenic organs.

[0063] like Figure 2As shown, overall, matrix remodeling was observed in the cell-collagen hybrid hydrogel after 7, 14, and 21 days of induction. The differentiated mesenchymal stem cells exhibited obvious chondrocyte characteristics and secreted cartilage matrix, which was stained with safranin dye. Among them, the spherical phenotype formed by the cell-collagen hydrogel after 14 days of induction showed a high degree of similarity to natural hyaline cartilage, which meets the aforementioned definition of cartilage organoids required in this invention.

[0064] Example 3: Cryo-directional fabrication of GelMA / HAp scaffolds and morphological characterization

[0065] The cryoplate was pre-cooled to -20°C, and a silicone mold with an inner diameter of 4 mm and a height of 6 mm was placed inside. The GelMA / HAp precursor solution described in Example 1 was injected into the mold and frozen into ice. The presolution was then photocrosslinked using 405 nm ultraviolet light. The resulting hydrogel scaffold was freeze-dried and cut open, and the cross-section was observed using a field emission scanning electron microscope.

[0066] from Figure 3 The directional channels formed during freezing are densely packed and clearly visible, with channel diameters between 40-60 μm, meeting the size requirements for material exchange and cell migration, demonstrating the feasibility of the method for preparing a scaffold with a directional structure described in this invention. Figure 4 Hydroxyapatite powder is widely and uniformly distributed on the surface of the hydrogel.

[0067] Example 4: Activity Characterization of Cartilage Organoids by Cryoforming

[0068] The aforementioned cartilage organoids were mixed with cryo-forming ink and frozen at -20°C and -80°C, respectively. After photocrosslinking, the organoids were excised from the hydrogel and cultured in a 37°C carbon dioxide incubator. On days 1, 3, and 7 after treatment, the cartilage organoids were sectioned and CCK8 cell viability was detected. Cartilage organoids with the same culture time but without cryo-forming treatment were used as the control group. Figure 5 As can be seen, on the first day after freezing, the activity of cartilage organoids frozen at -20℃ was reduced by approximately 25% compared to the untreated control group. However, in the tests on days 3 and 7, the cell activity was not significantly different from the control group. The cell activity of the experimental group frozen at -80℃ was lower than that of the experimental group frozen at -20℃ at all three time points, demonstrating that the selection of -20℃ as the freezing orientation temperature in this invention can preserve the activity of cartilage organoids to a greater extent while ensuring the acquisition of the desired oriented structure.

[0069] Example 5: Characterization of the internal directional structure of callus organoid precursors

[0070] The aforementioned cartilage organoids were mixed with GelMA / HAp cryogenic ink and uniformly extruded onto a cryoplate pre-cooled to -20°C. After complete freezing, the ink was cross-linked using 405nm ultraviolet light. The prepared scaffold was freeze-dried, cut perpendicular to the bottom surface, and the cross-sectional structure was observed using a field emission scanning electron microscope.

[0071] Figure 6 As can be seen, during the co-extrusion process, the internal structure of this interosseous organoid precursor generally maintains the aforementioned directional structure, such as... Figure 6 As shown in Figure A, after freeze-drying, the cross-sectional structure of the scaffold was observed. Around the circular grooves left after some organoids detached, it can be seen that the scaffold material still retained the expected directional structure, proving that the presence of organoids does not affect the freeze-direction results. Furthermore, the scaffold material at the connection points between different organoids (…) Figure 6 Part B) also retains the oriented scaffold structure. In summary, this cryo-orientation method has strong robustness. The GelMA / HAp ink maintains the preset oriented structure, and the size of the formed oriented channels is between 10 and 30 μm, which is sufficient to support material exchange and cell migration within callus organs.

[0072] Example 6: Cartilaginous organoids undergo osteogenic differentiation in vitro to transform into callus organoids.

[0073] The aforementioned precursors of callus organoids were cultured in vitro for 14 days using ordinary mesenchymal stem cell proliferation medium without the addition of any exogenous bone differentiation-promoting culture components (such as sodium β-glycerophosphate, BMP-2, etc.). The materials were collected, frozen, sectioned, and stained with alkaline phosphatase and alizarin red.

[0074] Alkaline phosphatase staining and alizarin red staining are both markers of the differentiation of mature cartilage organoids towards osteoblasts: alkaline phosphatase is a marker of osteoblasts, while calcium deposition refers to mineralized granules secreted by osteoblasts. From Figure 8 As can be seen, the cartilaginous organoids encapsulated in GelMA / HAp showed significant alkaline phosphatase staining after culture, indicating that the cells in the cartilaginous organoids had begun to differentiate into osteoblast cell lines. Figure 9 As can be seen, calcium nodules stained with alizarin red dye are visible from the contact surface between the organoid and the GelMA / HAp material into the organoid, indicating that osteoblasts differentiated from the cartilage organoid have the ability to secrete calcified matrix.

[0075] In summary, the cartilaginous organoids in this patent have the ability to undergo osteogenic formation in vitro under the induction of materials, and to combine with cryo-directed materials to form callus organoids with good mass transfer properties, thereby establishing a complete model of the endo-osteogenic process in vitro, which can be applied to fields such as drug screening and tissue repair.

Claims

1. A method for constructing in vitro callus organoids, characterized in that, Includes the following steps: Step 1. Collagen hydrogel presolution (1) Dissolve the freeze-dried collagen in hydrochloric acid to prepare a stock solution with a certain collagen concentration. (2) Under ice bath conditions, the cartilage induction culture medium and collagen solution were mixed by blowing and mixing, and then the solution was adjusted to pH=7. Finally, the collagen solution was diluted to a certain concentration. (3) The obtained collagen solution was placed in an ice bath for half an hour for prepolymerization. Step 2. Construction of cartilage organoids (1) Proliferate P2 generation mesenchymal stem cells to 60%-80% of the culture dish area, digest the cells with trypsin, and repeatedly rinse the culture dish with growth medium to collect the cells. Take a small amount of cell suspension and drop it onto a cell counting plate, count the number of cells under a microscope, and calculate the total number of collected cells. Discard the excess cell suspension according to the target number of organoid cells, and centrifuge to obtain pure mesenchymal stem cells. (2) Mix pure mesenchymal stem cells with the collagen hydrogel precursor solution after incubation in step 1, and resuspend them into a uniform cell-collagen hydrogel precursor solution mixture suspension. Then inject the mixture into a cylindrical mold and transfer it to a 37°C carbon dioxide constant temperature incubator for incubation until complete gelation. After that, transfer it to cartilage induction medium for culture. Step 3. Preparation of freeze-molding ink (1) Weigh out the gelatin and dissolve it in deionized water under a 50-degree water bath. After the gelatin is completely dissolved, add methacrylic anhydride (MA) in several portions to adjust the pH of the reaction system to around 9. (2) The GelMA aqueous solution after the reaction was freeze-dried into a white sponge. The freeze-dried sponge was soaked and washed, then redissolved and transferred to a dialysis bag for dialysis. The dialyzed solution was freeze-dried again to obtain the finished GelMA sponge. (3) The GelMA sponge was reconstituted in PBS phosphate buffer containing LAP photoinitiator to prepare a GelMA hydrogel precursor solution. (4) Add the inducible biomaterial to the GelMA hydrogel precursor solution according to a certain solid content ratio, disperse it evenly by ultrasonication, and obtain the GelMA / HAp precursor solution that can be used for photocrosslinking. Then add a certain amount of polysaccharide to prepare the freeze-molded ink. Step 4. Preparation of callus organoid precursors The aforementioned cartilaginous organoids were mixed with cryo-forming ink, with a specific proportion of organoids mounted on each scaffold. The mixture was then transferred to a room-temperature mold placed on a cryoplate for cryo-forming. After forming, the material on the cryoplate was irradiated with ultraviolet light to ensure complete cross-linking, resulting in a callus organoid precursor loaded with cartilaginous organoids for in vitro callus organoid formation. This callus organoid precursor was then cultured in vitro to form callus organoids.

2. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The collagen in step 1 is type I collagen.

3. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The hydrogel presol in step 1 can also be hyaluronic acid or decellularized extracellular matrix.

4. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The chondrogenic culture medium in step 2 contains vitamin C, dexamethasone, ITS liquid culture medium supplement, and TGF-β.

5. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: In step 3, the inducible biomaterial can be hydroxyapatite powder (HAp), tricalcium phosphate powder, or biphasic calcium phosphate powder.

6. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The polysaccharide in step 3 can be menotriose.

7. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The dimensions of the mold for measuring the bone defect to be cut in step 4 are approximately 2-8 mm in inner diameter and 2-10 mm in height.

8. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The number of cartilaginous organoids in step 4 is 10-25.

9. The method for constructing in vitro callus organoids as described in claim 1, characterized in that: The freezing temperature in step 4 is -20℃.

10. The method for constructing in vitro callus organoids as described in claim 1 and its application: The method for constructing in vitro callus organoids as described in any one of claims 1 to 9 provides the application of in vitro callus organoids in bone defect repair and drug screening.

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