In-vitro construction method of injectable tissue engineering cartilage

Abstract

The invention relates to the technical field of cartilage construction, in particular to an injectable tissue engineering cartilage in-vitro construction method which comprises the following steps: S1, micro-ecological cartilage environment simulation design; s2, self-assembly of the stent and space-time guidance; and S3, constructing a reversible tissue-degradation platform. A tissue engineering system highly simulating a natural cartilage microenvironment is constructed through the technology of combining bionic design and dynamic regulation and control, the proliferation activity and functional differentiation efficiency of cartilage cells can be effectively improved, and meanwhile, by means of intelligent support design and a growth factor delivery system, the tissue engineering system can be applied to the field of cartilage tissue engineering. Accurate release of factors and cell navigation are realized; the dynamic repair technology is combined with the non-contact mechanical stimulation and immune regulation strategy, it is ensured that transplanted tissue can be continuously generated and fully integrated with host tissue, the problems of immunological rejection or insufficient stent degradation and the like are solved, the success rate and long-term stability of cartilage regeneration are remarkably improved, and the method has good clinical application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of cartilage construction, and in particular to an in vitro construction method of injectable tissue engineering cartilage. Background Art

[0002] Tissue engineering cartilage is one of the important research directions in the field of regenerative medicine, which is used to repair cartilage damage caused by trauma, degenerative diseases or congenital defects. As a special connective tissue, cartilage has extremely limited regenerative capacity. Traditional treatment methods such as autologous transplantation or allogeneic transplantation have problems such as insufficient donors, immune rejection and poor repair effects. In recent years, the development of tissue engineering technology has provided new ideas for cartilage repair. By constructing engineered tissues with functional cartilage characteristics in vitro, the repair efficiency can be significantly improved and the patient's prognosis can be improved.

[0003] In cartilage tissue engineering, cell sources, scaffold materials and culture environment are the core elements. However, existing technologies face many challenges in these aspects. Commonly used chondrocyte culture methods are difficult to achieve efficient cell proliferation and differentiation, the scaffold materials are not biomimetic and biocompatible, and dynamic culture conditions are difficult to simulate the microenvironment of natural cartilage. In addition, the factor release in traditional methods often lacks precision, resulting in unstable cartilage tissue repair effects, and scaffold degradation and tissue integration issues further limit the success rate of cartilage regeneration.

[0004] The current tissue engineering cartilage construction methods have the following major problems in practical applications: 1. Traditional culture methods cannot effectively simulate the growth environment of natural cartilage, resulting in low proliferation activity and differentiation efficiency of chondrocytes, making it difficult to form functional tissues; 2. Existing scaffold materials are different from natural cartilage tissues in terms of mechanical properties, biocompatibility and degradation properties, and most of them cannot achieve intelligent factor delivery and cell navigation functions; 3. Traditional factor release technologies are mostly single releases, which make it difficult to achieve dynamic regulation, and the cell repair process therefore lacks precision and sustainability; 4. During the transplantation stage, due to insufficient scaffold degradation or immune rejection problems, the integration efficiency of the graft and the host tissue is low, affecting the long-term effect of cartilage regeneration.

[0005] To solve the above problems, we provide a method for constructing injectable tissue engineering cartilage in vitro. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the first object of the present invention is to provide an in vitro construction method of injectable tissue engineering cartilage to solve the problems in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] An in vitro method for constructing injectable tissue engineering cartilage comprises the following steps:

[0009] S1, microecological cartilage environment design;

[0010] S2, self-assembly scaffold and spatiotemporal guidance;

[0011] S3, reversible tissue construction-degradation platform;

[0012] S4. Dynamic regulation of cell temporal behavior;

[0013] S5, non-contact mechanical stimulation;

[0014] S6. Efficient growth factor utilization;

[0015] S7, Combination of biological signals and multimodal imaging;

[0016] S8. Real-time repair and sustainable optimization.

[0017] The present invention is further configured as follows: in the step S1, in the design of the microecological cartilage-mimicking environment:

[0018] S1.1. A biomimetic model of the cartilage microecological environment was constructed using a 3D bioprinter by using scanning electron microscopy multimodal imaging technology combined with high-resolution three-dimensional reconstruction data of cartilage tissue. The printing material can be gelatin methacrylate (GelMA, 5% concentration) as the matrix, combined with 10 μg / mL chondroitin sulfate and 1 mg / mL hyaluronic acid to simulate the composition of the extracellular matrix. The experimental environment was controlled at 37°C, the humidity was kept above 90%, and a 5% CO2 atmosphere was maintained in a CO2 incubator;

[0019] S1.2. Add probiotic strains of Lactobacillus to the culture system, use their lactic acid metabolites to regulate the acid-base balance of the microenvironment, and achieve spatial separation of microorganisms and chondrocytes through a microfluidic chip. The chip channel width is controlled at about 100 μm to prevent excessive bacterial diffusion. 3 g / L glucose is added to the culture medium as a microbial metabolic substrate, and the pH of the culture medium is maintained between 6.8 and 7.2;

[0020] S1.3. In the early stage of culture, IL-1β (10 ng / mL) was added to simulate the inflammatory environment of degenerative cartilage, and cyclosporine (1 μg / mL) was used as an immunosuppressant for control. In this environment, the frequency of culture medium replacement needs to be increased to once a day, and cell survival status is monitored by fluorescence microscopy to ensure that inflammation does not lead to large-scale cell apoptosis;

[0021] S1.4. Design a culture device with an oxygen concentration gradient. Use a gas microchannel controller to achieve a linear gradient distribution of oxygen from 20% (close to the external environment) to 1% (hypoxic zone). Place cell cultures in zones with different oxygen concentrations. Use fluorescently labeled HIF-1α antibodies to detect the response of cells to the hypoxic environment. The initial experimental period is 7 days.

[0022] The present invention is further configured as follows: in the step S2, self-assembling the bracket and the time-space guidance:

[0023] S2.1, disperse thermosensitive polymers such as poly (N-isopropylacrylamide) microparticles in the cell suspension, and trigger the self-assembly of the particles into a mesh scaffold at 37°C by increasing the temperature. The self-assembly process can be observed by real-time microscopy, and the pore size of the scaffold is controlled in the range of 50–100 μm to ensure uniform cell attachment and penetration;

[0024] S2.2. Titanium dioxide nanoparticles were embedded in the scaffold and blue light (wavelength 450 nm) was used to induce photocatalytic reaction, so that the nanoparticles locally released reactive oxygen species, thereby guiding chondrocytes to migrate to specific areas. The light intensity gradient (10–50 mW / cm 2 ) and timed switching of light sources to achieve accurate path navigation;

[0025] S2.3. Design a multifunctional scaffold that encapsulates TGF-β (initial release concentration 5 ng / mL, sustained release time 1 week) and BMP-2 (initial release concentration 3 ng / mL, release time 2 to 4 weeks), achieves time-sharing release through controllable degradation of poly(lactic-co-glycolic acid) copolymer, and detects the release rate by enzyme-linked immunosorbent assay;

[0026] S2.4. A microchannel network (channel diameter 20 μm) was constructed in the scaffold using microfluidic technology, and culture medium containing glucose (4.5 g / L) and amino acids was injected to simulate the blood nutrient transfer function of natural tissues. Fluorescent dyes were used to verify the uniform distribution of the fluid in the channel, and the stability of nutrient delivery in the channel was monitored using optical imaging technology.

[0027] The present invention is further configured as follows: in the step S3, reversible tissue construction-degradation platform:

[0028] S3.1. Polyethylene glycol diacrylate material with dynamic cross-linking properties was mixed with 5% gelatin matrix and exposed to ultraviolet light (365nm, intensity 10mW / cm 2 ) Cross-linking to form a temporary scaffold, the scaffold hardness is adjusted to 10-20 kPa to match the mechanical environment of cartilage, and chemical cross-linking agents are used after scaffold injection to ensure short-term stability;

[0029] S3.2, polyaniline conductive microspheres (particle size 100nm) were embedded in the scaffold, and an electrochemical reaction was triggered by applying a low voltage (0.5V) to release active substances to promote the degradation of the scaffold. This experiment required the use of a culture system containing PBS buffer, and the current change was monitored by an electrochemical analyzer to ensure a uniform reaction;

[0030] S3.3, by adding specific fibronectin (concentration 1 μg / mL), the production of adhesion proteins between chondrocytes was promoted, and the intercellular connection was strengthened by regulating the calcium ion concentration (1.8 mM). The cell construction process was dynamically observed by high-resolution fluorescence microscopy;

[0031] S3.4. Chitosan-gluconic acid double-layer nanoparticles (particle size 50 nm) were loaded into the scaffold. One layer provided growth factors that promoted repair, and the other layer regulated the release rate through sustained release (initial release of 30% in 24 hours). The release dynamics were analyzed by Fourier transform infrared spectroscopy and monitored in conjunction with cell behavior.

[0032] The present invention is further configured as follows: in the step S4, in the dynamic regulation of the cell timing behavior:

[0033] S4.1. Use a multi-stage culture system. In the early stage, bone marrow mesenchymal stem cells are the main culture medium. 10ng / mL TGF-β1 is added to the culture medium to promote chondrocyte differentiation. In the middle stage, 5% fetal bovine serum is added to reduce the TGF-β concentration to 2ng / mL to maintain the cartilage characteristics of the cells. In the later stage, serum-free culture medium is switched to reduce the intervention of exogenous factors and support the improvement of cell autocrine ability. The whole process is carried out in a CO2 incubator at a temperature of 37°C and a humidity of 95%.

[0034] S4.2. Design a 24-hour cycle culture system by adjusting oxygen concentration (5% to 20%), mechanical pressure (applying periodic pressure of 0.1 MPa, frequency of 1 Hz) and temperature (simulating the temperature difference between day and night, alternating between 37°C and 34°C), using the built-in pressure sensor and temperature monitoring system to adjust the parameters in real time, and analyze the expression changes of cartilage-specific genes (Col2a1) by RT-PCR;

[0035] S4.3. Use an automated culture medium replacement system to monitor the concentrations of glucose, lactate, and amino acids in the culture medium in real time, automatically adjust the nutrients according to the metabolic needs of the cells, use biosensors to monitor, ensure that the glucose concentration remains at 4-6mmol / L, and the lactate concentration is less than 2mmol / L, and combine regular detection of cell secretions such as glycosaminoglycans to evaluate the dynamic optimization effect of the culture medium;

[0036] S4.4. Based on microfluidic technology, a micro-chamber culture device was designed. Each chamber holds about 10 μL of culture medium and is embedded with a pressure control module to provide a micro-pressure stimulation of 0.01-0.05 MPa. The width of the micro-chamber is about 200 μm and the height is controlled at 100 μm. It is used to culture single cells or small colonies and observe the proliferation and differentiation characteristics of chondrocytes under different pressures.

[0037] The present invention is further configured as follows: in the step S5, non-contact mechanical stimulation:

[0038] S5.1, using an ultrasonic generator (frequency 40kHz, power density 1W / cm 2 ) Directional vibration stimulation was applied to chondrocytes, each stimulation lasted for 5 minutes, with an interval of 12 hours. During the culture period, mechanical stress induced by vibration promoted the secretion of extracellular matrix, and the expression of matrix protein (collagen type II) was detected by immunofluorescence technology;

[0039] S5.2. A controllable direct current electric field (voltage 1 V / cm) was embedded in the culture system. A periodic electric field was applied at both ends of the cartilage scaffold to generate a local pressure field to stimulate the mechanical receptors of the cells. The effect of pressure on cell morphology was observed by optical microscopy, and the changes in mechanical stimulation-related proteins (integrin β1) were detected by Western blot.

[0040] S5.3, embed metal oxide nanoparticles (zinc oxide, particle size about 30nm) into the scaffold, trigger the micro-oscillation of the particles by a low-frequency oscillating magnetic field (frequency 10Hz), and observe the effect of the oscillation on the distribution of cell membrane receptors by electron microscopy after 7 days of cell culture, and verify the upregulation of matrix production-related genes by RT-PCR;

[0041] S5.4. Magnetic iron oxide nanoparticles (particle size 50 nm) were embedded in the scaffold, and the cells were aggregated and arranged in a specific area through an external magnetic field (intensity 0.1 T). The dynamic behavior of the cells was monitored by a real-time imaging system, and the survival rate of the arranged cells was evaluated by flow cytometry.

[0042] The present invention is further configured as follows: in the step S6, efficient growth factor utilization:

[0043] S6.1. Thermosensitive chitosan microspheres (particle size of about 100 nm) were used as growth factor carriers to encapsulate TGF-β (concentration 10 ng / mL) and IGF-1 (concentration 5 ng / mL) in them. The growth factors were gradually released by temperature control (release at 37°C). The factor release curve was detected by UV-Vis spectroscopy, and the activity of the released factors was verified by ELISA.

[0044] S6.2. Use CRISPR / Cas9 technology to edit chondrocyte genes to increase the secretion of IGF-1 and introduce the regulatory gene of the anti-inflammatory factor IL-10. After gene editing, use fluorescent quantitative PCR to verify the expression efficiency of the gene, and evaluate the promotion of cartilage matrix production by bidirectional factor secretion through culture experiments.

[0045] S6.3, the factors were directly injected into the scaffold through a microneedle array (needle tip diameter of about 10 μm), providing high concentrations of TGF-β (50 ng / mL) in different regions, using infrared imaging technology to monitor the penetration depth of the microneedles, and combining microscopy to analyze the cell proliferation in the injection area;

[0046] S6.4. PLGA nanoparticles with different degradation rates were embedded in the scaffold to encapsulate multiple factors (VEGF, BMP-2, IGF-1). The release order of the factors was regulated by scaffold degradation, and the time curve of factor release was analyzed by high-performance liquid chromatography. Western blot was used to detect the activation of signal pathways after the action of the factors.

[0047] The present invention is further configured as follows: in the step S7, combining biological signals with multimodal imaging:

[0048] S7.1. Use high-resolution confocal microscopy to monitor the morphological changes of cells in the scaffold in real time, with an interval of 6 hours between each imaging, to observe the proliferation and distribution of cells, and use fluorescent probes labeled with collagen type II to evaluate the dynamics of extracellular matrix generation;

[0049] S7.2, introduce a bioluminescent imaging system to monitor the dynamics of cell secretion by labeling the chondroitin protein (CS-PG) secreted by chondrocytes. The intensity of the luminescent signal is proportional to the amount of matrix secretion. Combined with chemiluminescence detection technology, the secretion level at different time points is quantitatively analyzed;

[0050] S7.3. Use high-field magnetic resonance imaging (MRI, 7 Tesla) in vitro to observe the structural changes and cell distribution of the scaffold, perform image segmentation analysis on the cell density within the scaffold, and further evaluate the scaffold degradation in combination with the imaging results of specific markers;

[0051] S7.4. Use AI algorithms to perform real-time analysis of multimodal imaging data, extract characteristic parameters (cell density, matrix uniformity), automatically adjust culture conditions (oxygen concentration or growth factor concentration), establish a feedback loop, and optimize the entire culture process.

[0052] The present invention is further configured as follows: in the step S8, real-time repair and sustainable optimization:

[0053] S8.1. Use wearable devices to monitor pressure changes and temperature fluctuations at the implant site, combine with wireless sensors to measure local microenvironmental parameters (pH and oxygen concentration), and receive data in real time through mobile applications to adjust the pressure distribution of external assistive devices (support splints);

[0054] S8.2, using low energy laser (wavelength 630nm, power density 50mW / cm 2 ) Irradiate the transplanted area for 10 minutes each time, once a day. Laser stimulation promotes collagen synthesis by enhancing cell mitochondrial activity. Fluorescence microscopy is used to observe cell proliferation after laser treatment.

[0055] S8.3. Develop a "patch" scaffold based on minimally invasive injection, using a cross-linked gelatin scaffold (hardness 5-10 kPa) to locally fill the implantation failure area, detect the positioning accuracy of the patch through postoperative imaging, and evaluate the survival of cells in the patch;

[0056] S8.4. Monitor the immune inflammatory markers (CRP, IL-6) in the transplant area monthly. If abnormally elevated, inject low-dose immunomodulatory drugs (dexamethasone, concentration 0.1 mg / mL) through a micropump system, combined with immunohistochemical analysis, to evaluate the impact of long-term immune response on the transplant area.

[0057] Beneficial Effects

[0058] Compared with the known public technology, the technical solution provided by the present invention has the following advantages:

[0059] Beneficial effects:

[0060] The present invention uses a technology that combines bionic design with dynamic regulation to construct a tissue engineering system that highly simulates the natural cartilage microenvironment, which can effectively enhance the proliferation activity and functional differentiation efficiency of chondrocytes. At the same time, through intelligent scaffold design and growth factor delivery system, precise factor release and cell navigation are achieved. Dynamic repair technology is combined with non-contact mechanical stimulation and immune regulation strategies to ensure that the transplanted tissue can be continuously generated and fully integrated with the host tissue, avoiding problems such as immune rejection or insufficient scaffold degradation, thereby significantly improving the success rate and long-term stability of cartilage regeneration, and has good clinical application prospects. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The present invention will be further described below in conjunction with the embodiments.

[0063] Example 1

[0064] The present invention provides a technical solution: a method for constructing injectable tissue engineering cartilage in vitro, wherein in step S1, the microecological cartilage-mimicking environment is designed:

[0065] S1.1. A biomimetic model of the cartilage microecological environment was constructed using a 3D bioprinter by using scanning electron microscopy multimodal imaging technology combined with high-resolution three-dimensional reconstruction data of cartilage tissue. The printing material can be gelatin methacrylate (GelMA, 5% concentration) as the matrix, combined with 10 μg / mL chondroitin sulfate and 1 mg / mL hyaluronic acid to simulate the composition of the extracellular matrix. The experimental environment was controlled at 37°C, the humidity was kept above 90%, and a 5% CO2 atmosphere was maintained in a CO2 incubator;

[0066] S1.2. Add probiotic strains of Lactobacillus to the culture system, use their lactic acid metabolites to regulate the acid-base balance of the microenvironment, and achieve spatial separation of microorganisms and chondrocytes through a microfluidic chip. The chip channel width is controlled at about 100 μm to prevent excessive bacterial diffusion. 3 g / L glucose is added to the culture medium as a microbial metabolic substrate, and the pH of the culture medium is maintained between 6.8 and 7.2;

[0067] S1.3. In the early stage of culture, IL-1β (10 ng / mL) was added to simulate the inflammatory environment of degenerative cartilage, and cyclosporine (1 μg / mL) was used as an immunosuppressant for control. In this environment, the frequency of culture medium replacement needs to be increased to once a day, and cell survival status is monitored by fluorescence microscopy to ensure that inflammation does not lead to large-scale cell apoptosis;

[0068] S1.4. Design a culture device with an oxygen concentration gradient, use a gas microchannel controller to achieve a linear gradient distribution of oxygen from 20% (close to the external environment) to 1% (hypoxic zone), place cell cultures in different oxygen concentration zones, and use fluorescently labeled HIF-1α antibodies to detect the response of cells to the hypoxic environment. The initial experimental period is 7 days;

[0069] In the step S4, dynamic regulation of cell temporal behavior:

[0070] S4.1. Use a multi-stage culture system. In the early stage, bone marrow mesenchymal stem cells are the main culture medium. 10ng / mL TGF-β1 is added to the culture medium to promote chondrocyte differentiation. In the middle stage, 5% fetal bovine serum is added to reduce the TGF-β concentration to 2ng / mL to maintain the cartilage characteristics of the cells. In the later stage, serum-free culture medium is switched to reduce the intervention of exogenous factors and support the improvement of cell autocrine ability. The whole process is carried out in a CO2 incubator at a temperature of 37°C and a humidity of 95%.

[0071] S4.2. Design a 24-hour cycle culture system by adjusting oxygen concentration (5% to 20%), mechanical pressure (applying periodic pressure of 0.1 MPa, frequency of 1 Hz) and temperature (simulating the temperature difference between day and night, alternating between 37°C and 34°C), using the built-in pressure sensor and temperature monitoring system to adjust the parameters in real time, and analyze the expression changes of cartilage-specific genes (Col2a1) by RT-PCR;

[0072] S4.3. Use an automated culture medium replacement system to monitor the concentrations of glucose, lactate, and amino acids in the culture medium in real time, automatically adjust the nutrients according to the metabolic needs of the cells, use biosensors to monitor, ensure that the glucose concentration remains at 4-6mmol / L, and the lactate concentration is less than 2mmol / L, and combine regular detection of cell secretions such as glycosaminoglycans to evaluate the dynamic optimization effect of the culture medium;

[0073] S4.4. Based on microfluidic technology, a micro-chamber culture device was designed. Each chamber contained about 10 μL of culture medium and was embedded with a pressure control module to provide a micro-pressure stimulation of 0.01-0.05 MPa. The width of the micro-chamber was about 200 μm and the height was controlled at 100 μm. It was used to culture single cells or small colonies and observe the proliferation and differentiation characteristics of chondrocytes under different pressures.

[0074] In the step S7, combining biological signals with multimodal imaging:

[0075] S7.1. Use high-resolution confocal microscopy to monitor the morphological changes of cells in the scaffold in real time, with an interval of 6 hours between each imaging, to observe the proliferation and distribution of cells, and use fluorescent probes labeled with collagen type II to evaluate the dynamics of extracellular matrix generation;

[0076] S7.2, introduce a bioluminescent imaging system to monitor the dynamics of cell secretion by labeling the chondroitin protein (CS-PG) secreted by chondrocytes. The intensity of the luminescent signal is proportional to the amount of matrix secretion. Combined with chemiluminescence detection technology, the secretion level at different time points is quantitatively analyzed;

[0077] S7.3. Use high-field magnetic resonance imaging (MRI, 7 Tesla) in vitro to observe the structural changes and cell distribution of the scaffold, perform image segmentation analysis on the cell density within the scaffold, and further evaluate the scaffold degradation in combination with the imaging results of specific markers;

[0078] S7.4. Use AI algorithms to perform real-time analysis of multimodal imaging data, extract characteristic parameters (cell density, matrix uniformity), automatically adjust culture conditions (oxygen concentration or growth factor concentration), establish a feedback loop, and optimize the entire culture process.

[0079] In this embodiment, the microenvironment of natural cartilage tissue is simulated through bionic design and dynamic environmental regulation, including extracellular matrix (reconstructed by combining GelMA with hyaluronic acid), oxygen gradient (adjusting 1% to 20% oxygen concentration through a gas microchannel controller), and mechanical stimulation (using non-contact ultrasound or periodic pressure). Combined with real-time imaging monitoring of biological signals (such as fluorescence microscopy, MRI) and artificial intelligence algorithm analysis, the culture conditions are dynamically adjusted to optimize cell proliferation, differentiation and matrix production. Through the high bionics and dynamic optimization of the microenvironment, the activity of chondrocytes and the biological function of tissue engineering scaffolds are improved, laying the foundation for subsequent cartilage regeneration.

[0080] Example 2

[0081] The present invention provides a technical solution: a method for constructing injectable tissue engineering cartilage in vitro, wherein in step S2, the self-assembly scaffold and the time-space guidance:

[0082] S2.1, disperse thermosensitive polymers such as poly (N-isopropylacrylamide) microparticles in the cell suspension, and trigger the self-assembly of the particles into a mesh scaffold at 37°C by increasing the temperature. The self-assembly process can be observed by real-time microscopy, and the pore size of the scaffold is controlled in the range of 50–100 μm to ensure uniform cell attachment and penetration;

[0083] S2.2. Titanium dioxide nanoparticles were embedded in the scaffold and blue light (wavelength 450 nm) was used to induce photocatalytic reaction, so that the nanoparticles locally released reactive oxygen species, thereby guiding chondrocytes to migrate to specific areas. The light intensity gradient (10–50 mW / cm 2 ) and timed switching of light sources to achieve accurate path navigation;

[0084] S2.3. Design a multifunctional scaffold that encapsulates TGF-β (initial release concentration 5 ng / mL, sustained release time 1 week) and BMP-2 (initial release concentration 3 ng / mL, release time 2 to 4 weeks), achieves time-sharing release through controllable degradation of poly(lactic-co-glycolic acid) copolymer, and detects the release rate by enzyme-linked immunosorbent assay;

[0085] S2.4. A microchannel network (channel diameter 20 μm) was constructed in the scaffold using microfluidic technology, and a culture medium containing glucose (4.5 g / L) and amino acids was injected to simulate the blood nutrient transfer function of natural tissues. Fluorescent dyes were used to verify the uniform distribution of fluid in the channel, and the stability of nutrient delivery in the channel was monitored using optical imaging technology.

[0086] In the step S6, efficient growth factor utilization:

[0087] S6.1. Thermosensitive chitosan microspheres (particle size of about 100 nm) were used as growth factor carriers to encapsulate TGF-β (concentration 10 ng / mL) and IGF-1 (concentration 5 ng / mL) in them. The growth factors were gradually released by temperature control (release at 37°C). The factor release curve was detected by UV-Vis spectroscopy, and the activity of the released factors was verified by ELISA.

[0088] S6.2. Use CRISPR / Cas9 technology to edit chondrocyte genes to increase the secretion of IGF-1 and introduce the regulatory gene of the anti-inflammatory factor IL-10. After gene editing, use fluorescent quantitative PCR to verify the expression efficiency of the gene, and evaluate the promotion of cartilage matrix production by bidirectional factor secretion through culture experiments.

[0089] S6.3, the factors were directly injected into the scaffold through a microneedle array (needle tip diameter of about 10 μm), providing high concentrations of TGF-β (50 ng / mL) in different regions, using infrared imaging technology to monitor the penetration depth of the microneedles, and combining microscopy to analyze the cell proliferation in the injection area;

[0090] S6.4. PLGA nanoparticles with different degradation rates were embedded in the scaffold to encapsulate multiple factors (VEGF, BMP-2, IGF-1). The release order of the factors was regulated by scaffold degradation, and the time curve of factor release was analyzed by high-performance liquid chromatography. Western blot was used to detect the activation of signal pathways after the action of the factors.

[0091] In this embodiment, a self-assembled scaffold constructed based on thermosensitive polymers and nanoparticles realizes precise navigation and functional zoning of cells through light, temperature or magnetic field stimulation. The scaffold design includes a multifunctional channel network for delivering nutrients and growth factors. At the same time, growth factors utilize intelligent strategies, such as the time-sharing release of TGF-β, IGF-1, etc. using PLGA nanoparticles, or gene-edited cells to achieve balanced output of autocrine pro-chondrogenic factors and anti-inflammatory factors. Through the synergistic effect of the scaffold and the growth factor system, the functional differentiation of chondrocytes is enhanced, while the dosage of exogenous factors is reduced, thereby reducing possible side effects and costs.

[0092] Example 3

[0093] The present invention provides a technical solution: a method for constructing injectable tissue engineering cartilage in vitro, wherein in step S3, the reversible tissue construction-degradation platform:

[0094] S3.1. Polyethylene glycol diacrylate material with dynamic cross-linking properties was mixed with 5% gelatin matrix and exposed to ultraviolet light (365nm, intensity 10mW / cm 2 ) Cross-linking to form a temporary scaffold, the scaffold hardness is adjusted to 10-20 kPa to match the mechanical environment of cartilage, and chemical cross-linking agents are used after scaffold injection to ensure short-term stability;

[0095] S3.2, polyaniline conductive microspheres (particle size 100nm) were embedded in the scaffold, and an electrochemical reaction was triggered by applying a low voltage (0.5V) to release active substances to promote the degradation of the scaffold. This experiment required the use of a culture system containing PBS buffer, and the current change was monitored by an electrochemical analyzer to ensure a uniform reaction;

[0096] S3.3, by adding specific fibronectin (concentration 1 μg / mL), the production of adhesion proteins between chondrocytes was promoted, and the intercellular connection was strengthened by regulating the calcium ion concentration (1.8 mM). The cell construction process was dynamically observed by high-resolution fluorescence microscopy;

[0097] S3.4, chitosan-gluconic acid double-layer nanoparticles (particle size 50 nm) were loaded into the scaffold, one layer provided growth factors that promoted repair, and the other layer regulated the release rate by sustained release (initial release of 30% in 24 hours), and the release dynamics were analyzed by Fourier transform infrared spectroscopy and linked to cell behavior monitoring;

[0098] In the step S5, non-contact mechanical stimulation:

[0099] S5.1, using an ultrasonic generator (frequency 40kHz, power density 1W / cm 2 ) Directional vibration stimulation was applied to chondrocytes, each stimulation lasted for 5 minutes, with an interval of 12 hours. During the culture period, mechanical stress induced by vibration promoted the secretion of extracellular matrix, and the expression of matrix protein (collagen type II) was detected by immunofluorescence technology;

[0100] S5.2. A controllable direct current electric field (voltage 1 V / cm) was embedded in the culture system. A periodic electric field was applied at both ends of the cartilage scaffold to generate a local pressure field to stimulate the mechanical receptors of the cells. The effect of pressure on cell morphology was observed by optical microscopy, and the changes in mechanical stimulation-related proteins (integrin β1) were detected by Western blot.

[0101] S5.3, embed metal oxide nanoparticles (zinc oxide, particle size about 30nm) into the scaffold, trigger the micro-oscillation of the particles by a low-frequency oscillating magnetic field (frequency 10Hz), and observe the effect of the oscillation on the distribution of cell membrane receptors by electron microscopy after 7 days of cell culture, and verify the upregulation of matrix production-related genes by RT-PCR;

[0102] S5.4, embed magnetic iron oxide nanoparticles (particle size 50 nm) in the scaffold, achieve cell aggregation and arrangement in a specific area through an external magnetic field (intensity 0.1 T), monitor the dynamic behavior of cells through a real-time imaging system, and evaluate the survival rate of cells after arrangement by flow cytometry;

[0103] In the step S8, real-time repair and sustainable optimization:

[0104] S8.1. Use wearable devices to monitor pressure changes and temperature fluctuations at the implant site, combine with wireless sensors to measure local microenvironmental parameters (pH and oxygen concentration), and receive data in real time through mobile applications to adjust the pressure distribution of external assistive devices (support splints);

[0105] S8.2, using low energy laser (wavelength 630nm, power density 50mW / cm 2 ) Irradiate the transplanted area for 10 minutes each time, once a day. Laser stimulation promotes collagen synthesis by enhancing cell mitochondrial activity. Fluorescence microscopy is used to observe cell proliferation after laser treatment.

[0106] S8.3. Develop a "patch" scaffold based on minimally invasive injection, using a cross-linked gelatin scaffold (hardness 5-10 kPa) to locally fill the implantation failure area, detect the positioning accuracy of the patch through postoperative imaging, and evaluate the survival of cells in the patch;

[0107] S8.4. Monitor the immune inflammatory markers (CRP, IL-6) in the transplant area monthly. If abnormally elevated, inject low-dose immunomodulatory drugs (dexamethasone, concentration 0.1 mg / mL) through a micropump system, combined with immunohistochemical analysis, to evaluate the impact of long-term immune response on the transplant area.

[0108] In this embodiment, a dynamically degradable scaffold is used to realize the "construction-repair-replacement" function of cartilage tissue. The new cartilage tissue gradually replaces the scaffold through reversible materials and electrochemically triggered degradation technology. At the same time, external mechanical stimulation (such as magnetic field induction) and laser-promoted regeneration technology are used to continuously repair the implanted site. Combined with immune regulation strategies (such as monitoring inflammatory markers and injecting low-dose immune regulation drugs), long-term biocompatibility is ensured, and continuous repair and long-term stability of cartilage are achieved, avoiding repair failure caused by insufficient degradation of scaffold materials or immune rejection, and ensuring functional recovery after transplantation.

[0109] In actual use, the present invention reconstructs the natural cartilage microenvironment through bionic design, utilizes high-resolution 3D printing technology and multimodal imaging technology, combines gelatin methacrylate (GelMA, 5% concentration), chondroitin sulfate (10μg / mL) and hyaluronic acid (1mg / mL), and simulates the physicochemical properties of the extracellular matrix. Furthermore, Lactobacillus probiotics are introduced into the culture system, and the acid-base balance of the culture microenvironment is regulated by its metabolites. At the same time, the precise spatial separation of chondrocytes and probiotics is achieved through a microfluidic chip to ensure the stability of the microenvironment. In the early stage of cell culture, inflammatory conditions are simulated by adding IL-1β (10ng / mL), and excessive immune response is suppressed by cyclosporin (1μg / mL), while dynamic regulation of different oxygen concentrations is achieved with the help of an oxygen gradient culture device. Real-time fluorescence imaging and artificial intelligence algorithms are used to monitor the growth and metabolic dynamics of chondrocytes, and environmental parameters are adjusted according to cell behavior, ultimately optimizing cell proliferation and cartilage matrix generation capabilities.

[0110] Next, a self-assembling scaffold was constructed using a thermosensitive polymer (N-isopropylacrylamide), and the temperature was controlled to 37°C to trigger the microparticles to form a mesh scaffold with a pore size of 50–100 μm, ensuring uniform distribution and attachment of cells. At the same time, titanium dioxide nanoparticles were embedded in the scaffold to release local reactive oxygen species through a photocatalytic reaction at a wavelength of 450 nm, guiding cell migration and differentiation. The scaffold also integrates PLGA microparticles with different degradation rates for the phased release of growth factors (TGF-β and BMP-2) to achieve dynamic nutrient supply. In addition, CRISPR / Cas9 technology was used to edit cell genes to secrete IGF-1 and IL-10, achieving balanced regulation of endogenous chondrogenic factors and anti-inflammatory factors. Through the synergistic effect of the intelligent scaffold and the factor delivery system, the efficiency of cell differentiation is improved, the amount of exogenous factors is reduced, and the side effects are effectively reduced.

[0111] In the process of cartilage construction, the dynamic degradable scaffold provides a multi-stage support function of "construction-repair-replacement". Polyethylene glycol diacrylate material is used to regulate the hardness of the scaffold (10-20kPa) through ultraviolet cross-linking to match the mechanical properties of cartilage; at the same time, the degradation of the scaffold is triggered by electrochemistry to make room for the formation of new tissue. In the repair stage, external non-contact mechanical stimulation, such as ultrasound (40kHz), magnetic field induced oscillation (frequency 10Hz) and low-energy laser (wavelength 630nm), is combined to enhance the secretion of extracellular matrix and the stability of cartilage tissue. In addition, wireless sensors at the implantation site are used to monitor changes in pressure, pH value and oxygen concentration, and immunomodulatory drugs (dexamethasone) are injected through a micropump system to prevent inflammatory response. Finally, the whole set of technical solutions ensures the long-term stability of cartilage function after transplantation through dynamic repair and immune regulation, and effectively avoids the problem of repair failure caused by immune rejection or insufficient scaffold degradation.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing injectable tissue engineering cartilage in vitro, characterized in that: The following steps are involved: S1, microecological cartilage environment design; S2, self-assembly scaffold and spatiotemporal guidance; S3, reversible tissue construction-degradation platform; S4. Dynamic regulation of cell temporal behavior; S5, non-contact mechanical stimulation; S6. Efficient growth factor utilization; S7, Combination of biological signals and multimodal imaging; S8. Real-time repair and sustainable optimization.

2. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S1, the microecological cartilage-mimicking environment design: S1.

1. A biomimetic model of the cartilage microecological environment was constructed using a 3D bioprinter by using scanning electron microscopy multimodal imaging technology combined with high-resolution three-dimensional reconstruction data of cartilage tissue. The printing material can be gelatin methacrylate (GelMA, 5% concentration) as the matrix, combined with 10 μg / mL chondroitin sulfate and 1 mg / mL hyaluronic acid to simulate the composition of the extracellular matrix. The experimental environment was controlled at 37°C, the humidity was kept above 90%, and a 5% CO2 atmosphere was maintained in a CO2 incubator; S1.

2. Add probiotic strains of Lactobacillus to the culture system, use their lactic acid metabolites to regulate the acid-base balance of the microenvironment, and achieve spatial separation of microorganisms and chondrocytes through a microfluidic chip. The chip channel width is controlled at about 100 μm to prevent excessive bacterial diffusion. 3 g / L glucose is added to the culture medium as a microbial metabolic substrate, and the pH of the culture medium is maintained between 6.8 and 7.2; S1.

3. In the early stage of culture, IL-1β (10 ng / mL) was added to simulate the inflammatory environment of degenerative cartilage, and cyclosporine (1 μg / mL) was used as an immunosuppressant for control. In this environment, the frequency of culture medium replacement needs to be increased to once a day, and cell survival status is monitored by fluorescence microscopy to ensure that inflammation does not lead to large-scale cell apoptosis; S1.

4. Design a culture device with an oxygen concentration gradient. Use a gas microchannel controller to achieve a linear gradient distribution of oxygen from 20% (close to the external environment) to 1% (hypoxic zone). Place cell cultures in zones with different oxygen concentrations. Use fluorescently labeled HIF-1α antibodies to detect the response of cells to the hypoxic environment. The initial experimental period is 7 days.

3. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S2, self-assembly of the support and time-space guidance: S2.1, disperse thermosensitive polymers such as poly (N-isopropylacrylamide) microparticles in the cell suspension, and trigger the self-assembly of the particles into a mesh scaffold at 37°C by increasing the temperature. The self-assembly process can be observed by real-time microscopy, and the pore size of the scaffold is controlled in the range of 50–100 μm to ensure uniform cell attachment and penetration; S2.

2. Titanium dioxide nanoparticles were embedded in the scaffold and blue light (wavelength 450 nm) was used to induce photocatalytic reaction, so that the nanoparticles locally released reactive oxygen species, thereby guiding chondrocytes to migrate to specific areas. The light intensity gradient (10–50 mW / cm 2 ) and timed switching of light sources to achieve accurate path navigation; S2.

3. Design a multifunctional scaffold that encapsulates TGF-β (initial release concentration 5 ng / mL, sustained release time 1 week) and BMP-2 (initial release concentration 3 ng / mL, release time 2 to 4 weeks), achieves time-sharing release through controllable degradation of poly(lactic-co-glycolic acid) copolymer, and detects the release rate by enzyme-linked immunosorbent assay; S2.

4. A microchannel network (channel diameter 20 μm) was constructed in the scaffold using microfluidic technology, and culture medium containing glucose (4.5 g / L) and amino acids was injected to simulate the blood nutrient transfer function of natural tissues. Fluorescent dyes were used to verify the uniform distribution of the fluid in the channel, and the stability of nutrient delivery in the channel was monitored using optical imaging technology.

4. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S3, reversible tissue construction-degradation platform: S3.

1. Polyethylene glycol diacrylate material with dynamic cross-linking properties was mixed with 5% gelatin matrix and exposed to ultraviolet light (365nm, intensity 10mW / cm 2 ) Cross-linking to form a temporary scaffold, the scaffold hardness is adjusted to 10-20 kPa to match the mechanical environment of cartilage, and chemical cross-linking agents are used after scaffold injection to ensure short-term stability; S3.2, polyaniline conductive microspheres (particle size 100nm) were embedded in the scaffold, and an electrochemical reaction was triggered by applying a low voltage (0.5V) to release active substances to promote the degradation of the scaffold. This experiment required the use of a culture system containing PBS buffer, and the current change was monitored by an electrochemical analyzer to ensure a uniform reaction; S3.3, by adding specific fibronectin (concentration 1 μg / mL), the production of adhesion proteins between chondrocytes was promoted, and the intercellular connection was strengthened by regulating the calcium ion concentration (1.8 mM). The cell construction process was dynamically observed by high-resolution fluorescence microscopy; S3.

4. Chitosan-gluconic acid double-layer nanoparticles (particle size 50 nm) were loaded into the scaffold. One layer provided growth factors that promoted repair, and the other layer regulated the release rate through sustained release (initial release of 30% in 24 hours). The release dynamics were analyzed by Fourier transform infrared spectroscopy and monitored in conjunction with cell behavior.

5. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S4, dynamic regulation of cell temporal behavior: S4.

1. Use a multi-stage culture system. In the early stage, bone marrow mesenchymal stem cells are the main culture medium. 10ng / mL TGF-β1 is added to the culture medium to promote chondrocyte differentiation. In the middle stage, 5% fetal bovine serum is added to reduce the TGF-β concentration to 2ng / mL to maintain the cartilage characteristics of the cells. In the later stage, serum-free culture medium is switched to reduce the intervention of exogenous factors and support the improvement of cell autocrine ability. The whole process is carried out in a CO2 incubator at a temperature of 37°C and a humidity of 95%. S4.

2. Design a 24-hour cycle culture system by adjusting oxygen concentration (5% to 20%), mechanical pressure (applying periodic pressure of 0.1 MPa, frequency of 1 Hz) and temperature (simulating the temperature difference between day and night, alternating between 37°C and 34°C), using the built-in pressure sensor and temperature monitoring system to adjust the parameters in real time, and analyze the expression changes of cartilage-specific genes (Col2a1) by RT-PCR; S4.

3. Use an automated culture medium replacement system to monitor the concentrations of glucose, lactate, and amino acids in the culture medium in real time, automatically adjust the nutrients according to the metabolic needs of the cells, use biosensors to monitor, ensure that the glucose concentration remains at 4-6mmol / L, and the lactate concentration is less than 2mmol / L, and combine regular detection of cell secretions such as glycosaminoglycans to evaluate the dynamic optimization effect of the culture medium; S4.

4. Based on microfluidic technology, a micro-chamber culture device was designed. Each chamber holds about 10 μL of culture medium and is embedded with a pressure control module to provide a micro-pressure stimulation of 0.01-0.05 MPa. The width of the micro-chamber is about 200 μm and the height is controlled at 100 μm. It is used to culture single cells or small colonies and observe the proliferation and differentiation characteristics of chondrocytes under different pressures.

6. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S5, non-contact mechanical stimulation: S5.1, using an ultrasonic generator (frequency 40kHz, power density 1W / cm 2 ) Directional vibration stimulation was applied to chondrocytes, each stimulation lasted for 5 minutes, with an interval of 12 hours. During the culture period, mechanical stress induced by vibration promoted the secretion of extracellular matrix, and the expression of matrix protein (collagen type II) was detected by immunofluorescence technology; S5.

2. A controllable direct current electric field (voltage 1 V / cm) was embedded in the culture system. A periodic electric field was applied at both ends of the cartilage scaffold to generate a local pressure field to stimulate the mechanical receptors of the cells. The effect of pressure on cell morphology was observed by optical microscopy, and the changes in mechanical stimulation-related proteins (integrin β1) were detected by Western blot. S5.3, embed metal oxide nanoparticles (zinc oxide, particle size about 30nm) into the scaffold, trigger the micro-oscillation of the particles by a low-frequency oscillating magnetic field (frequency 10Hz), and observe the effect of the oscillation on the distribution of cell membrane receptors by electron microscopy after 7 days of cell culture, and verify the upregulation of matrix production-related genes by RT-PCR; S5.

4. Magnetic iron oxide nanoparticles (particle size 50 nm) were embedded in the scaffold, and the cells were aggregated and arranged in a specific area through an external magnetic field (intensity 0.1 T). The dynamic behavior of the cells was monitored by a real-time imaging system, and the survival rate of the arranged cells was evaluated by flow cytometry.

7. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S6, efficient growth factor utilization: S6.

1. Thermosensitive chitosan microspheres (particle size of about 100 nm) were used as growth factor carriers to encapsulate TGF-β (concentration 10 ng / mL) and IGF-1 (concentration 5 ng / mL) in them. The growth factors were gradually released by temperature control (release at 37°C). The factor release curve was detected by UV-Vis spectroscopy, and the activity of the released factors was verified by ELISA. S6.

2. Use CRISPR / Cas9 technology to edit chondrocyte genes to increase the secretion of IGF-1 and introduce the regulatory gene of the anti-inflammatory factor IL-10. After gene editing, use fluorescent quantitative PCR to verify the expression efficiency of the gene, and evaluate the promotion of cartilage matrix production by bidirectional factor secretion through culture experiments. S6.3, the factors were directly injected into the scaffold through a microneedle array (needle tip diameter of about 10 μm), providing high concentrations of TGF-β (50 ng / mL) in different regions, using infrared imaging technology to monitor the penetration depth of the microneedles, and combining microscopy to analyze the cell proliferation in the injection area; S6.

4. PLGA nanoparticles with different degradation rates were embedded in the scaffold to encapsulate multiple factors (VEGF, BMP-2, IGF-1). The release order of the factors was regulated by scaffold degradation, and the time curve of factor release was analyzed by high-performance liquid chromatography. Western blot was used to detect the activation of signal pathways after the action of the factors.

8. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S7, combining biological signals with multimodal imaging: S7.

1. Use high-resolution confocal microscopy to monitor the morphological changes of cells in the scaffold in real time, with an interval of 6 hours between each imaging, to observe the proliferation and distribution of cells, and use fluorescent probes labeled with collagen type II to evaluate the dynamics of extracellular matrix generation; S7.2, introduce a bioluminescent imaging system to monitor the dynamics of cell secretion by labeling the chondroitin protein (CS-PG) secreted by chondrocytes. The intensity of the luminescent signal is proportional to the amount of matrix secretion. Combined with chemiluminescence detection technology, the secretion level at different time points is quantitatively analyzed; S7.

3. Use high-field magnetic resonance imaging (MRI, 7 Tesla) in vitro to observe the structural changes and cell distribution of the scaffold, perform image segmentation analysis on the cell density within the scaffold, and further evaluate the scaffold degradation in combination with the imaging results of specific markers; S7.

4. Use AI algorithms to perform real-time analysis of multimodal imaging data, extract characteristic parameters (cell density, matrix uniformity), automatically adjust culture conditions (oxygen concentration or growth factor concentration), establish a feedback loop, and optimize the entire culture process.

9. The method for constructing injectable tissue engineering cartilage in vitro according to claim 1, characterized in that: In the step S8, real-time repair and sustainable optimization: S8.

1. Use wearable devices to monitor pressure changes and temperature fluctuations at the implant site, combine with wireless sensors to measure local microenvironmental parameters (pH and oxygen concentration), and receive data in real time through mobile applications to adjust the pressure distribution of external assistive devices (support splints); S8.2, using low energy laser (wavelength 630nm, power density 50mW / cm 2 ) Irradiate the transplanted area for 10 minutes each time, once a day. Laser stimulation promotes collagen synthesis by enhancing cell mitochondrial activity. Fluorescence microscopy is used to observe cell proliferation after laser treatment. S8.

3. Develop a "patch" scaffold based on minimally invasive injection, using a cross-linked gelatin scaffold (hardness 5-10 kPa) to locally fill the implantation failure area, detect the positioning accuracy of the patch through postoperative imaging, and evaluate the survival of cells in the patch; S8.

4. Monitor the immune inflammatory markers (CRP, IL-6) in the transplant area monthly. If abnormally elevated, inject low-dose immunomodulatory drugs (dexamethasone, concentration 0.1 mg / mL) through a micropump system, combined with immunohistochemical analysis, to evaluate the impact of long-term immune response on the transplant area.