Preparation method of acellular tissue-engineered double-network interpenetrating cartilage matrix graft

Abstract

The present invention relates to a preparation method of a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft in the field of interface repair biomaterials. Cartilage cells and gelatin microspheres are co-resuspended in a photocrosslinkable hydrogel monomer solution and crosslinked by ultraviolet light irradiation to form a crosslinked hydrogel, thereby forming a three-dimensional cell culture construct; the three-dimensional cell culture construct is cultured at a constant temperature, and a microporous structure is formed inside the hydrogel. The cartilage cells grow and proliferate inside the gel and secrete a matrix with cartilage characteristics to obtain a tissue-engineered double-network interpenetrating cartilage matrix graft; then, through multi-stage decellularization treatment, a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft is prepared. The histological characteristics of the decellularized matrix graft in the present invention macroscopically have a double-interpenetrating network structure of a photocrosslinkable hydrogel and an extracellular matrix secreted by cartilage cells, and microscopically have clear cartilage lacuna structures and the expression of type I collagen and type II collagen proteins, and have applications in interface tissue repair.

Description

Technical Field

[0001] The present invention belongs to the field of interfacial repair biomaterials, and particularly relates to a method for preparing a decellularized tissue-engineered dual-network interpenetrating cartilage matrix graft. Background Art

[0002] The extracellular matrix (ECM) is mainly a hydrophilic and porous three-dimensional matrix composed of collagen fibers and proteoglycans, etc., and is the main matrix component of biomaterials. The ECM not only provides a structural scaffold for living tissues, but also has an important influence on the biochemical and biomechanical signals of processes such as cell growth, proliferation, differentiation, morphogenesis, and homeostasis. Natural polymer hydrogels, derived from biological tissues or generated by microorganisms, have good biocompatibility, can simulate multiple aspects of the natural microenvironment, and are often used as scaffold materials for tissue regeneration to load cells and growth factors. Interfacial tissues exist at the junctions of soft tissues (tendons, cartilage, ligaments) and hard tissues (bone) and are ubiquitous throughout the musculoskeletal system.

[0003] In the prior art, between soft tissues and hard tissues, due to the complex gradient structure of matrix components, tissue structure arrangements, and cell types, and also due to the function of effectively transmitting mechanical stress between interfacial tissues to maintain interfacial homeostasis, it is extremely easy to cause interfacial damage, and it is difficult to repair after damage. Between such structures with large differences in soft and hard tissues, there is often a lack of biomaterial designs that can meet the unique functional requirements of interfacial tissues, and a lack of active grafts with multi-component gradient structures for guidance, making interfacial tissue repair one of the greatest challenges in the field of regenerative repair. Therefore, there is an urgent need to develop a multi-component biomimetic active graft using tissue engineering strategies to improve interfacial tissue repair. Summary of the Invention

[0004] The present invention aims at the problem that it is difficult to synchronously regenerate the multi-component extracellular matrix gradient structure in interfacial tissue repair in the prior art, and provides a method for preparing a decellularized tissue-engineered dual-network interpenetrating cartilage matrix graft.

[0005] The object of the present invention is achieved as follows. A method for preparing a decellularized tissue-engineered dual-network interpenetrating cartilage matrix graft includes the following steps:

[0006] (1) Construct a three-dimensional cell culture system: Mix a photo-crosslinkable hydrogel monomer solution with chondrocytes and gelatin microspheres evenly to form a hydrogel solution encapsulating cells and gelatin microspheres, inject it into a porous silica gel plate mold with a pore size of 2 - 10 mm and a pore depth of 2 - 6 mm, and obtain a crosslinked hydrogel by ultraviolet light irradiation crosslinking to form a three-dimensional cell culture system;

[0007] (2) Preparation of tissue-engineered double-network interpenetrating cartilage matrix graft: Add the cross-linked hydrogel prepared in step (1) to cell culture medium, place it in an incubator at 37 °C for culture for 2 - 6 weeks to form a cartilage matrix graft with interpenetrating extracellular matrix network secreted by hydrogel and chondrocytes, namely the tissue-engineered double-network interpenetrating cartilage matrix graft;

[0008] (3) Preparation of acellular tissue-engineered double-network interpenetrating cartilage matrix graft: Subject the tissue-engineered double-network interpenetrating cartilage matrix graft prepared in step (2) to multiple cycles of freeze-thaw treatment first, and then perform acellular treatment with cell eluent to finally obtain the acellular tissue-engineered double-network interpenetrating cartilage matrix graft.

[0009] The present invention takes advantage of the easy controllability of forming and the ability to support cell growth of natural hydrogels. Using natural hydrogels as scaffold materials, a three-dimensional cell culture system in vitro is constructed, and a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft is prepared. In the present invention, chondrocytes and gelatin microspheres are co-resuspended in a photo-crosslinkable hydrogel monomer solution. After ultraviolet light irradiation, free radical-initiated polymerization crosslinks to form a crosslinked hydrogel, and the chondrocytes and gelatin microsphere porogens are uniformly encapsulated in the gelatin-based photo-crosslinkable hydrogel, thus constructing a three-dimensional cell culture system. The three-dimensional cell culture system is incubated at a constant temperature. The gelatin microspheres dissolve and elute at a temperature of 37°C, forming a microporous structure inside the hydrogel. The chondrocytes grow and proliferate inside the gel and secrete matrix with cartilage characteristics. The regenerated matrix deposits on the hydrogel to form a second network structure. Therefore, macroscopically, a hydrogel and extracellular matrix interpenetrating network matrix system secreted by chondrocytes is formed, that is, a tissue-engineered double-network interpenetrating cartilage matrix graft is obtained. The tissue-engineered double-network interpenetrating cartilage matrix graft is subjected to multi-stage decellularization treatment to remove cell-based immunogenic substances, and a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft is constructed, still well retaining the internal structure and mechanical properties of the graft. In the present invention, hollow gelatin microspheres are used as porogens in the hydrogel, making the inside of the hydrogel have multiple interconnected micron-sized cavity structures. The microporous space is not only conducive to the diffusion and exchange of nutrients, metabolic wastes, and electrolytes, but also suitable for cell infiltration, migration, and matrix secretion. The decellularization method is mild and effective, not only removing most of the immunogenic cell components in the tissue, but also maintaining the histological structure in the original tissue, and largely retaining the tissue matrix components, providing a basis for realizing the interfacial tissue repair of multiple components. The decellularized tissue-engineered double-network interpenetrating cartilage matrix graft prepared by the present invention has a double interpenetrating network structure of a photo-crosslinked gel and an extracellular matrix secreted by chondrocytes. Macroscopically, it has a double interpenetrating network structure of a photo-crosslinked gel and an extracellular matrix secreted by chondrocytes. Microscopically, it has clear cartilage lacuna structures and the expression of type I and type II collagen proteins, and has potential applications in interfacial tissue repair. The decellularized tissue-engineered double-network interpenetrating cartilage matrix graft prepared by the present invention has good biosafety and is not easily degraded, which is beneficial for storage at room temperature and long-distance transportation.

[0010] Further, step (3) specifically includes the following sub-steps:

[0011] (3.1) Freeze the tissue-engineered double-network interpenetrating cartilage matrix graft prepared in step (1) at -80°C for 3 hours, then thaw at room temperature for 4 - 6 hours, and repeat the freeze-thaw cycle 3 - 5 times in sequence to obtain a pre-product;

[0012] (3.2) The pre-product obtained in step (3.1) is successively subjected to decellularization treatment with the following cell eluents: Tris-HCl solution, Triton X-100 solution, phosphate buffer solution (PBS), NaOH solution, PBS solution, nuclease solution composed of deoxyribonuclease and ribonuclease, and PBS solution. The elution conditions of the cell eluents are as follows: the temperature is 37°C, the oscillation speed is 50 - 200 rpm / min, PBS is eluted 3 - 5 times, and the elution time for each stage of cell eluent is 3 - 5 hours, finally obtaining a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft.

[0013] Further, in step (2), the culture conditions are as follows: the temperature is 37°C, the CO2 concentration is 5%, the relative humidity is 95%, and the horizontal shaker speed is 20 - 60 rpm / min.

[0014] Further, in step (1), the photocrosslinkable hydrogel monomer is one or more of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated chondroitin sulfate, and methacrylated chitosan.

[0015] Further, in step (1), the chondrocytes are articular chondrocytes derived from the knee joints of 4 - 8-month-old pigs, the number of chondrocytes is 0.5 - 2×10 7 cells / mL of the hydrogel solution, the amount of gelatin microspheres is 0.1 - 0.4 g / mL of the hydrogel solution in wet weight, and the gelatin microspheres are hollow gelatin microspheres and solid gelatin microspheres with a particle size of 50 - 300 μm.

[0016] Further, in step (1), the ultraviolet wavelength is 250 nm - 450 nm, the temperature is 15 - 37°C, and the light irradiation time is 3 - 8 minutes.

[0017] Further, in step (1), the photocrosslinkable hydrogel monomer solution contains an initiator, and the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone or lithium phenyl-2,4,6-trimethylbenzoylphosphinate, with a concentration of 0.01% - 0.5%.

[0018] Further, in step (2), the cell culture medium contains 10.03 g / L of DMEM powder, 2.775 g / L of sodium bicarbonate, 15% - 20% fetal bovine serum, 1% penicillin-streptomycin, 1 mM HEPES, 1 mM sodium pyruvate, 0.05 g / L of vitamin C, and 0.046 g / L of proline.

[0019] Further, in step (3), the PBS solution contains 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, with a pH value of 7.3 ± 0.1. The Tris-HCl solution contains 5 - 15 mM Tris-HCl and 0.05% - 0.2% disodium ethylenediaminetetraacetate (w / v). The Triton X-100 solution is a Tris-HCl solution containing 0.5% - 3% Triton X-100. The NaOH solution is a 0.1% - 0.8% solution. The nuclease solution is a solution containing 100 μg / mL deoxyribonuclease and 10 μg / mL ribonuclease. Description of the Drawings

[0020] Figure 1 Schematic diagram of the construction method of the acellular tissue-engineered double-network interpenetrating cartilage matrix graft.

[0021] Figure 2 Pictures of dry and wet gelatin microspheres.

[0022] Figure 3 Pictures of chondrocytes under an optical microscope.

[0023] Figure 4 Scanning electron microscope images of gelMA hydrogel, gelMA microporous hydrogel, and gelMA microporous hydrogel loaded with chondrocytes.

[0024] Figure 5 Histological staining images of gelMA hydrogel, gelMA microporous hydrogel, and gelMA microporous hydrogel loaded with chondrocytes.

[0025] Figure 6 Diagrams showing the mechanical properties of gelMA hydrogel, gelMA microporous hydrogel, and gelMA microporous hydrogel loaded with chondrocytes.

[0026] Figure 7 Immunofluorescence staining images of the tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) and the acellular tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN).

[0027] Figure 8 Diagrams showing the mechanical properties of the tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) and the acellular tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN).

[0028] Figure 9 Biocompatibility images of the acellular tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN). Detailed implementation manners

[0029] The present invention will be further analyzed, described and compared through specific examples and comparative examples below.

[0030] Example 1

[0031] All hydrogel precursor solutions were prepared in PBS solution. 1×10 7 chondrocytes were resuspended in 1 mL of 20% (w / v) gelMA solution containing 0.5% Irgacure 2959, and then mixed with 0.3 g of gelatin microspheres. 100 μL of the mixed solution was dropped into a cylindrical mold (diameter 6 mm, height 2.8 mm), and crosslinked by ultraviolet irradiation for 5 minutes (365 nm, 30 mW / cm 2 ²). The mold was removed, and the hydrogel construct was transferred to a culture plate. Three-dimensional cell culture medium was added, and the conditions of the incubator were a temperature of 37 °C, 5% CO₂, a relative humidity of 95%, and a horizontal shaker speed of 40 rpm / min. After culturing for 28 days, a tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) was obtained.

[0032] Each sample of gelMA PTCC was placed in a 2 mL tube and subjected to 3 cycles of freeze-thawing, freezing at -80 °C for 3 hours and then thawing at room temperature for 4 hours, which was one cycle. Then, in a 37 °C constant temperature shaker, the rotation speed was set at 120 rpm / min, and it was washed with 10 mM Tris-HCl solution (pH 8.0) for 24 h; washed with 1% Triton X-100 solution for 48 h at 37 °C; washed 3 times with PBS, 3 h each time at 37 °C; washed in 0.4% NaOH solution for 45 min at 4 °C; washed 3 times with PBS, 3 h each time at 37 °C; digested with a nuclease solution composed of deoxyribonuclease and ribonuclease (0.5 mg / mL deoxyribonuclease and 0.05 mg / mL ribonuclease) for 3 h at 37 °C; washed 3 times with PBS, 3 h each time at 37 °C. Until the decellularization process was completed, a graft with an interpenetrating network structure of gelMA hydrogel and extracellular matrix secreted by chondrocytes was obtained, which was called a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN). The samples were placed in PBS solution and stored in a 4 °C refrigerator.

[0033] Example 2

[0034] All hydrogel precursor solutions were prepared in PBS solution. 1×10 7Chondrocytes were resuspended in 1 mL of 15% (w / v) gelMA solution containing 0.5% Irgacure 2959, and then mixed with 0.3 g of gelatin microspheres. 100 μL of the mixed solution was dropped into a cylindrical mold (diameter 6 mm, height 2.8 mm), and crosslinked by ultraviolet irradiation for 5 minutes (365 nm, 20 mW / cm 2 ). The mold was removed, and the hydrogel construct was transferred to a culture plate. Three-dimensional cell culture medium was added, and the culture conditions were a temperature of 37 °C, 5% CO2, a relative humidity of 95%, and a horizontal shaker speed of 40 rpm / min. After 28 days of culture, a tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) was obtained.

[0035] Each sample of gelMA PTCC was placed in a 2 mL tube and subjected to 3 freeze-thaw cycles, freezing at -80 °C for 3 hours and then thawing at room temperature for 4 hours, which was one cycle. Then, in a 37 °C constant temperature shaking incubator, the rotation speed was set at 120 rpm / min, and it was washed with 10 mM Tris-HCl solution (pH 8.0) for 24 h; washed in 1% Triton X-100 solution for 48 h at 37 °C; washed 3 times with PBS for 3 h each time at 37 °C; washed in 0.4% NaOH solution for 45 min at 4 °C; washed 3 times with PBS for 3 h each time at 37 °C; digested with a nuclease solution composed of deoxyribonuclease and ribonuclease (0.5 mg / mL deoxyribonuclease and 0.05 mg / mL ribonuclease) for 3 h at 37 °C; washed 3 times with PBS for 3 h each time at 37 °C. Until the decellularization process was completed, a graft with an interpenetrating network structure of gelMA hydrogel and the extracellular matrix secreted by chondrocytes was obtained, which was called a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN). The samples were placed in PBS solution and stored in a 4 °C refrigerator.

[0036] Comparative Example 1

[0037] All hydrogel precursor solutions were prepared in PBS solution. 1×10 7 chondrocytes were resuspended in 1 mL of 20% (w / v) gelMA solution containing 0.5% Irgacure 2959, and then mixed with 0.3 g of gelatin microspheres. 100 μL of the mixed solution was dropped into a cylindrical mold (diameter 6 mm, height 2.8 mm), and crosslinked by ultraviolet irradiation for 5 minutes (365 nm, 30 mW / cm 2)。The prepared hydrogel construct was cultured in a three-dimensional cell culture medium. The incubator conditions were a temperature of 37 °C, 5% CO2, a relative humidity of 95%, and a horizontal shaker speed of 40 rpm / min. It was cultured for 2 days to dissolve the encapsulated gelatin microspheres, and it was named gelatin methacrylate microporous hydrogel loaded with chondrocytes (gelMA Microporous-Cell).

[0038] For convenient comparison, a hydrogel without chondrocytes was prepared simultaneously here, named gelatin methacrylate microporous hydrogel (gelMA Microporous), and a hydrogel without gelatin microspheres and chondrocytes was prepared, named gelatin methacrylate hydrogel (gelMA) as a control.

[0039] Figure 1 Schematic diagram of the construction method of a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft: The chondrocytes were resuspended in an appropriate amount of gelMA hydrogel monomer solution at a density of 1×10 7 cells / mL, and the wet gelatin microspheres were weighed at 0.3 g / mL. It was added dropwise to a silicone plate mold and irradiated with ultraviolet light at a wavelength of 365 nm for 5 min. The mold was removed, and the crosslinked hydrogel was transferred to a culture plate. Cell culture medium was added, and the gelatin microspheres dissolved in the 37 °C environment, forming a microporous structure in the hydrogel. The chondrocytes grew and proliferated in the microporous hydrogel. After culturing for 28 days, a hydrogel-chondrocyte-extracellular matrix secreted by chondrocytes mixed system was prepared, called tissue-engineered double-network interpenetrating cartilage matrix graft (gelMAPTCC). After decellularization treatment, a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMAPTCC) was obtained.

[0040] Figure 2 Scanning electron microscope and optical microscope pictures of gelatin microspheres. The scanning electron microscope pictures of dry gelatin microspheres had a particle size in the range of 100-150 μm, and the sphere sizes were uniform. The optical microscope pictures of wet gelatin microspheres showed that the diameter of the gelatin microspheres was 100-150 μm and they were in a dispersed state one by one.

[0041] Figure 3 Optical microscope pictures of chondrocytes. The chondrocytes were polygonal or oval. The gray cells adhered to the culture plate and grew with a polygonal shape; the bright cells adhered to the cells on the culture plate and grew with an oval shape.

[0042] Figure 4Morphologies of the gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes prepared in Comparative Example 1: Cross-sectional images were taken using a scanning electron microscope. It can be seen from the images that the gelMA gel is relatively dense, with multiple small pores distributed on the cross-section, and the pore diameter is about 50 μm; the gelMA microporous gel is relatively loose and porous, with small cavities of about 50 μm on the cross-section, and has multiple interconnected larger cavity structures, where the pore diameter is 100 - 150 μm; the image of the gelMA microporous gel loaded with chondrocytes shows that oval chondrocytes adhere to the gel micropores, and the larger microporous structures provide space for chondrocyte growth, proliferation, and subsequent matrix secretion.

[0043] Figure 5 Histological section staining of the gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes prepared in Comparative Example 1: The gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes were fixed with paraformaldehyde, sectioned using a cryostat, and then the above samples were stained with hematoxylin-eosin (HE), safranin-O (Saf-O), and Masson's trichrome (Masson). It can be seen from the images that the gelatin microspheres dissolve inside the gel, forming interconnected microporous structures. HE staining shows that the gelatin is bright red, Masson staining shows that the gelatin is blue, and Saf-O staining shows that the gelatin is light pink, indicating that the main component of the gelatin is extracellular matrix such as collagen fibers.

[0044] Figure 6 Determination of the dynamic oscillation frequency, ultraviolet light kinetics, and static compression modulus of the gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes prepared in Comparative Example 1: The gel liquids of the gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes were respectively measured for the storage modulus (G′) and loss modulus (G″) through a rheometer in oscillation mode with a fixed strain of 1% and an oscillation frequency of 0.1 Hz - 10 Hz. As Figure 6 can be seen, there are no significant differences in the storage modulus and loss modulus of the gel liquids of the three. There are also no significant differences in the storage modulus and loss modulus measured after ultraviolet light irradiation, indicating that the microporous structure caused by the gelatin microspheres does not affect the dynamic modulus of the hydrogel. The crosslinked hydrogels, including the gelMA gel, gelMA microporous gel, and gelMA microporous gel loaded with chondrocytes, were subjected to a compression test through the compression module of the rheometer at a compression speed of 1 mm / min until the strain reached 90%, and the compression modulus and elastic modulus were measured. As Figure 6It can be seen that the compressive modulus of the gelMA gel containing micropores is 30% of that of the gelMA gel, indicating that the gel does have a microporous structure microscopically, which causes the compressive resistance of the gel to decrease, but it still has a supporting effect macroscopically.

[0045] Figure 7 Histological immunofluorescence staining of the tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) and the decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN) prepared in Example 1: Fix gelMA PTCC and dECM-gelMA IPN with paraformaldehyde, section them using a cryostat, and then perform staining for type I collagen (Collagen Type I, Col I), type II collagen (Collagen Type II, Col II), fibronectin (Fibronectin, Fn), and cell nuclei (DAPI) on the above samples. As Figure 7 It can be seen that in gelMA PTCC, cells proliferate along the edges of the gel pores and connect with each other, and extracellular matrix connective tissues are formed around the pores. After decellularization, in dECM-gelMA IPN, the cells are completely removed without residue. The staining results show that the components of type I collagen and fibronectin in the decellularized graft are evenly distributed, and type II collagen is mainly distributed on the surface layer and a small amount is in the interior of the gel, forming a macroscopic interpenetrating network structure between the gel and the extracellular matrix secreted by chondrocytes, and having obvious cartilage lacuna structures microscopically.

[0046] Table 1 shows the biochemical analysis of gelMA, gelMA PTCC, and dECM-gelMA IPN: Wash gelMA, gelMA PTCC, and dECM-gelMA IPN in PBS, weigh them, and freeze-dry them. Digest them with papain solution at 60 °C for 12 h. The amount of sulfated glycosaminoglycan (sGAG) is quantified using the dimethylmethylene blue dye-binding assay and chondroitin sulfate standards. The amount of remaining DNA is quantified using the Hoechst 33258 dye-binding assay and calf thymus standards. Digest half of the digestion solution in 6 M hydrochloric acid at 105 °C for 18 h, and determine the total collagen content by measuring the hydroxyproline content. As can be seen from Table 1, the DNA is basically completely removed, the glycosaminoglycans are lost during the decellularization process, and the collagen content increases.

[0047] Table 1

[0048] Component gelMA (μg / mg) gelMA PTCC (μg / mg) dECM - gelMA IPN (μg / mg) DNA 7.921719667 16.38302 9.156614667 Glycosaminoglycan 0 255.42516 6.2672534 Collagen 915.8484667 704.7889333 969.31395

[0049] Figure 8Mechanical property tests of the tissue-engineered double-network interpenetrating cartilage matrix graft (gelMA PTCC) and decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN) prepared in Example 1: Using a rheometer in oscillatory mode, fix the strain of gelMA PTCC and dECM-gelMA IPN at 1%, and the oscillation frequency at 0.1 Hz - 10 Hz, and measure the storage modulus (G′) and loss modulus (G″). In compression mode, measure the compressive mechanical properties of gelMA PTCC and dECM-gelMA IPN. When the compression speed is 1 mm / min and the compression reaches a strain of 90%, record the compression modulus and elastic modulus. As Figure 8 It can be seen that the G′ and G″ of gelMA PTCC before decellularization are one order of magnitude higher than those of dECM-gelMA IPN after decellularization. gelMA PTCC before decellularization was crushed when the strain reached 50%, while it would be flattened but not fragmented after decellularization, and the elastic modulus remained 20%.

[0050] Figure 9 Biocompatibility determination of the decellularized tissue-engineered double-network interpenetrating cartilage matrix graft (dECM-gelMA IPN) prepared in Example 1: After subculturing mouse epithelial-like fibroblasts (L929 cells), prepare a cell suspension of 2×10 4 cells / mL with the culture medium. Add 100 μL of the cell suspension to each well of a 96-well plate and place it in an incubator for 24 h. Discard the original culture medium, add 100 μL of the extract of the sample material (sample group) and the culture medium (blank control group) to each well, and place it in the incubator for 7 days. At four time points of 1, 3, 5, and 7 days, use a CCK-8 kit and an enzyme-linked immunosorbent assay reader to measure the absorbance value at a wavelength of 450 nm to reflect the proliferation of L929 cells. As Figure 9 It can be seen that in the first 1 - 5 days is the cell proliferation period, and the 7th day reaches the proliferation plateau period. There is no difference in cell proliferation between the experimental group and the blank control group, indicating that the decellularized tissue-engineered double-network interpenetrating cartilage matrix graft has good cytocompatibility.

Claims

1. An acellular tissue-engineered double-network interpenetrating cartilage matrix graft, characterized in that, Suspend chondrocytes and gelatin microspheres together in a photocrosslinkable hydrogel monomer solution, and crosslink and form a crosslinked hydrogel by ultraviolet light irradiation, namely constructing a three-dimensional cell culture system; incubate the three-dimensional cell culture system at a constant temperature, the gelatin solution dissolves, and a microporous structure is formed inside the hydrogel. The chondrocytes grow and proliferate inside the gel and secrete a matrix with cartilage characteristics to obtain a tissue-engineered double-network interpenetrating cartilage matrix graft; subject the tissue-engineered double-network interpenetrating cartilage matrix graft to multi-stage decellularization treatment to construct a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft; the photocrosslinkable hydrogel monomer is one or more of gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and chitosan methacrylate; the chondrocytes are hyaline chondrocytes derived from the knee joint of 4- to 8-month-old pigs, and the number of the chondrocytes is 0.5-2×10 7 cells / mL of the hydrogel solution, the amount of the gelatin microspheres is 0.1-0.4 g / mL of the hydrogel solution in wet weight, and the gelatin microspheres are hollow gelatin microspheres or solid gelatin microspheres with a particle size of 50-300 μm.

2. A preparation method of the acellular tissue-engineered double-network interpenetrating cartilage matrix graft as described in claim 1, characterized in that, It includes the following steps: (1) Construct a three-dimensional cell culture system: Mix the photocrosslinkable hydrogel monomer solution with chondrocytes and gelatin microspheres evenly to form a hydrogel solution encapsulating cells and gelatin microspheres, and inject it into a porous silica gel plate mold with a pore size of 2 - 10 mm and a pore depth of 2 - 6 mm. After crosslinking and forming by ultraviolet light irradiation, a crosslinked hydrogel is obtained, that is, a three-dimensional cell culture system is constructed; (2) Prepare a tissue-engineered double-network interpenetrating cartilage matrix graft: Add the crosslinked hydrogel prepared in step (1) to a cell culture medium and place it in an incubator at 37 °C for culture for 2 - 6 weeks to form a hydrogel and an extracellular matrix network interpenetrating cartilage matrix graft secreted by chondrocytes, that is, a tissue-engineered double-network interpenetrating cartilage matrix graft; (3) Prepare a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft: First, perform multiple cycles of freeze-thaw treatment on the tissue-engineered double-network interpenetrating cartilage matrix graft prepared in step (2), and then perform decellularization treatment with a cell eluent to finally obtain a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft.

3. The preparation method of an acellular tissue-engineered double-network interpenetrating cartilage matrix graft according to claim 2, characterized in that, Step (3) specifically includes the following sub-steps: (3.1) Freeze the tissue-engineered double-network interpenetrating cartilage matrix graft prepared in step (2) at -80 °C for 3 hours, then thaw it at room temperature for 4 - 6 hours, and repeat the freeze-thaw process 3 - 5 times in sequence to obtain a pre-product; (3.2) Perform decellularization treatment on the pre-product obtained in step (3.1) successively with the following cell eluents: Tris-HCl solution, Triton X-100 solution, phosphate buffer solution (PBS solution), NaOH solution, PBS solution, a nuclease solution composed of deoxyribonuclease and ribonuclease, and PBS solution. The elution conditions of the cell eluent are: the temperature is 37 °C, the oscillation speed is 50 - 200 rpm / min, each PBS solution is eluted 3 - 5 times, and the elution time for each stage of the cell eluent is 3 - 5 hours. Finally, a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft is obtained.

4. The preparation method of an acellular tissue-engineered double-network interpenetrating cartilage matrix graft according to claim 2, wherein, In step (2), the culture conditions are: the temperature is 37 °C, the CO2 concentration is 5%, the relative humidity is 95%, and the horizontal shaker rotation speed is 20 - 60 rpm / min.

5. According to the method for preparing a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft as claimed in claim 2, in step (1), the ultraviolet wavelength is 250 nm - 450 nm, the temperature is 15 - 37 °C, and the light irradiation time is 3 - 8 minutes.

6. According to the method for preparing a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft as claimed in claim 5, the photocrosslinkable hydrogel monomer solution in step (1) contains an initiator, and the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone or lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and the concentration is 0.01% - 0.5%.

7. A preparation method of a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft according to claim 6, in step (2), the cell culture medium contains 10.03 g / L DMEM powder, 2.775 g / L sodium bicarbonate, 15%-20% fetal bovine serum, 1% penicillin-streptomycin, 1 mM HEPES, 1 mM sodium pyruvate, 0.05 g / L vitamin C, and 0.046 g / L proline.

8. A preparation method of a decellularized tissue-engineered double-network interpenetrating cartilage matrix graft according to claim 7, in step (3), the PBS solution contains 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, with a pH value of 7.3 ± 0.1, the Tris-HCl solution contains 5-15 mM Tris-HCl, 0.05%-0.2% disodium ethylenediaminetetraacetate (w / v), the Triton X-100 solution is a Tris-HCl solution containing 0.5%-3% Triton X-100, the NaOH solution is a 0.1%-0.8% solution, and the nuclease solution is a solution containing 100 μg / mL deoxyribonuclease and 10 μg / mL ribonuclease.

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