Cartilage regeneration and repair method and system based on irreversible electroporation technology

Abstract

The invention discloses a cartilage regeneration and repair method and system based on an irreversible electroporation technology. The method comprises the following steps: establishing an electroporation model of a cell membrane, simulating the distribution of an electric field and the potential change of the cell membrane, and predicting a membrane perforation effect; analyzing potential change according to an electroporation model, and combining energy consumption change of the cell membrane to obtain a critical condition of cell membrane pore formation; in combination with critical conditions, establishing an adaptive adjustment feedback mechanism, controlling the adjustment of electroporation parameters, and performing cartilage related gene transfection and cartilage repair; setting a comprehensive evaluation function, evaluating the repairing effect of the cartilage tissue, and adjusting electroporation parameters and a gene transfection strategy. The system comprises an electroporation model modeling module, a critical analysis module, an adaptive mechanism establishment module, a transfection repair module and an evaluation adjustment module. According to the method, the transfection efficiency of cartilage repair related genes can be improved and cartilage regeneration can be promoted by accurately regulating and controlling electroporation parameters and combining a self-adaptive feedback mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of irreversible electroporation, and in particular to a cartilage regeneration and repair method and system using irreversible electroporation technology. Background Art

[0002] Cartilage damage and degeneration are common clinical problems, especially cartilage defects caused by trauma, arthritis or aging. Existing cartilage repair technologies mainly include autologous cartilage transplantation, allogeneic cartilage transplantation and cartilage regeneration drug treatment, but these methods still face problems such as unsatisfactory repair effect, low cell survival rate, long repair cycle and high cost. Therefore, how to efficiently repair damaged cartilage and promote cartilage regeneration has become the focus of current research.

[0003] The limitations of traditional cartilage repair methods are that autologous cartilage transplantation and allogeneic cartilage transplantation are highly invasive, have a long recovery period after surgery, and have problems such as insufficient donors and poor adaptability of the transplant site. Although cartilage regeneration drug treatment is effective in some cases, the effect is relatively limited, and it is difficult to maintain the repair effect for a long time. In traditional gene transfection methods, viral vectors or chemical reagents are often used to carry genes, but these methods may lead to immune responses or low transfection efficiency. Traditional gene transfection methods are difficult to accurately control the amount and location of gene introduction, and are prone to uneven or ineffective transfection. In addition, although traditional electroporation technology can improve the efficiency of gene transfection, there are problems such as imprecise control of operating parameters and greater cell damage. Especially in the transfection of chondrocytes, due to the characteristics of chondrocytes (such as low proliferation and differentiation ability), improper electroporation parameters can easily lead to cell death and poor repair effect.

[0004] The application of electroporation technology in cartilage repair is restricted by many factors, such as the complexity of electric field distribution, the structural characteristics of cell membrane, the control of required energy, etc. Therefore, it is difficult for traditional electroporation methods to fine-tune the special needs of chondrocytes. Electroporation parameters (such as voltage, pulse width, frequency, etc.) are extremely sensitive to the influence of cell membranes. Incorrect parameter settings may not only cause cell death, but also affect the effect of gene transfection. Therefore, there is a need for a mechanism to adaptively adjust electroporation parameters to ensure the maximum promotion of chondrocyte survival and gene expression.

[0005] In the existing technology, although some methods can repair cartilage, they lack real-time and comprehensive evaluation of the repair effect. It is often impossible to provide real-time feedback on the electric field distribution and cell response during electroporation, so it is impossible to effectively adjust the electroporation parameters and gene transfection strategy. Most existing systems are difficult to achieve dynamic monitoring and optimization of the cartilage repair process, making it impossible to adjust the repair effect in a timely and effective manner. Summary of the invention

[0006] The purpose of the present invention is to provide a cartilage regeneration and repair method and system based on irreversible electroporation technology to solve at least one of the above-mentioned technical problems. It can improve the transfection efficiency of cartilage repair-related genes and promote cartilage regeneration by precisely regulating electroporation parameters and combining an adaptive feedback mechanism.

[0007] The embodiment of the present invention is achieved as follows:

[0008] A cartilage regeneration and repair method using irreversible electroporation technology, comprising:

[0009] A cell membrane electroporation model was established to predict the membrane perforation effect by simulating the distribution of the electric field and the potential changes of the cell membrane.

[0010] According to the electroporation model, the critical conditions for the formation of cell membrane pores are obtained by analyzing the change in electric potential and combining the change in energy consumption of the cell membrane.

[0011] In combination with the critical conditions, an adaptive adjustment feedback mechanism is established to control the regulation of electroporation parameters.

[0012] According to the set electroporation parameters, cartilage-related gene transfection and cartilage repair are performed.

[0013] A comprehensive evaluation function was set up to evaluate the repair effect of cartilage tissue, and the electroporation parameters and gene transfection strategy were adjusted according to the real-time evaluation results.

[0014] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair, the establishment of a cell membrane electroporation model, by simulating the distribution of the electric field and the potential change of the cell membrane, predicting the membrane perforation effect includes:

[0015] The relationship between electric field distribution and electric field strength is used to represent the electric potential of the cell membrane, and a cell membrane electroporation model is established to describe how the electric field is distributed on the cell membrane over time t.

[0016] Among them, t′ is the integral variable, representing every moment from 0 to the current time t.

[0017] V(t) is the cell membrane potential, which indicates the change in charge distribution at different locations on the cell membrane.

[0018] E(t) is the electric field intensity, which represents the time variation of the electric field and is a function of the current pulse.

[0019] r is the distribution direction and amplitude of the electric field on the cell membrane, indicating the intensity of the electric field at different locations on the cell membrane surface.

[0020] Its technical effect is to establish a mathematical model of electric field distribution and potential changes, accurately simulate the effect of electric field on cell membrane, quantify the change of electric field strength over time, and the charge distribution and membrane potential changes at different positions on the cell membrane. Through simulation data, it is predicted whether the cell membrane will be perforated under specific electric field conditions, and the degree and distribution of perforations are predicted. The model quantitatively analyzes the electric field distribution and potential changes, thereby optimizing the selection of parameters such as different electric field strengths, pulse durations, and frequencies.

[0021] In a preferred embodiment of the present invention, in the above-mentioned cartilage regeneration and repair method of irreversible electroporation technology, the critical conditions for the formation of cell membrane pores are obtained by analyzing the potential changes and combining the energy consumption changes of the cell membrane according to the electroporation model, including:

[0022] When the cell membrane potential V(t) exceeds the critical potential V threshold When electroporation is performed, the integrity of the cell membrane is destroyed, cell membrane pores are formed, and irreversible electroporation occurs.

[0023] Set the critical condition formula for the formation of cell membrane pores V(t) ≥ V threshold .

[0024] Modeling energy consumption in cell membranes

[0025] Among them, ΔE is the energy consumption in the process of cell membrane pore formation, which represents the energy accumulation of the cell membrane under the action of the electric field.

[0026] C m is the capacitance of the cell membrane, reflecting the response characteristics of the membrane to the electric field.

[0027] V(t) is the cell membrane potential, which changes with the electric field.

[0028] When the energy ΔE accumulated by the cell membrane under the action of the electric field reaches the energy consumption threshold of the cell membrane, E threshold , which can cause the formation of cell membrane pores.

[0029] Set the energy critical condition formula for cell membrane pore formation ΔE≥E threshold .

[0030] Its technical effect is: combining the change of electric potential with the energy consumption of the cell membrane to clarify the critical conditions for the formation of cell membrane pores. The energy consumption accumulation of the cell membrane is closely related to factors such as the intensity of the electric field and the pulse time. When the energy accumulated by the cell membrane under the action of the electric field reaches the threshold, pores will occur. Through precise control, cell damage caused by excessive perforation during electroporation can be avoided. By adjusting the electric field intensity and the time of electric field action, the formation of cell membrane pores can be ensured, which can effectively promote gene introduction, improve gene transfection efficiency, and avoid excessive damage to cells.

[0031] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair method, the step of combining the critical conditions to establish an adaptive adjustment feedback mechanism to control the adjustment of electroporation parameters includes:

[0032] Monitor the cell membrane potential V(t) and the energy consumption ΔE in real time, and adjust the electroporation parameters to meet the critical potential condition formula V(t)≥V threshold The energy critical condition formula for the formation of cell membrane pores is ΔE≥E threshold At least one of .

[0033] According to the relationship between repair efficiency and current intensity, the repair efficiency used to describe the repair rate of cartilage tissue under the action of the electric field is set Among them, α is an empirical constant reflecting the degree of cell response to current, β is an empirical constant determining the attenuation rate of the repair process, and I pulse (t) is the instantaneous current intensity, I max is the maximum current intensity.

[0034] Set the cell survival rate S to reflect the cell repair effect survival (t) and cell proliferation S proliferation (t).

[0035] Set the feedback function f eval (t) = λ1·S survival (t)+λ2·S proliferation (t)+λ3·RepairEfficiency(t), where is the comprehensive evaluation value of the repair effect, and λ1, λ2 and λ3 are weight coefficients.

[0036] According to the evaluation function f eval The value of (t) adjusts the electroporation parameters. If the repair efficiency is low, the current intensity and pulse width are increased. If the repair efficiency is high, the current intensity and pulse width are reduced.

[0037] Its technical effect is: real-time monitoring of key variables in the electroporation process, including cell membrane potential and energy consumption, to ensure that electroporation parameters can be optimized and adjusted according to the actual response of the cells. When the current intensity is low, the repair effect is poor, and when the current intensity is too high, it may cause cell damage or death. Through the adaptive feedback mechanism, when the repair efficiency is low, the current intensity and pulse width are automatically increased to improve the repair efficiency; when the repair efficiency is high, the current intensity and pulse width are automatically reduced to avoid excessive electroporation.

[0038] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology cartilage regeneration and repair method, according to the set electroporation parameters, the cartilage-related gene transfection and cartilage repair include:

[0039] Predetermine the electroporation parameters based on the goal of cartilage repair and the cell status.

[0040] Formula for calculating gene transfection efficiency Among them, γ is a constant used to identify the response ability of cells to electric fields, δ is a constant representing the time decay rate during transfection, P(t) is the instantaneous current pulse intensity, and P max is the maximum current intensity.

[0041] According to the relationship between electroporation parameters and gene transfection efficiency, the gene transfection efficiency can be maximized by dynamically adjusting the electroporation parameters.

[0042] Select gene vectors suitable for cartilage repair.

[0043] The chondrocytes or related tissues are mixed with a gene vector containing the target gene.

[0044] A preset electric field pulse is applied, and the current pulse intensity and pulse duration are used to form pores in the cell membrane, allowing the gene vector to enter the cell.

[0045] Molecular biology techniques were used to verify whether the target gene was successfully expressed.

[0046] Its technical effect is that the gene transfection efficiency formula combines current intensity, pulse duration and other parameters, and dynamically adjusts them through a feedback mechanism, so that the effect of the electric field on the cells during the transfection process is maintained at an optimal level, thereby improving the gene transfection efficiency and ensuring that the target gene can successfully enter the cell and play a role. One of the keys to electroporation technology is to form temporary pores on the cell membrane through electric field pulses, so that the gene carrier can enter the cell. For the purpose of cartilage repair, select a suitable gene carrier and mix it with chondrocytes or related tissues to ensure that the gene carrier can smoothly enter the cell with the help of electroporation during the repair process. Through the preset electroporation parameters, ensure that the intensity of the electric field and the pulse duration can form appropriate pores in the cell membrane, successfully allow the gene carrier to enter the cell, efficiently deliver the target gene, and reduce non-specific cell damage.

[0047] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair method, the comprehensive evaluation function is set to evaluate the repair effect of cartilage tissue, and the electroporation parameters and gene transfection strategy are adjusted according to the real-time evaluation results, including:

[0048] Through histological analysis and cell counting, the number of cells in the cartilage repair area is regularly detected, and the number of cartilage repair cells C is obtained by counting repair (t).

[0049] By using fluorescent labeling and / or PCR and / or Western blot technology, evaluate whether the transfected repair gene is successfully introduced and expressed, and calculate the gene transfection efficiency T gene (t).

[0050] According to the number of cartilage repair cells C repair (t) and the gene transfection efficiency T gene (t), set the comprehensive evaluation function Among them, C max is the maximum number of repair cells, η and ζ are empirical constants.

[0051] If the value of the comprehensive evaluation function is lower than the preset value, it means that the cell repair and proliferation is not ideal and the transfection efficiency is low. By increasing the current intensity and pulse width, the gene transfection efficiency is improved to promote cartilage repair.

[0052] Its technical effect is that the comprehensive evaluation function combines the number of cartilage repair cells with the gene transfection efficiency to evaluate the overall effect of cartilage repair from multiple dimensions. Through histological analysis (such as immunohistochemical staining) and cell counting, the number of cells in the cartilage repair area is regularly monitored to reflect the proliferation of cells during the repair process; through fluorescent labeling, PCR and Western blot technology, it is evaluated whether the transfected repair genes (such as TGF-β, VEGF, etc.) are successfully introduced and expressed, and the gene transfection efficiency is calculated. By real-time evaluation of the number of repair cells and gene transfection efficiency, the electroporation parameters and gene transfection strategy are dynamically adjusted to optimize the proliferation and differentiation process of the repair cells, avoiding cell damage or death caused by improper electroporation parameter settings (such as excessive current or too long pulses).

[0053] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation method for cartilage regeneration and repair, if the gene transfection efficiency of the selected gene is T gene If the value of (t) is lower than the preset range, other gene vectors are selected or the vector concentration is increased.

[0054] Its technical effect is: when the transfection efficiency of the selected gene vector is low due to some reasons (such as poor cell membrane permeability, poor vector stability, etc.), by replacing a more efficient vector or increasing the concentration of the vector, the probability of gene introduction into the cell is increased, ensuring that the repair gene (such as TGF-β, VEGF, etc.) smoothly enters the chondrocyte and plays a role, improving the gene expression level in the cartilage repair process, and accelerating the repair process. Different types of gene vectors (such as plasmid vectors, viral vectors, etc.) have different cell penetration capabilities and expression efficiencies. By selecting other vectors when the gene transfection efficiency is low, a more suitable vector type can be selected according to the characteristics of different cell types, making gene transfection more efficient.

[0055] In a preferred embodiment of the present invention, in the above-mentioned cartilage regeneration and repair method using irreversible electroporation technology, in the comprehensive evaluation function, It reflects the proportion of repair cells and indicates the cell proliferation in the current repair process. The higher the proportion of repair cells, the closer the number of repair cells is to the maximum repair capacity, and the better the repair effect.

[0056] In the comprehensive evaluation function, It reflects the effect of gene transfection efficiency on the repair effect. The higher the transfection efficiency, the more obvious the gene's promoting effect on repair and the better the repair effect.

[0057] Its technical effect is: using the proportion of repair cells as an evaluation standard to guide the adjustment of electroporation parameters during the repair process to ensure that the number of repair cells reaches the maximum repair capacity. When the proliferation ability of cells increases, the number of cells in the repair process increases, and the cartilage repair effect will improve accordingly. By adjusting the electroporation parameters in real time, the proliferation of repair cells is promoted to make them closer to the maximum repair capacity, ensuring better repair effect and faster repair speed of cartilage tissue. By reflecting the gene transfection efficiency, the effect of genes on the repair effect is quantified. The higher the gene transfection efficiency, the higher the expression level of repair genes (such as TGF-β, VEGF, etc.) in chondrocytes, which can more effectively regulate cell proliferation, differentiation and angiogenesis in the process of cartilage repair, and further promote repair.

[0058] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair method, if the selected gene has a preset optimal promoting effect on repair and the cell proliferation has reached a preset good range, the use of gene vectors is reduced or stopped to focus on further promoting cell repair.

[0059] The technical effect is that when the gene transfection efficiency has reached an ideal state, focusing resources on further promoting cell repair can more effectively promote the repair process of cartilage tissue. At this time, the proliferation and differentiation of repair cells have been well promoted by the action of genes, reducing the use of gene carriers, and shifting the focus of treatment to how to continue to promote cell proliferation and cartilage tissue regeneration through other methods (such as optimizing electroporation parameters, cell culture conditions, etc.), thereby improving the repair efficiency.

[0060] A cartilage regeneration and repair system using irreversible electroporation technology, comprising:

[0061] The electroporation model modeling module is used to establish the electroporation model of the cell membrane and predict the membrane perforation effect by simulating the distribution of the electric field and the potential changes of the cell membrane.

[0062] The critical analysis module is used to obtain the critical conditions for the formation of cell membrane pores by analyzing the change of electric potential in combination with the change of energy consumption of the cell membrane according to the electroporation model.

[0063] The adaptive mechanism establishment module is used to establish an adaptive adjustment feedback mechanism in combination with the critical conditions to control the regulation of electroporation parameters.

[0064] The transfection and repair module is used to perform cartilage-related gene transfection and cartilage repair according to the set electroporation parameters.

[0065] The evaluation and adjustment module is used to set a comprehensive evaluation function, evaluate the repair effect of cartilage tissue, and adjust the electroporation parameters and gene transfection strategy according to the real-time evaluation results.

[0066] The beneficial effects of the embodiments of the present invention are:

[0067] The present invention establishes an electroporation model of the cell membrane, simulates the electric field distribution and potential changes, and accurately predicts the membrane perforation effect. By real-time monitoring of the cell membrane potential and energy consumption changes, the critical conditions for the formation of membrane pores are dynamically analyzed, thereby achieving precise control of the cell membrane perforation process. By setting the critical potential and energy threshold, it is ensured that the cell membrane will not excessively damage the cells while achieving the best repair effect, which not only optimizes the repair effect of cartilage tissue, but also reduces cell death and unnecessary damage that may occur during the electroporation process.

[0068] The present invention monitors the electric potential and energy consumption of the cell membrane in real time. When the membrane potential exceeds the critical value or the energy consumption reaches the threshold, it can automatically adjust the electroporation parameters, such as current intensity, pulse width, etc., to ensure that the electroporation process is both effective and safe, avoid unnecessary over-stimulation or improper operation, and make the cartilage repair process more accurate and efficient.

[0069] The present invention can maximize the gene transfection efficiency by dynamically adjusting the relationship between gene transfection efficiency and electroporation parameters. The optimization of gene transfection efficiency takes into account the electric field response ability of cells and the time decay effect during the transfection process, thereby making the transfection of related repair genes such as TGF-β, VEGF, etc. in the cartilage repair process more efficient. The number of repair cells and gene transfection efficiency are monitored in real time, and the electroporation parameters or the use of gene vectors are adjusted in time to ensure the maximization of the repair effect.

[0070] The present invention quantitatively evaluates the cartilage repair effect by setting a comprehensive evaluation function and combining multiple factors such as cell proliferation and gene transfection efficiency. The design of the evaluation function allows the cell survival rate, proliferation and gene transfection effect to interact and influence each other during the repair process, thereby providing a scientific basis for adjusting electroporation parameters and gene transfection strategies. By real-time monitoring of the repair effect, the system can adjust the current intensity, pulse width and use of gene vectors according to the evaluation results to ensure that the repair effect reaches the expected level.

[0071] The present invention can dynamically adjust the different stages of cartilage repair by monitoring the proliferation and repair effect of cartilage repair cells. If the evaluation results show that the number of repair cells is insufficient or the transfection efficiency is low, the current intensity or pulse width will be automatically increased to improve the gene transfection efficiency and cell proliferation effect; when the repair effect has reached the preset range, the electroporation intensity will be reduced, the use of gene carriers will be reduced, and the focus will be on further repair and growth of cells. Through a precise adjustment mechanism, excessive intervention is avoided to ensure that the cartilage repair process is more stable and effective.

[0072] The present invention optimizes the selection of gene vectors and repair strategies. According to the needs of cartilage repair and the state of cells, appropriate gene vectors are selected and introduced into the repair cells through precise electric field regulation. The selection of genes that promote cartilage repair, such as TGF-β and VEGF, can effectively stimulate the repair processes such as chondrocyte proliferation, differentiation, and angiogenesis, thereby further promoting the cartilage repair effect. The effect of gene transfection is evaluated in real time, and the vector concentration is adjusted or the vector type is changed when necessary to achieve the best repair effect.

[0073] The present invention combines histological analysis with molecular biology techniques, such as PCR, Western blot, etc., to comprehensively evaluate the effect of cartilage repair. Through histological staining and cartilage morphological analysis, the changes in the cartilage repair area can be observed intuitively; molecular biological methods are used to quantitatively detect the transfection effect and expression level of the repair gene, thereby providing data support for subsequent adjustments to the repair process. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0075] Figure 1 The figure is a flow chart of the cartilage regeneration and repair method using the irreversible electroporation technology of the present invention. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0077] Please refer to Figure 1The first embodiment of the present invention provides a cartilage regeneration and repair method using irreversible electroporation technology, including: a cartilage regeneration and repair method using irreversible electroporation technology, which includes: establishing an electroporation model of a cell membrane, and predicting the membrane perforation effect by simulating the distribution of an electric field and changes in the electric potential of the cell membrane; according to the electroporation model, by analyzing changes in electric potential and combining changes in energy consumption of the cell membrane, obtaining critical conditions for the formation of cell membrane pores; in combination with the critical conditions, establishing an adaptive adjustment feedback mechanism to control the adjustment of electroporation parameters; performing cartilage-related gene transfection and cartilage repair according to the set electroporation parameters; setting a comprehensive evaluation function to evaluate the repair effect of cartilage tissue, and adjusting electroporation parameters and gene transfection strategies according to real-time evaluation results.

[0078] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair, the establishment of a cell membrane electroporation model, by simulating the distribution of the electric field and the change of the electric potential of the cell membrane, predicting the membrane perforation effect includes: representing the electric potential of the cell membrane by the relationship between the electric field distribution and the electric field strength, establishing a cell membrane electroporation model that describes how the electric field is distributed on the cell membrane over time t Among them, t′ is the integral variable, representing every moment from 0 to the current time t; V(t) is the cell membrane potential, representing the change in charge distribution at different positions on the cell membrane; E(t) is the electric field intensity, representing the time change of the electric field, using the function of the current pulse; r is the distribution direction and amplitude of the electric field on the cell membrane, representing the intensity of the electric field at different positions on the cell membrane surface.

[0079] Its technical effect is to establish a mathematical model of electric field distribution and potential changes, accurately simulate the effect of electric field on cell membrane, quantify the change of electric field strength over time, and the charge distribution and membrane potential changes at different positions on the cell membrane. Through simulation data, it is predicted whether the cell membrane will be perforated under specific electric field conditions, and the degree and distribution of perforations are predicted. The model quantitatively analyzes the electric field distribution and potential changes, thereby optimizing the selection of parameters such as different electric field strengths, pulse durations, and frequencies.

[0080] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology cartilage regeneration and repair method, the critical condition for the formation of cell membrane pores is obtained by analyzing the potential change and combining the energy consumption change of the cell membrane according to the electroporation model, including: when the cell membrane potential V(t) exceeds the critical potential V threshold When the cell membrane integrity is destroyed, cell membrane pores are formed, and irreversible electroporation occurs; the critical condition formula for the formation of cell membrane pores is set as V(t)≥V threshold ; Establish energy consumption model of cell membrane Among them, ΔE is the energy consumption in the process of cell membrane pore formation, which represents the energy accumulation of cell membrane under the action of electric field; C m is the capacitance of the cell membrane, reflecting the response characteristics of the membrane to the electric field; V(t) is the cell membrane potential, which changes with the change of the electric field; when the energy ΔE accumulated by the cell membrane under the action of the electric field reaches the energy consumption threshold of the cell membrane, E threshold , which can cause the formation of cell membrane pores; set the energy critical condition formula for the formation of cell membrane pores ΔE≥E threshold .

[0081] Its technical effect is: combining the change of electric potential with the energy consumption of the cell membrane to clarify the critical conditions for the formation of cell membrane pores. The energy consumption accumulation of the cell membrane is closely related to factors such as the intensity of the electric field and the pulse time. When the energy accumulated by the cell membrane under the action of the electric field reaches the threshold, pores will occur. Through precise control, cell damage caused by excessive perforation during electroporation can be avoided. By adjusting the electric field intensity and the time of electric field action, the formation of cell membrane pores can be ensured, which can effectively promote gene introduction, improve gene transfection efficiency, and avoid excessive damage to cells.

[0082] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology cartilage regeneration and repair method, the combination of the critical condition, the establishment of an adaptive adjustment feedback mechanism, and the control of the adjustment of the electroporation parameters include: real-time monitoring of the cell membrane potential V(t) and the energy consumption ΔE, and adjusting the electroporation parameters to meet the potential critical condition formula V(t) ≥ V threshold The energy critical condition formula for the formation of cell membrane pores is ΔE≥E threshold If at least one of the above conditions is met, it means that the cell membrane has been irreversibly perforated and has entered the repair stage. By calculating the cell membrane potential V(t) and the energy consumption ΔE in real time, it is ensured that the cell membrane forms pores within a safe range. According to the relationship between the repair efficiency and the current intensity, the repair efficiency is set to describe the repair rate of the cartilage tissue under the action of the electric field. Among them, α is an empirical constant reflecting the degree of cell response to current, β is an empirical constant determining the attenuation rate of the repair process, and I pulse (t) is the instantaneous current intensity, I max is the maximum current intensity; set the cell survival rate S that reflects the cell repair effect survival (t) and cell proliferation S proliferation (t); Set the feedback function f eval (t) = λ1·S survival (t)+λ2·S proliferation(t)+λ3·Repair Efficiency(t), where λ1, λ2 and λ3 are weight coefficients; according to the evaluation function f eval The value of (t) adjusts the electroporation parameters. If the repair efficiency is low, the current intensity and pulse width are increased. If the repair efficiency is high, the current intensity and pulse width are reduced.

[0083] Its technical effect is: real-time monitoring of key variables in the electroporation process, including cell membrane potential and energy consumption, to ensure that electroporation parameters can be optimized and adjusted according to the actual response of the cells. When the current intensity is low, the repair effect is poor, and when the current intensity is too high, it may cause cell damage or death. Through the adaptive feedback mechanism, when the repair efficiency is low, the current intensity and pulse width are automatically increased to improve the repair efficiency; when the repair efficiency is high, the current intensity and pulse width are automatically reduced to avoid excessive electroporation.

[0084] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology cartilage regeneration and repair method, according to the set electroporation parameters, cartilage-related gene transfection and cartilage repair include: presetting electroporation parameters according to the target of cartilage repair and cell state; calculating the gene transfection efficiency formula Among them, γ is a constant used to identify the response ability of cells to electric fields, δ is a constant representing the time decay rate during transfection, P(t) is the instantaneous current pulse intensity, and P max is the maximum current intensity; according to the relationship between electroporation parameters and gene transfection efficiency, the electroporation parameters are dynamically adjusted to maximize the gene transfection efficiency; gene vectors suitable for cartilage repair are selected, such as TGF-β (transforming growth factor-β), VEGF (vascular endothelial growth factor) and other genes, which can promote cartilage repair by regulating cell proliferation, differentiation, angiogenesis and other processes. Gene vectors are usually plasmids or viral vectors containing the required target genes; chondrocytes or related tissues are mixed with gene vectors containing target genes; a preset electric field pulse is applied, and the current pulse intensity and pulse duration are used to form pores in the cell membrane to allow the gene vector to enter the cell; molecular biology techniques are used to verify whether the target gene is successfully expressed, and histological staining, cartilage morphology analysis and other methods can be used to observe the repair situation.

[0085] Its technical effect is that the gene transfection efficiency formula combines current intensity, pulse duration and other parameters, and dynamically adjusts them through a feedback mechanism, so that the effect of the electric field on the cells during the transfection process is maintained at an optimal level, thereby improving the gene transfection efficiency and ensuring that the target gene can successfully enter the cell and play a role. One of the keys to electroporation technology is to form temporary pores on the cell membrane through electric field pulses, so that the gene carrier can enter the cell. For the purpose of cartilage repair, select a suitable gene carrier and mix it with chondrocytes or related tissues to ensure that the gene carrier can smoothly enter the cell with the help of electroporation during the repair process. Through the preset electroporation parameters, ensure that the intensity of the electric field and the pulse duration can form appropriate pores in the cell membrane, successfully allow the gene carrier to enter the cell, efficiently deliver the target gene, and reduce non-specific cell damage.

[0086] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology cartilage regeneration and repair method, the comprehensive evaluation function is set to evaluate the repair effect of cartilage tissue, and the electroporation parameters and gene transfection strategy are adjusted according to the real-time evaluation results, including: regularly detecting the number of cells in the cartilage repair area through histological analysis (such as immunohistochemical staining) and cell counting, and statistically obtaining the number of cartilage repair cells C repair (t); By using fluorescent labeling and / or PCR and / or Western blot technology, evaluate whether the transfected repair gene (such as TGF-β, VEGF, etc.) is successfully introduced and expressed, and calculate the gene transfection efficiency T gene (t); According to the number of cartilage repair cells C repair (t) and the gene transfection efficiency T gene (t), set the comprehensive evaluation function Among them, C max is the maximum number of repair cells, η and ζ are empirical constants; if the value of the comprehensive evaluation function is lower than the preset value, it means that the cell repair and proliferation is not ideal and the transfection efficiency is low. By increasing the current intensity and pulse width, the gene transfection efficiency is improved and cartilage repair is promoted.

[0087] Its technical effect is that the comprehensive evaluation function combines the number of cartilage repair cells with the gene transfection efficiency to evaluate the overall effect of cartilage repair from multiple dimensions. Through histological analysis (such as immunohistochemical staining) and cell counting, the number of cells in the cartilage repair area is regularly monitored to reflect the proliferation of cells during the repair process; through fluorescent labeling, PCR and Western blot technology, it is evaluated whether the transfected repair genes (such as TGF-β, VEGF, etc.) are successfully introduced and expressed, and the gene transfection efficiency is calculated. By real-time evaluation of the number of repair cells and gene transfection efficiency, the electroporation parameters and gene transfection strategy are dynamically adjusted to optimize the proliferation and differentiation process of the repair cells, avoiding cell damage or death caused by improper electroporation parameter settings (such as excessive current or too long pulses).

[0088] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation method for cartilage regeneration and repair, if the gene transfection efficiency of the selected gene is T gene If the value of (t) is lower than the preset range, other gene vectors are selected or the vector concentration is increased.

[0089] Its technical effect is: when the transfection efficiency of the selected gene vector is low due to some reasons (such as poor cell membrane permeability, poor vector stability, etc.), by replacing a more efficient vector or increasing the concentration of the vector, the probability of gene introduction into the cell is increased, ensuring that the repair gene (such as TGF-β, VEGF, etc.) smoothly enters the chondrocyte and plays a role, improving the gene expression level in the cartilage repair process, and accelerating the repair process. Different types of gene vectors (such as plasmid vectors, viral vectors, etc.) have different cell penetration capabilities and expression efficiencies. By selecting other vectors when the gene transfection efficiency is low, a more suitable vector type can be selected according to the characteristics of different cell types, making gene transfection more efficient.

[0090] In a preferred embodiment of the present invention, in the above-mentioned cartilage regeneration and repair method using irreversible electroporation technology, in the comprehensive evaluation function, Reflects the proportion of repair cells, indicating the cell proliferation in the current repair process. The higher the proportion of repair cells, the closer the number of repair cells is to the maximum repair capacity, and the better the repair effect. In the comprehensive evaluation function, It reflects the effect of gene transfection efficiency on the repair effect. The higher the transfection efficiency, the more obvious the gene's promoting effect on repair and the better the repair effect.

[0091] Its technical effect is: using the proportion of repair cells as an evaluation standard to guide the adjustment of electroporation parameters during the repair process to ensure that the number of repair cells reaches the maximum repair capacity. When the proliferation ability of cells increases, the number of cells in the repair process increases, and the cartilage repair effect will improve accordingly. By adjusting the electroporation parameters in real time, the proliferation of repair cells is promoted to make them closer to the maximum repair capacity, ensuring better repair effect and faster repair speed of cartilage tissue. By reflecting the gene transfection efficiency, the effect of genes on the repair effect is quantified. The higher the gene transfection efficiency, the higher the expression level of repair genes (such as TGF-β, VEGF, etc.) in chondrocytes, which can more effectively regulate cell proliferation, differentiation and angiogenesis in the process of cartilage repair, and further promote repair.

[0092] In a preferred embodiment of the present invention, in the above-mentioned irreversible electroporation technology for cartilage regeneration and repair method, if the selected gene has a preset optimal promoting effect on repair and the cell proliferation has reached a preset good range, the use of gene vectors is reduced or stopped to focus on further promoting cell repair.

[0093] The technical effect is that when the gene transfection efficiency has reached an ideal state, focusing resources on further promoting cell repair can more effectively promote the repair process of cartilage tissue. At this time, the proliferation and differentiation of repair cells have been well promoted by the action of genes, reducing the use of gene carriers, and shifting the focus of treatment to how to continue to promote cell proliferation and cartilage tissue regeneration through other methods (such as optimizing electroporation parameters, cell culture conditions, etc.), thereby improving the repair efficiency.

[0094] The second embodiment of the present invention provides a cartilage regeneration and repair system using irreversible electroporation technology, which includes: an electroporation model building module, which is used to establish an electroporation model of the cell membrane, and predict the membrane perforation effect by simulating the distribution of the electric field and the potential changes of the cell membrane; a critical analysis module, which is used to obtain the critical conditions for the formation of cell membrane pores based on the electroporation model by analyzing the potential changes and combining the energy consumption changes of the cell membrane; an adaptive mechanism establishment module, which is used to establish an adaptive adjustment feedback mechanism in combination with the critical conditions to control the adjustment of electroporation parameters; a transfection and repair module, which is used to perform cartilage-related gene transfection and cartilage repair according to the set electroporation parameters; an evaluation and adjustment module, which is used to set a comprehensive evaluation function, evaluate the repair effect of cartilage tissue, and adjust the electroporation parameters and gene transfection strategy according to the real-time evaluation results.

[0095] The computer program product of the cartilage regeneration and repair method and device using irreversible electroporation technology provided in the embodiments of the present invention includes a computer-readable storage medium storing program code. The instructions included in the program code can be used to execute the method in the previous method embodiment. The specific implementation can be found in the method embodiment, which will not be repeated here.

[0096] Specifically, the storage medium can be a general storage medium, such as a mobile disk, a hard disk, etc. When the computer program on the storage medium is run, it can execute the cartilage regeneration and repair method of the above-mentioned irreversible electroporation technology, thereby improving the transfection efficiency of cartilage repair-related genes and promoting cartilage regeneration by precisely controlling the electroporation parameters and combining an adaptive feedback mechanism.

[0097] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a non-volatile computer-readable storage medium that is executable by a processor. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

[0098] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for cartilage regeneration and repair using irreversible electroporation technology, characterized in that: include: Establish a cell membrane electroporation model and predict the membrane perforation effect by simulating the distribution of the electric field and the potential changes of the cell membrane; According to the electroporation model, by analyzing the change in electric potential and combining the change in energy consumption of the cell membrane, the critical conditions for the formation of cell membrane pores are obtained; In combination with the critical conditions, an adaptive adjustment feedback mechanism is established to control the regulation of electroporation parameters; Cartilage-related gene transfection and cartilage repair are performed according to the set electroporation parameters; A comprehensive evaluation function was set up to evaluate the repair effect of cartilage tissue, and the electroporation parameters and gene transfection strategy were adjusted according to the real-time evaluation results.

2. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 1, characterized in that: The electroporation model of the cell membrane is established to predict the membrane perforation effect by simulating the distribution of the electric field and the potential change of the cell membrane, including: The relationship between electric field distribution and electric field strength is used to represent the electric potential of the cell membrane, and a cell membrane electroporation model is established to describe how the electric field is distributed on the cell membrane over time t. Among them, t′ is the integral variable, representing every moment from 0 to the current time t; V(t) is the cell membrane potential, which indicates the change in charge distribution at different locations on the cell membrane; E(t) is the electric field intensity, which represents the time variation of the electric field and is a function of the current pulse; r is the distribution direction and amplitude of the electric field on the cell membrane, indicating the intensity of the electric field at different locations on the cell membrane surface.

3. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 2, characterized in that: According to the electroporation model, by analyzing the change in electric potential and combining the change in energy consumption of the cell membrane, the critical conditions for the formation of cell membrane pores include: When the cell membrane potential V(t) exceeds the critical potential V threshold When the cell membrane integrity is destroyed, cell membrane pores are formed, and irreversible electroporation occurs; Set the critical condition formula for the formation of cell membrane pores V(t) ≥ V threshold ; Modeling energy consumption in cell membranes Among them, ΔE is the energy consumption in the process of cell membrane pore formation, which represents the energy accumulation of the cell membrane under the action of the electric field; C m is the capacitance of the cell membrane, reflecting the response characteristics of the membrane to the electric field; V(t) is the cell membrane potential, which changes with the electric field; When the energy ΔE accumulated by the cell membrane under the action of the electric field reaches the energy consumption threshold of the cell membrane, E threshold , which can cause the formation of cell membrane pores; Set the energy critical condition formula for cell membrane pore formation ΔE≥E threshold .

4. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 3, characterized in that: The step of establishing an adaptive adjustment feedback mechanism in combination with the critical conditions to control the adjustment of electroporation parameters includes: Monitor the cell membrane potential V(t) and the energy consumption ΔE in real time, and adjust the electroporation parameters to meet the critical potential condition formula V(t)≥V threshold The energy critical condition formula for the formation of cell membrane pores is ΔE≥E threshold At least one of; According to the relationship between repair efficiency and current intensity, the repair efficiency used to describe the repair rate of cartilage tissue under the action of the electric field is set Among them, α is an empirical constant reflecting the degree of cell response to current, β is an empirical constant determining the attenuation rate of the repair process, and I pulse (t) is the instantaneous current intensity, I max is the maximum current intensity; Set the cell survival rate S to reflect the cell repair effect survival (t) and cell proliferation S proliferation (t); Set the feedback function f eval (t) = λ1·S survival (t)+λ2·S proliferation (t)+λ3·Repair Efficiency(t), where is the comprehensive evaluation value of the repair effect, λ1, λ2 and λ3 are weight coefficients; According to the evaluation function f eval The value of (t) adjusts the electroporation parameters. If the repair efficiency is low, the current intensity and pulse width are increased. If the repair efficiency is high, the current intensity and pulse width are reduced.

5. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 1, characterized in that: The step of performing cartilage-related gene transfection and cartilage repair according to the set electroporation parameters includes: Electroporation parameters are pre-set according to the goal of cartilage repair and cell status; Formula for calculating gene transfection efficiency Among them, γ is a constant used to identify the response ability of cells to electric fields, δ is a constant representing the time decay rate during transfection, P(t) is the instantaneous current pulse intensity, and P max is the maximum current intensity; According to the relationship between electroporation parameters and gene transfection efficiency, the gene transfection efficiency can be maximized by dynamically adjusting the electroporation parameters; Select gene vectors suitable for cartilage repair; Mixing chondrocytes or related tissues with a gene vector containing a target gene; Applying a preset electric field pulse, using current pulse intensity and pulse duration, to form pores in the cell membrane, allowing the gene vector to enter the cell; Molecular biology techniques were used to verify whether the target gene was successfully expressed.

6. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 5, characterized in that: The comprehensive evaluation function is set to evaluate the repair effect of cartilage tissue, and the electroporation parameters and gene transfection strategy are adjusted according to the real-time evaluation results, including: Through histological analysis and cell counting, the number of cells in the cartilage repair area is regularly detected, and the number of cartilage repair cells C is obtained by counting repair (t); By using fluorescent labeling and / or PCR and / or Western blot technology, evaluate whether the transfected repair gene is successfully introduced and expressed, and calculate the gene transfection efficiency T gene (t); According to the number of cartilage repair cells C repair (t) and the gene transfection efficiency T gene (t), set the comprehensive evaluation function Among them, C max is the maximum number of repair cells, η and ζ are empirical constants; If the value of the comprehensive evaluation function is lower than the preset value, it means that the cell repair and proliferation is not ideal and the transfection efficiency is low. By increasing the current intensity and pulse width, the gene transfection efficiency is improved to promote cartilage repair.

7. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 6, characterized in that: If the gene transfection efficiency of the selected gene is T gene If the value of (t) is lower than the preset range, other gene vectors are selected or the vector concentration is increased.

8. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 6, characterized in that: In the comprehensive evaluation function, Reflects the proportion of repair cells, indicating the cell proliferation in the current repair process. The higher the proportion of repair cells, the closer the number of repair cells is to the maximum repair capacity, and the better the repair effect. In the comprehensive evaluation function, It reflects the effect of gene transfection efficiency on the repair effect. The higher the transfection efficiency, the more obvious the gene's promoting effect on repair and the better the repair effect.

9. The method for cartilage regeneration and repair using irreversible electroporation technology according to claim 8, characterized in that: If the selected gene has a preset optimal promoting effect on repair and the cell proliferation has reached a preset good range, the use of the gene vector is reduced or stopped to focus on further promoting cell repair.

10. A cartilage regeneration and repair system using irreversible electroporation technology, characterized in that: include: The electroporation model modeling module is used to establish the electroporation model of the cell membrane and predict the membrane perforation effect by simulating the distribution of the electric field and the potential change of the cell membrane; A critical analysis module, for obtaining critical conditions for cell membrane pore formation by analyzing potential changes in combination with changes in cell membrane energy consumption according to the electroporation model; An adaptive mechanism establishment module, used to establish an adaptive adjustment feedback mechanism in combination with the critical conditions to control the adjustment of electroporation parameters; A transfection and repair module, used for performing cartilage-related gene transfection and cartilage repair according to the set electroporation parameters; The evaluation and adjustment module is used to set a comprehensive evaluation function, evaluate the repair effect of cartilage tissue, and adjust the electroporation parameters and gene transfection strategy according to the real-time evaluation results.

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