Transparent-calcified bilayered cartilage organoid and preparation method and application thereof

Abstract

The present application relates to a kind of transparent-calcified double-layer cartilage organoids and its preparation method and application, including the transparent cartilage layer in upper layer and the calcified cartilage layer in lower layer, by ultraviolet light crosslinking transparent cartilage layer and calcified cartilage layer form double-layer cartilage organoids, transparent cartilage layer is obtained by using DNA-methacrylated silk fibroin hydrogel culture bone marrow mesenchymal stem cells, for promoting transparent cartilage-like matrix deposition;Calcified cartilage layer is obtained by using DNA-methacrylated alginate hydrogel loaded with calcium carbonate culture chondrocyte, for inducing chondrocyte hypertrophy and mineralized matrix formation.Compared with prior art, the transparent-calcified double-layer cartilage organoids formed by the present application forms the mechanical gradient similar to natural tissue, can better simulate the layered structure of natural bone-cartilage interface, can significantly improve the tissue filling, surface flatness, cartilage matrix deposition and interface integration effect in cartilage defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a transparent-calcified bilayer cartilage organoid, its preparation method and application. Background Technology

[0002] Articular cartilage damage is a crucial pathological basis for osteoarthritis and post-traumatic joint degeneration. Because articular cartilage lacks blood vessels, nerves, and lymphatic tissue, and has a limited number of chondrocytes with low metabolic activity, spontaneous repair after injury is usually difficult. Current clinical treatments include microfracture surgery, autologous chondrocyte transplantation, and osteochondral transplantation, but problems remain, such as poor quality of repaired tissue, fibrocartilage formation, donor site damage, limited donor availability, and insufficient long-term mechanical stability.

[0003] Natural articular cartilage is not a homogeneous tissue but rather possesses a highly layered osteochondral interface structure. Its outer layer is primarily composed of hyaline cartilage rich in COL II and Aggrecan, capable of withstanding joint loads and maintaining low-friction movement. The deeper layer contains a calcified cartilage layer, rich in hypertrophic chondrocytes and mineralized matrix, playing a crucial transitional role between hyaline cartilage and subchondral bone. This calcified cartilage layer not only enhances the mechanical anchoring between cartilage and subchondral bone but also regulates material exchange and tissue homeostasis at the cartilage-bone interface. However, most current cartilage tissue engineering strategies focus primarily on hyaline cartilage regeneration, neglecting the role of the calcified cartilage layer in osteochondral interface reconstruction. This leads to insufficient integration of the repaired tissue with the host tissue, limiting long-term stability.

[0004] In recent years, organoid technology has provided new research avenues for tissue regeneration. Organoids are three-dimensional micro-tissues formed by cells through self-organization or induction in vitro, capable of mimicking the structure and function of natural tissues to a certain extent. Compared to traditional cell suspensions or single scaffolds, organoids possess higher cell density, richer extracellular matrix, and a microenvironment more closely resembling that of natural tissues, thus showing promising application potential in the repair of tissues such as bone, nerves, intestines, and cartilage. Furthermore, numerous studies have demonstrated that hyaline cartilage organoids can promote cartilage-like matrix formation and improve the repair of cartilage defects. However, single hyaline cartilage organoids still struggle to fully mimic the layered structure of the natural bone-cartilage interface, particularly lacking the contribution of the calcified cartilage layer to interface integration and mechanical support. Therefore, developing cartilage organoids with a dual-layer structure of hyaline cartilage and calcified cartilage is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing cartilage tissue engineering materials, such as difficulty in simultaneously reconstructing the hyaline cartilage layer and the calcified cartilage layer, insufficient integration of repair tissue interfaces, and limited mechanical stability, thereby providing a hyaline-calcified bilayer cartilage organoid, its preparation method, and its application.

[0006] The objective of this invention can be achieved through the following technical solutions: One technical solution of the present invention is to provide a transparent-calcified bilayered cartilaginous organoid, comprising an upper layer of transparent cartilage and a lower layer of calcified cartilage, wherein the transparent cartilage layer and the calcified cartilage layer are cross-linked by ultraviolet light to form a bilayered cartilaginous organoid, wherein... The hyaline cartilage layer was constructed by culturing bone marrow mesenchymal stem cells in DNA-methacrylamide silk fibroin hydrogel, which was used to promote the deposition of hyaline cartilage-like matrix. The calcified cartilage layer was constructed by culturing chondrocytes in a DNA-methacrylated alginate hydrogel loaded with calcium carbonate, and was used to induce chondrocyte hypertrophy and mineralized matrix formation.

[0007] The second technical solution of the present invention is to provide a method for preparing a transparent-calcified bilayer cartilage organoid as described in one of the above technical solutions, comprising the following steps: S1. Prepare a DNA-methacrylamide alginate premix loaded with calcium carbonate and add chondrocytes to obtain a calcified cartilage layer printing precursor solution. S2. Prepare DNA-methacrylamide silk fibroin premix, add bone marrow mesenchymal stem cells to obtain hyaline cartilage layer printing precursor solution; S3. Using digital light processing bioprinting technology, the calcified cartilage layer printing precursor solution from step S1 and the hyaline cartilage layer printing precursor solution from step S2 are printed sequentially to obtain a double-layer structure with a calcified cartilage layer in the lower layer and a hyaline cartilage layer in the upper layer. S4. The bilayer structure obtained in step S3 is placed in chondrogenic induction medium and cultured to obtain a transparent-calcified bilayer cartilage organoid.

[0008] In some specific embodiments, in step S1, the DNA-methacrylated alginate premix loaded with calcium carbonate is prepared by the following steps: Y-type and L-type DNA single strands were dissolved in buffer solution and mixed to form a DNA supramolecular network. Methacrylamide alginate, photoinitiator and calcium carbonate particles were added and mixed thoroughly to obtain a DNA-methacrylamide alginate premix loaded with calcium carbonate.

[0009] In some specific embodiments, in step S1, the concentration of methacrylamide alginate in the calcium carbonate-loaded DNA-methacrylamide alginate premix is ​​5-15 wt%, the concentration of photoinitiator is 0.1-0.5 wt%, the concentration of calcium carbonate is 0.5-2 wt%, and the total concentration of single-stranded DNA is 100-1000 nmol / mL; wherein, The Y-type DNA single strand includes DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; the molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1; The L-type DNA single strand includes DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:1. The total molar ratio of the Y-type DNA single strand to the L-type DNA single strand is 1:1 to 1:2.

[0010] In this invention, the DNA single strands Y1, Y2, and Y3 each have three sticky ends, and the DNA single strands L1 and L2 each have sticky ends at both ends. The L-type DNA single strand is completely complementary to the sticky ends of the Y-type DNA single strand.

[0011] In some specific embodiments, in step S2, the DNA-methacrylamide silk fibroin premix is ​​prepared by the following steps: Y-type and L-type DNA single strands were dissolved in buffer solution and mixed to form a DNA supramolecular network. Methacrylated silk fibroin and a photoinitiator were added and mixed to obtain DNA-methacrylated silk fibroin premix.

[0012] In some specific embodiments, in step S2, the concentration of methacrylamide silk fibroin in the DNA-methacrylamide silk fibroin premix is ​​5-15 wt%, the concentration of photoinitiator is 0.1-0.5 wt%, and the total concentration of single-stranded DNA is 100-1000 nmol / mL; wherein, The Y-type DNA single strand includes DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; the molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1; The L-type DNA single strand includes DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:1. The total molar ratio of the Y-type DNA single strand to the L-type DNA single strand is 1:1 to 1:2.

[0013] In this invention, the DNA single strands Y1, Y2, and Y3 each have three sticky ends, and the DNA single strands L1 and L2 each have sticky ends at both ends. The L-type DNA single strand is completely complementary to the sticky ends of the Y-type DNA single strand.

[0014] In some specific embodiments, in step S1, the ratio of the calcium carbonate-loaded DNA-methacrylated alginate premix to chondrocytes is 1 mL: (1,000,000~30,000,000) cells; In step S2, the ratio of the DNA-methacrylamide silk fibroin premix to bone marrow mesenchymal stem cells is 1 mL: (1,000,000~30,000,000) cells.

[0015] In some specific implementations, the printing parameters for step S3 are: The ultraviolet light wavelength for ultraviolet crosslinking is 365 nm, and the light intensity is 3-20 mW / cm². 2 The photocrosslinking time is 10-60 s.

[0016] In some specific embodiments, in step S4, the chondrogenic culture medium consists of: DMEM high-glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / mL transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS cell culture additive and 1 μM ascorbic acid-2-phosphate.

[0017] In some specific implementations, step S4 involves a culture period of 2 to 4 weeks at a temperature of 37°C.

[0018] The third technical solution of the present invention is to provide an application of the transparent-calcified bilayer cartilage organoid as described in one of the above technical solutions, selected from one of the following applications: (1) Used to prepare cartilage tissue repair materials; (2) Used for drug screening; (3) Used for disease model construction.

[0019] Compared with the prior art, the present invention has the following advantages: (1) This invention uses DNA-methacrylated silk fibroin hydrogel to construct a hyaline cartilage layer, which can promote the differentiation of bone marrow mesenchymal stem cells into a stable hyaline cartilage phenotype. A calcified cartilage layer is constructed using DNA-methacrylated alginate hydrogel loaded with calcium carbonate, which can promote the differentiation of bone marrow mesenchymal stem cells into a stable hyaline cartilage phenotype. 2+ Release promotes chondrocyte hypertrophy and mineralized cartilage formation. Therefore, this invention constructs a bilayered cartilage organoid containing both a hyaline cartilage layer and a calcified cartilage layer, forming a mechanical gradient similar to natural tissue, which can better simulate the layered structure of the natural osteochondral interface.

[0020] (2) This invention achieves spatial assembly of transparent cartilage layer and calcified cartilage layer through DLP bioprinting, which can form biomimetic cartilage organoids with structural and mechanical gradients.

[0021] (3) The transparent-calcified bilayer cartilage organoids of the present invention can significantly improve tissue filling, surface smoothness, cartilage matrix deposition and interface integration in the repair of cartilage defects. Attached Figure Description

[0022] Figure 1 This is an actual image of a transparent, calcified, double-layered cartilaginous organoid.

[0023] Figure 2 A CT image of a transparent, calcified, double-layered cartilaginous organoid.

[0024] Figure 3 The surface roughness of transparent calcified bilayered cartilaginous organoids.

[0025] Figure 4 This image shows HE, Safranin-Fix Green, and Alcian Blue staining of the hyaline cartilage layer in a double-layered cartilaginous organoid.

[0026] Figure 5 Immunofluorescence staining of COLX, COL II, and ACAN proteins in the hyaline cartilage layer of a bilayered cartilaginous organoid.

[0027] Figure 6 HE, ARS, and ALP staining images of the calcified cartilage layer in a double-layered cartilaginous organoid.

[0028] Figure 7 Immunofluorescence staining of COL X, COL II, and OPN proteins in the hyaline cartilage layer of a bilayered cartilaginous organoid.

[0029] Figure 8 Macroscopic images of double-layered cartilage organoids used for cartilage repair at 3 and 6 weeks.

[0030] Figure 9Histological staining images of newly formed cartilage at 3 and 6 weeks after the use of bilayered cartilage organoids for cartilage repair.

[0031] Figure 10 Immunohistochemical staining images of newly formed cartilage at 3 and 6 weeks after the use of bilayered cartilage organoids for cartilage repair. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0034] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0035] Example 1: This embodiment provides a transparent-calcified bilayered cartilage organoid, the preparation method of which includes the following steps: (1) Preparation of bio-ink from calcified cartilage (1-1) Preparation of DNA-methacrylamide premixed solution loaded with calcium carbonate Y-type and L-type DNA single strands were dissolved in PBS buffer at pH 7.4 and mixed at 37 °C to form a DNA supramolecular network. Methacrylamide alginate, photoinitiator LAP, and calcium carbonate particles were then added and thoroughly mixed to obtain a calcium carbonate-loaded DNA-methacrylated alginate premix. The concentrations of methacrylamide alginate, LAP, and calcium carbonate were 10 wt%, 0.25 wt%, 1 wt%, and the total concentration of Y-type and L-type DNA single strands was 500 nmol / mL.

[0036] Y-type DNA single strands include DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3. The nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3. The molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1. L-type DNA single strands include DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4 and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:1. The total molar ratio of Y-type DNA single strands to L-type DNA single strands is 1:1. (1-2) Set the cell density to 1×10 7 Chondrocytes of approximately 100 cells / mL were added to a DNA-methacrylamide alginate premix loaded with calcium carbonate to obtain calcified cartilage layer bio-ink.

[0037] (2) Preparation of transparent cartilage layer bio-ink (2-1) Preparation of DNA-methacrylamide silk fibroin premix Y-type and L-type DNA single strands were dissolved in PBS buffer at pH 7.4 and mixed at 37 °C to form a DNA supramolecular network. Methacrylamide silk fibroin and photoinitiator LAP were then added and thoroughly mixed to obtain the DNA-methacrylamide silk fibroin precursor solution. The concentration of methacrylamide silk fibroin was 10 wt%, the concentration of LAP was 0.25 wt%, and the total concentration of Y-type and L-type DNA single strands was 500 nmol / mL.

[0038] Y-type DNA single strands include DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3. The nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3. The molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1. L-type DNA single strands include DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4 and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:1. The total molar ratio of Y-type DNA single strands to L-type DNA single strands is 1:1. (2-2) Set the cell density to 1×10 7 Bone marrow mesenchymal stem cells of approximately 100 cells / mL were added to a DNA-methacrylamide silk fibroin premix to obtain a hyaline cartilage layer bio-ink.

[0039] (3) Preparation of transparent-calcified bilayered cartilage organoids (3-1) Using digital light processing bioprinting technology, the bio-ink of calcified cartilage in step (1) and the bio-ink of transparent cartilage layer in step (2) are printed sequentially to obtain a double-layer connected structure with the calcified cartilage layer in the lower layer and the transparent cartilage layer in the upper layer. The printing parameters are: the ultraviolet wavelength of the ultraviolet light during ultraviolet light crosslinking is 365 nm, and the light intensity is 12 mW / cm². 2 The exposure time is 13 seconds, the number of base layers is 2, and the exposure time of the base layer is 15 seconds.

[0040] (3-2) The bilayered connective structures were cultured in chondrogenic induction medium at 37 ℃, with the medium changed every 2 days. Macroscopic photographs were taken of the transparent calcified bilayered cartilaginous organoids at different stages of culture (1 week, 2 weeks, 3 weeks, and 4 weeks). Figure 1 As shown.

[0041] The chondrogenic culture medium consisted of DMEM high-glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / mL transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS cell culture additive, and 1 μM ascorbic acid-2-phosphate. The 1% penicillin-streptomycin consisted of 100 U / mL penicillin and 100 μg / mL streptomycin. The ITS cell culture additive was from Cyagen Biosciences, catalog number ITSS-10201.

[0042] The prepared transparent-calcified bilayered cartilage organoids were characterized as follows: (1) Clear-calcified bilayered cartilaginous organoids cultured for 1, 2, 3, and 4 weeks were collected and subjected to CT and surface roughness imaging. Micro-CT was used to examine the calcification distribution of the clear-calcified bilayered cartilaginous organoids at different time points. The CT results showed the following: Figure 2 As shown, a clear stratified interface is formed between the hyaline cartilage layer and the calcified cartilage layer. With increasing culture time, the density within the calcified cartilage layer gradually increases, while the hyaline cartilage layer maintains a relatively uniform low density. This indicates that the stratified design can maintain the unique microenvironment of each layer, which is beneficial for the differentiation of different cell types. The result that the upper layer of the hyaline-calcified bilayer organoid has no calcification while the lower layer gradually increases in calcification proves that regardless of the length of culture time, the structure of the hyaline-calcified bilayer organoid already possesses the characteristics of natural cartilage tissue.

[0043] (2) The surface roughness of the transparent-calcified bilayer organoid was characterized by ULTRA DOF 3D DIGITAL MICROSCOPE. The surface roughness of cartilage affects its friction properties.

[0044] The results are as follows Figure 3 As shown, after one week of culture, the surface height of the transparent-calcified bilayer organoids is uniformly distributed with a small range of variation. As the culture time increases, the surface roughness increases, forming a microscopic heterogeneous structure, which provides a basis for subsequent tissue functionalization.

[0045] (3) The hyaline cartilage layer in the hyaline-calcified bilayer organoids cultured for 1, 2, 3 and 4 weeks was stained with HE, Safranin O Fast Green (S / F) and Alcian Blue (AB).

[0046] The clear-calcified bilayer organoids were gently washed three times with PBS to remove residual culture medium. Then, 4% paraformaldehyde was added to fix the organoids for 24 h. After fixation, the organoids were thoroughly washed with PBS. Subsequently, the organoids were subjected to a gradient (70%, 80%, 90%, 100%) ethanol dehydration, xylene clearing, and paraffin embedding. The clear cartilage layer of the organoid samples was cut into continuous sections approximately 5 μm thick using a paraffin microtome, attached to glass slides, and baked at 60℃ for 1-2 h. Before staining, the clear cartilage layer sections were dewaxed with xylene and rehydrated sequentially through a gradient (70%, 80%, 90%, 100%) ethanol to distilled water.

[0047] HE staining procedure: After rehydration, hyaline cartilage sections are stained in hematoxylin solution for 3-5 min, rinsed with running water, and then subjected to differentiation and blueing treatment. Subsequently, eosin solution is added for staining for 1-3 min, followed by rapid rinsing with distilled water. After staining, the sections are dehydrated with a gradient of 70%, 80%, 90%, and 100% ethanol, cleared with xylene, and mounted with neutral resin.

[0048] The procedure for safranin-bone green staining is as follows: After rehydration, hyaline cartilage sections are first stained with Weigert iron hematoxylin for 5-10 min, then rinsed with running water. Next, Fast Green solution is added for staining for 3-5 min, followed by rapid differentiation with 1% acetic acid for 10-15 s to remove background staining. Afterward, Safranin O solution is added for staining for 5-10 min to detect glycosaminoglycan deposition. Following staining, the sections are rapidly dehydrated with a gradient of 70%, 80%, 90%, and 100% ethanol, cleared with xylene, and mounted with neutral resin.

[0049] The procedure for Alcian Blue staining is as follows: After rehydration, the hyaline cartilage sections are equilibrated in 3% acetic acid solution for 3 min, followed by staining with 1% Alcian Blue solution for 20-30 min. After staining, the sections are gently rinsed with 3% acetic acid to remove non-specific background, and then washed with distilled water. Subsequently, the sections are dehydrated by gradient (70%, 80%, 90%, 100%) ethanol, cleared with xylene, and mounted with neutral resin.

[0050] All stained sections were observed and photographed under an optical microscope, and the results are as follows: Figure 4 As shown in the figure. HE staining results showed that with the extension of culture time, the distribution of chondrocytes differentiated from bone marrow mesenchymal stem cells in the hyaline cartilage layer organoids gradually became more uniform, and the tissue structure gradually became denser. At 1 week of culture, the distribution of chondrocytes was relatively sparse; by 4 weeks, the number of chondrocytes increased significantly, forming a continuous and uniform tissue-like structure, indicating that chondrocytes can survive and proliferate continuously inside the hydrogel, while promoting tissue remodeling and maturation. Safranin-Fix-Green staining results showed that only a weak red staining signal was observed in the early stage of culture, but the staining intensity gradually increased with the extension of culture time, especially at 3 and 4 weeks, when obvious red matrix deposition appeared. This result indicates that glycosaminoglycans continue to accumulate in the hyaline cartilage layer, and the extracellular matrix of chondrocytes continues to form. Further evaluation of acidic mucopolysaccharide deposition by alicin blue staining showed that with the increase of culture time, the blue staining area gradually expanded and the staining intensity increased, indicating that cartilage-specific matrix continued to be secreted and accumulated, further confirming that the hyaline-calcified bilayer cartilage organoids gradually formed mature hyaline cartilage-like tissue during the culture process.

[0051] (4) Immunological staining of the hyaline cartilage layer in hyaline-calcified bilayer organoids cultured for 1, 2, 3 and 4 weeks.

[0052] First, the hyaline cartilage layer of the hyaline-calcified bilayer organoid was fixed in 4% paraformaldehyde solution, then permeated with 0.3% Triton X-100 solution for 10 minutes, blocked with 10% goat serum for 2 hours, and incubated overnight with primary antibodies (Col II, ACAN, and COLX). Finally, it was incubated with secondary antibody (1:5000) and TRITC Phalloidin at room temperature for 1 hour for DAPI staining, and finally images were acquired using an inverted fluorescence microscope.

[0053] The results are as follows Figure 5 As shown, after 3 weeks of culture, the hyaline cartilage layer exhibits higher expression of ACAN and COL II in the cartilage matrix, while lower expression of the cartilage hypertrophy protein COLX. However, by 4 weeks, COLX expression begins to increase, while ACAN and COL II expression decreases. These results indicate that the hyaline cartilage layer reaches optimal maturity at 3 weeks.

[0054] (5) The calcified cartilage layer in the transparent calcified bilayer organoids cultured for 1, 2, 3 and 4 weeks was stained with HE, Alizarin Red (ARS) and Alkaline Phosphatase (ALP).

[0055] First, the clear-calcified bilayer organoids were gently washed three times with PBS to remove residual culture medium from the surface. Then, 4% paraformaldehyde was added to the clear-calcified bilayer organoids for fixation for 24 h. After fixation, the clear-calcified bilayer organoids were thoroughly washed with PBS. Subsequently, the clear-calcified bilayer organoids were sequentially dehydrated with a gradient (70%, 80%, 90%, 100%) of ethanol, cleared with xylene, and embedded in paraffin. Using a paraffin microtome, the calcified cartilage layer of the clear-calcified bilayer organoid samples was cut into continuous sections approximately 5 μm thick, attached to glass slides, and baked at 60℃ for 1-2 h. Before staining, the calcified cartilage layer sections were dewaxed with xylene and rehydrated sequentially with a gradient (70%, 80%, 90%, 100%) of ethanol to distilled water.

[0056] HE staining: Rehydrated calcified cartilage sections were stained in hematoxylin for 3-5 min, rinsed with running water, and then subjected to differentiation and blueing treatment. Eosin staining was then added for 1-3 min, followed by rapid rinsing with distilled water. After staining, the sections were dehydrated with a gradient of 70%, 80%, 90%, and 100% ethanol, cleared with xylene, and mounted with neutral resin.

[0057] ARS staining: After rehydration, calcified cartilage sections were stained with 2% Alizarin Red S staining solution, the pH was adjusted to 4.1-4.3, and stained at room temperature in the dark for 20-30 minutes. After staining, the sections were gently rinsed repeatedly with deionized water until the background color was basically removed. The sections were then dehydrated with a gradient of 70%, 80%, 90%, and 100% ethanol, cleared with xylene, and mounted with neutral resin.

[0058] ALP staining: Prepare the BCIP / NBT working solution according to the ALP staining kit (Beyotime, P0321S) instructions and add it to the calcified cartilage section, ensuring the section is completely covered by the staining solution. Incubate at room temperature in the dark for 15-30 min, observing the color development during this time. Once a clear blue-purple positive stain appears on the calcified cartilage layer, discard the staining solution and rinse thoroughly with deionized water to terminate the reaction.

[0059] After all staining was completed, the samples were observed and photographed under an optical microscope.

[0060] The results are as follows Figure 6As shown, HE staining results revealed that the calcified cartilage layer maintained a relatively intact circular structure at different culture times, with hypertrophic chondrocytes differentiated from chondrocytes evenly distributed within the hydrogel. With prolonged culture time, the distribution of hypertrophic chondrocytes and matrix in the organoid periphery gradually increased, indicating the gradual formation of tissue-like structures. ARS staining results showed that mineralization deposition within the calcified cartilage layer gradually increased with culture time. Only weak staining was visible at 1 week, the staining area expanded after 2 weeks, and the red mineralization signal significantly increased at 3 and 4 weeks, indicating that the DNA-methacrylamide alginate hydrogel loaded with calcium carbonate could continuously induce calcium salt deposition. ALP staining results further showed that the ALP-positive area gradually increased with prolonged culture time, with the strongest staining at 4 weeks, indicating a gradual increase in osteogenic / mineralization activity related to calcified cartilage.

[0061] (6) Immunological staining of the calcified cartilage layer in transparent-calcified bilayer organoids cultured for 1, 2, 3 and 4 weeks.

[0062] First, the calcified cartilage layer in the clear-calcified bilayer organoid was fixed in 4% paraformaldehyde solution, then permeated with 0.3% Triton X-100 solution for 10 minutes, blocked with 10% goat serum for 2 hours, and incubated overnight with primary antibodies (Col II, OPN and COL X). Finally, it was incubated with secondary antibody (1:5000) and TRITC Phalloidin at room temperature for 1 hour. After DAPI staining, images were acquired using an inverted fluorescence microscope.

[0063] The results are as follows Figure 7 As shown, the expression of chondroitin COL II in the calcified cartilage layer decreases with increasing culture time, while the expression of hypertrophic protein COLX increases with increasing culture time. However, the osteogenic matrix expression is low in the first 3 weeks but increases in the 4th week. These results indicate that at 3 weeks, the expression of COLX in the calcified cartilage layer is enhanced while the expression of OPN remains low, suggesting that a stable and moderately mature calcified cartilage phenotype can be obtained at this time point.

[0064] Example 2 The transparent-calcified bilayered cartilage organoids prepared in Example 1 were used for cartilage defect repair.

[0065] Establish a rat cartilage defect model: SD male rats aged 7-8 weeks (weighing about 250 g) were taken, and after general anesthesia, the joint capsule was cut to expose the femoral trochlea. A cartilage defect with a diameter of 2 mm and a depth of 1.5 mm was constructed in the midline using a punch.

[0066] The groups are as follows: BCO group: In a rat cartilage defect model, transparent-calcified bilayer organoids cultured for 3 weeks in Example 1 were transplanted in situ into the cartilage defects and sutured layer by layer. HCO group: Transplantation of transparent cartilage organoids into cartilage defects in rat cartilage defect model in situ, followed by suturing layer by layer; Control group: No repair treatment was performed on the rat cartilage defect model; A separate Sham group was set up: sham surgery was performed, which did not cause any cartilage damage when constructing a rat cartilage defect model.

[0067] Rats were fed normally after surgery, and sacrificed 6 weeks later to analyze the repair effect.

[0068] The results are as follows Figure 8 As shown, the femurs were observed by the naked eye at weeks 3 and 6 of the animal experiments. Compared with the control group and the single-layer hyaline cartilage organoid group, the hyaline-calcified double-layer cartilage organoid group showed almost no cartilage defects and was closest to the sham surgery group. The results show that the hyaline-calcified double-layer cartilage organoid group can promote cartilage regeneration and repair, and the therapeutic effect is better than that of the single-layer hyaline cartilage organoid.

[0069] The newly formed cartilage was fixed in 4% paraformaldehyde for 48 hours, followed by decalcification, embedding, and sectioning. Histological analysis was performed using HE and Safranin-Fixed Green (SF / O) staining, and the results are as follows: Figure 9 As shown, HE and SF / O staining demonstrated that the hyaline-calcified bilayered cartilage organoids promoted cartilage regeneration and exhibited a highly ordered chondrocyte arrangement and intact tissue structure similar to natural cartilage. Chondrocytes were evenly distributed in the extracellular matrix without significant inflammatory cell infiltration. In contrast, the control group and the monolayered hyaline cartilage organoid group showed fibrotic repair characteristics (disordered cell arrangement and loose matrix) at 3 weeks, and immature tissue areas and matrix heterogeneity were still visible at 6 weeks.

[0070] Immunohistochemical staining of newly formed cartilage: Newly formed cartilage tissue sections were incubated overnight at 4°C with anti-ACAN or anti-Col II antibodies. After incubation with secondary antibodies at room temperature, the sections were stained with 3,3′-diaminobenzidine for visualization. All tissue sections were photographed under a microscope, and the results are as follows: Figure 10 The ACAN and Col II immunohistochemical staining shown also revealed the significant role of the hyaline-calcified bilayer cartilage organoids in cartilage formation. At 3 weeks, it already showed a high density of Col II positive signal (brown), and as the repair time increased (to 6 weeks), its distribution uniformity and staining intensity were significantly close to those of natural cartilage, indicating the orderly reconstruction of the collagen network of the extracellular matrix.

[0071] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A transparent-calcified bilayered cartilaginous organoid, characterized in that, It comprises an upper layer of hyaline cartilage and a lower layer of calcified cartilage. The hyaline cartilage and calcified cartilage are cross-linked using ultraviolet light to form a bilayered cartilaginous organoid. The hyaline cartilage layer was constructed by culturing bone marrow mesenchymal stem cells in DNA-methacrylamide silk fibroin hydrogel, which was used to promote the deposition of hyaline cartilage-like matrix. The calcified cartilage layer was constructed by culturing chondrocytes in a DNA-methacrylated alginate hydrogel loaded with calcium carbonate, and was used to induce chondrocyte hypertrophy and mineralized matrix formation.

2. A method for preparing a transparent-calcified bilayered cartilage organoid as described in claim 1, characterized in that, Includes the following steps: S1. Prepare a DNA-methacrylamide alginate premix loaded with calcium carbonate and add chondrocytes to obtain a calcified cartilage layer printing precursor solution. S2. Prepare DNA-methacrylamide silk fibroin premix, add bone marrow mesenchymal stem cells to obtain hyaline cartilage layer printing precursor solution; S3. Using digital light processing bioprinting technology, the calcified cartilage layer printing precursor solution from step S1 and the hyaline cartilage layer printing precursor solution from step S2 are printed sequentially to obtain a double-layer structure with a calcified cartilage layer in the lower layer and a hyaline cartilage layer in the upper layer. S4. The bilayer structure obtained in step S3 is placed in chondrogenic induction medium and cultured to obtain a transparent-calcified bilayer cartilage organoid.

3. The method for preparing transparent-calcified bilayered cartilaginous organoids according to claim 2, characterized in that, In step S1, the DNA-methacrylated alginate premix loaded with calcium carbonate is prepared by the following steps: Y-type and L-type DNA single strands were dissolved in buffer solution and mixed to form a DNA supramolecular network. Methacrylamide alginate, photoinitiator and calcium carbonate particles were added and mixed thoroughly to obtain a DNA-methacrylamide alginate premix loaded with calcium carbonate.

4. The method for preparing a transparent-calcified bilayered cartilaginous organoid according to claim 3, characterized in that, The concentration of methacrylamide alginate in the calcium carbonate-loaded DNA-methacrylamide alginate premix is ​​5-15 wt%, the concentration of photoinitiator is 0.1-0.5 wt%, the concentration of calcium carbonate is 0.5-2 wt%, and the total concentration of Y-type and L-type DNA single strands is 100-1000 nmol / mL; wherein, The Y-type DNA single strand includes DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; the molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1; The L-type DNA single strand includes DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:

1. The total molar ratio of the Y-type DNA single strand to the L-type DNA single strand is 1:1 to 1:

2.

5. The method for preparing a transparent-calcified bilayered cartilaginous organoid according to claim 2, characterized in that, In step S2, the DNA-methacrylamide silk fibroin premix is ​​prepared by the following steps: Y-type and L-type DNA single strands were dissolved in buffer solution and mixed to form a DNA supramolecular network. Methacrylated silk fibroin and a photoinitiator were added and mixed to obtain DNA-methacrylated silk fibroin premix.

6. The method for preparing a transparent-calcified bilayered cartilaginous organoid according to claim 5, characterized in that, The concentration of methacrylamide silk fibroin in the DNA-methacrylamide silk fibroin premix is ​​5-15 wt%, the concentration of photoinitiator is 0.1-0.5 wt%, and the total concentration of Y-type DNA single strands and L-type DNA single strands is 100-1000 nmol / mL; wherein, The Y-type DNA single strand includes DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; the molar ratio of DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3 is 1:1:1; The L-type DNA single strand includes DNA single strand L1 and DNA single strand L2, wherein the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand L1 to DNA single strand L2 is 1:

1. The total molar ratio of the Y-type DNA single strand to the L-type DNA single strand is 1:1 to 1:

2.

7. The method for preparing transparent-calcified bilayered cartilaginous organoids according to claim 2, characterized in that, In step S1, the ratio of the calcium carbonate-loaded DNA-methacrylated alginate premix to chondrocytes is 1 mL: (1,000,000~30,000,000) cells. In step S2, the ratio of the DNA-methacrylamide silk fibroin premix to bone marrow mesenchymal stem cells is 1 mL: (1,000,000~30,000,000) cells.

8. The method for preparing a transparent-calcified bilayered cartilaginous organoid according to claim 2, characterized in that, Step S3, the printing parameters are: The ultraviolet light wavelength for ultraviolet crosslinking is 365 nm, and the light intensity is 3-20 mW / cm². 2 The photocrosslinking time is 10-60s.

9. The method for preparing a transparent-calcified bilayered cartilaginous organoid according to claim 2, characterized in that, In step S4, the chondrogenic culture medium consists of: DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / mL transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS cell culture additive and 1 μM ascorbic acid-2-phosphate; The incubation period is 2 to 4 weeks, and the temperature is 37 ℃.

10. An application of the transparent-calcified bilayered cartilaginous organoid as described in claim 1, characterized in that, Choose from one of the following applications: (1) Used to prepare cartilage tissue repair materials; (2) Used for drug screening; (3) Used for disease model construction.

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