A method for preparing a gradient-degrading cold gel for skeletal muscle repair

Abstract

The application discloses a preparation method of a gradient-degradation cold gel for repairing skeletal muscle, and a host side and a defect center side end element precursor liquid with different methacrylating double bond molar concentrations are prepared and configured, and a continuous composition field is formed through complementary flow output, static mixing and mold thickness direction filling; a freezing front is started by the host side before significant diffusion homogenization, and low-temperature cross-linking is completed in the frozen concentrated micro-phase; the obtained cold gel has no discrete layer interface, the effective cross-linking density and connected pore diameter continuously increase from the host side to the defect center side, and the host side occurs enzymatic mass loss earlier than the defect center side, which is used for jointly calibrating the formula and the freezing procedure, and is suitable for directional repair of volumetric skeletal muscle defects.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials and tissue engineering scaffolds, and more particularly to a method for preparing a gradient-degradable skeletal muscle repair cryogel. Background Technology

[0002] Volumetric skeletal muscle defects disrupt the continuity of muscle fibers, microvessels, and extracellular matrix. In the early stages of repair, scaffolds need to be implanted to maintain the defect space and resist deformation caused by muscle contraction. Subsequently, the scaffolds need to gradually make room to allow host-derived blood vessels and myogenic cells to enter and form new tissue structures.

[0003] Existing cryogels typically employ a homogeneous polymer composition and cross-linking conditions throughout the entire structure. When the degree of cross-linking is too low, the material may experience rapid mass loss and collapse of the supporting structure in the early stages of repair; conversely, when the degree of cross-linking is too high, the material may remain for a long time, increasing the risk of foreign body reactions and collagen deposition. Simply changing the pore size may also negate the regulatory effect of chemical cross-linking on the degradation rate due to changes in mass transfer and enzyme entry rates, making it difficult to stably obtain a degradation front that propagates from the surviving muscle tissue side towards the defect center.

[0004] Existing technologies lack a preparation process that can simultaneously establish a continuous composition gradient, an effective crosslinking density gradient, and a directional interconnected pore gradient within a cold gel without discrete layer interfaces, and jointly calibrate the formulation and freezing procedure by measuring the enzymatic mass loss sequence. Summary of the Invention

[0005] The purpose of this invention is to address the problem that uniformly cross-linked cryogels struggle to simultaneously provide early structural support and later tissue relocation. To this end, this invention utilizes two end-member precursor solutions with different compositions and double bond concentrations to form a compositional field that continuously varies along the thickness direction. Using the freezing front crossing time being less than the diffusion homogenization characteristic time as a gradient locking rule, controlled freezing is initiated from the host side, and free radical low-temperature cross-linking is completed in the frozen concentrated microphase. Through joint calibration of the formulation-freezing procedure, the synergistic effect of chemical cross-linking and pore size mass transfer is ensured to maintain the order of degradation—first on the host side and then later on the defective center side—thus obtaining a gradient-degrading cryogel without discrete layer interfaces.

[0006] This invention provides a method for preparing a gradient-degrading skeletal muscle repair cryogel, comprising: S1, preparing a host-side endmember precursor solution H and a defect-center-side endmember precursor solution C, both containing methacrylated fucoidan, methacrylated gelatin, and a free radical initiation system, such that the mass ratio of methacrylated fucoidan to methacrylated gelatin and the molar concentration of methacrylated double bonds in the host-side endmember precursor solution H are less than the corresponding values ​​in the defect-center-side endmember precursor solution C, thereby obtaining an endmember precursor solution group; S2, outputting the endmember precursor solution group at complementary flow rates, and after static mixing, filling the mold along the thickness direction, such that the volume fraction of the host-side endmember precursor solution H decreases from the host side to the defect side. S3. The freezing front continuously decreases at the center side to form a composition field; S4. The freezing front is initiated and adjusted from the host side to obtain a frozen gradient body by making the freezing time less than the diffusion homogenization characteristic time when the freezing time is less than the ...

[0007] Optionally, S1 includes: determining the degree of substitution of methacrylamide fucoidan and methacrylamide gelatin respectively, and calculating the molar concentration of methacrylamide double bonds in each end-member precursor solution based on the degree of substitution and the feed mass;

[0008] The host-side endmember precursor solution H and the defect-center-side endmember precursor solution C were prepared using the same batch of buffer solution and free radical initiation system, so that the total polymer mass, initiator amount and buffer salt amount per unit volume of the two endmember precursor solutions were the same.

[0009] The mass ratio of the methacrylamide fucoidan to the methacrylamide gelatin is calculated based on the mass of the feed, and the mass ratio and the molar concentration of the methacrylamide double bond are used as the formula input parameters for the two metering units.

[0010] Furthermore, it also includes: setting at least three candidate levels for the mass ratio and the molar concentration of the methacrylamide double bond, preparing candidate end-member samples and measuring the equilibrium swelling ratio and enzymatic mass loss curve, and determining the effective crosslinking density of each candidate end-member sample based on the equilibrium swelling ratio;

[0011] From the candidate end-member samples, select a formulation combination in which the effective crosslinking density of the host-side end-member sample is less than that of the defective center-side end-member sample, and the time taken for the host-side end-member sample to reach the same proportion is less than the corresponding time taken for the defective center-side end-member sample. Write the mass ratio and molar concentration of the methacrylated double bond in the formulation combination into the formulation control record.

[0012] Optionally, S2 includes: setting a first metering unit and a second metering unit respectively connected to the host-side end-member precursor fluid H and the defect-center-side end-member precursor fluid C; setting the sum of the instantaneous flow rates of the first metering unit and the second metering unit as a constant total flow rate; setting the ratio of the instantaneous flow rate of the host-side end-member precursor fluid H to the constant total flow rate as a flow rate function that continuously decreases from 1 to 0 with the normalized filled mold volume; and setting the instantaneous flow rate of the defect-center-side end-member precursor fluid C as the difference between the constant total flow rate and the instantaneous flow rate of the host-side end-member precursor fluid H.

[0013] The flow function is time-shifted and compensated based on the retention volume of the static mixer so that the end-member volume fraction at the outlet of the static mixer corresponds to the thickness position in the mold.

[0014] The mass fraction of locally methacrylated fucoidan collected along the thickness direction of the mold was used as the verification data of the composition field, and the metered flow rate, cumulative filling volume and corresponding thickness position were recorded.

[0015] Optionally, S3 includes: at the same temperature and ionic strength as the molding process, measuring the difference between the upper and lower bounds of the mass fraction of methacrylamide fucoidan and the difference between the upper and lower bounds of the molar concentration of methacrylamide double bonds in the gradient region, respectively, taking the time required for each difference to decay to one-third of its initial value as a candidate feature time, and determining the first candidate feature time after arranging the two candidate feature times in ascending order of numerical values ​​as the diffusion homogenization feature time;

[0016] By recording the moment when each temperature measuring point first reaches the freezing temperature of the precursor liquid by setting temperature measuring points along the thickness direction of the mold, the time difference between the arrival time of the host-side measuring point and the defect center-side measuring point is determined as the time for the freezing front to cross the gradient region.

[0017] Based on the comparison results of the two times, the temperature change curves of each temperature control zone of the mold are set so that the time for the freezing front to cross the gradient zone is less than the diffusion homogenization characteristic time, and the temperature drop per unit time of the host side temperature control zone is greater than the temperature drop per unit time of the defect center side temperature control zone.

[0018] Furthermore, it also includes: preparing calibration samples using multiple constant freezing front advance velocities, calculating the freezing front advance velocity based on the arrival time of the temperature measurement points of each calibration sample, and determining the corresponding median of the equivalent connected aperture, and establishing a calibration curve with the freezing front advance velocity as the input and the median of the equivalent connected aperture as the output.

[0019] The median profile of the candidate equivalent connecting aperture, which monotonically increases from the host side to the defect center side, is used as the initial target profile. Based on the initial target profile, the advance velocity of the target freezing front between each adjacent temperature measurement point is calculated from the calibration curve. The absolute value of the difference between the measured advance velocity of the freezing front and the advance velocity of the target freezing front is used as the velocity deviation.

[0020] When the speed deviation is greater than the allowable deviation determined by the temperature measurement uncertainty and the distance between measuring points, the freezing abnormality interlock is triggered, the batch is stopped from entering step S4 and the arrival time of each temperature measuring point is saved. After the temperature change curve of the corresponding temperature control zone is corrected, step S3 is re-executed.

[0021] If the degradation sequence verification in step S5 fails, the median profile of the candidate equivalent connected aperture is corrected according to the degradation sequence parameter deviation at each sampling location, and the corrected profile that has passed the verification in step S5 is written into the target aperture process parameters.

[0022] Optionally, S4 includes: using a redox initiation system composed of persulfate and tertiary amine, allowing free radical crosslinking of the methacrylamide double bonds in the frozen concentrated microphase while the precursor solution is kept frozen, and determining the gel fraction at the plateau region of the gel fraction change curve with crosslinking time as the crosslinking endpoint;

[0023] After thawing, the material was washed with buffer solution. The upper limit of the concentration of unreacted initiator that did not produce a decrease in cell viability in the pre-cell compatibility test was determined as the safety threshold for the biomaterial.

[0024] If the gel fraction does not reach the crosslinking endpoint or the concentration of unreacted initiator in the wash solution is greater than the safety threshold of the biomaterial, the treatment cycle is extended by the same duration as the original crosslinking or washing time and the test is repeated. If the retest still fails to meet the standard, the batch is discarded and does not proceed to step S5.

[0025] The effective crosslinking density at each sampling location along the thickness direction is determined based on the equilibrium swelling data. The median of the equivalent connected pore diameter at each sampling location is determined based on the three-dimensional pore structure image. The composition distribution along the thickness direction is examined using continuous cross-sectional imaging with a scanning step size no greater than one-twentieth of the gradient region thickness. When the mass fraction of locally methacrylated fucoidan and the molar concentration of locally methacrylated double bonds are both located between their respective endmember values ​​and change monotonically, and there is no compositional abrupt interface extending along the cross-section of the cold gel, it is determined that the cold gel has no discrete layer interface.

[0026] Optionally, S5 includes: setting multiple sampling positions along the thickness direction of the cold gel at the same interval, including sampling positions on the host side and sampling positions on the defect center side; using a mixed enzyme system containing collagenase and fucoidan degrading enzyme; using the number of enzyme activity units contained in a unit volume of enzyme solution as the enzyme concentration calibrator; and obtaining data on the change of mass loss over time at each sampling position under the same enzyme concentration, temperature and oscillation conditions.

[0027] The in vitro enzymatic culture period preset according to the target repair cycle is determined as the verification time limit. All sampling locations within the range of 20% to 40% mass loss ratio can reach the same ratio within the verification time limit. The time for each sampling location to reach the same ratio is used as the degradation sequence parameter, and the degradation sequence parameter is verified to increase monotonically from the host side to the defect center side.

[0028] If the same ratio cannot be selected within the verification time limit, extend the verification time limit by one time limit and retest. If it still cannot be selected after retesting, record the reason for selection failure, adjust the mass ratio of the end-member precursor solution or the molar concentration of the methacrylamide double bond, and go back to step S1 to prepare and verify again.

[0029] When the verification result does not meet the monotonically increasing constraint, record the reason for the abnormality, adjust the mass ratio of the end-member precursor liquid or the molar concentration of the methacrylamide double bond and the temperature change curve of the corresponding temperature control zone, and go back to step S1 to prepare and verify again.

[0030] Furthermore, it also includes: before verifying the order of enzymatic mass loss, performing cyclic compression tests, equilibrium swelling tests, and three-dimensional pore structure tests on each sampling location; determining the effective crosslinking density of each sampling location based on the equilibrium swelling test results; and determining the median of the equivalent connected pore size of each sampling location based on the three-dimensional pore structure test results.

[0031] The ratio of the peak contractile stress measured before implantation of the target defect to the upper limit of the physiological contractile strain measurement range of the surviving muscle tissue in the target defect is determined as the support modulus threshold, and the upper limit of the physiological contractile strain measurement range is used as the set contractile strain for no less than one thousand cycles of compression.

[0032] The following conditions are set together as the pass conditions for joint calibration: the initial compression modulus of the host-side sampling position is not less than the support modulus threshold;

[0033] The compression modulus retention rate after cyclic compression is not less than 80%, and the cold gel has no through cracks.

[0034] Both the effective crosslinking density and the median equivalent interconnected pore size increase monotonically from the host side to the defect center side.

[0035] The degradation sequence parameter increases monotonically from the host side to the defect center side;

[0036] The feeding mass and degree of substitution of the two end-member precursor liquids in step S1 are used as the formulation record field, and the moment when each temperature measuring point first reaches the freezing temperature of the precursor liquid in step S3 is used as the temperature record field. The formulation record field, temperature record field, pore structure test results, cyclic compression test results, enzymatic mass loss data and abnormal reasons are written into the batch verification record.

[0037] Furthermore, it also includes: after meeting the pass conditions of the joint calibration, forming a notch or flange on the host-side edge of the cold gel that is asymmetrical with the shape of the defect center-side edge, and writing the shape code of the notch or flange and the correspondence with the host-side orientation into the batch verification record to form a directional implantation error prevention mark.

[0038] The beneficial effects of this invention are:

[0039] 1. By using complementary metering of the two-terminal precursor liquids, static mixing, and continuous molding along the thickness direction, a continuous composition field is formed in the cold gel, avoiding the interlayer interface caused by discrete layered structures.

[0040] 2. By initiating directional freezing before significant diffusion homogenization and crosslinking in the frozen concentrated microphase, the effective crosslinking density and directional connectivity pore size continuously change from the host side to the defect center side, taking into account both the early mechanical requirements of the host side and the continuous support and mass transfer pathway of the center side.

[0041] 3. By combining the calibration of end-member formulations and freezing procedures with the measured enzymatic mass loss sequence along the thickness direction as constraints, the influence of pore size mass transfer effect on the chemical degradation trend can be corrected, so that the degradation front advances in a predetermined direction.

[0042] 4. By forming asymmetrical anti-mistake markers on the host side and recording their directional correspondence, it is easy to directionally implant the cryogel towards the surviving muscle tissue with the host side facing it. Attached Figure Description

[0043] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0044] Figure 1 This is a flowchart of a method for preparing a gradient degradation skeletal muscle repair cryogel according to the present invention.

[0045] Figure 2 This is a flowchart of the S3 directional freezing and gradient locking sub-process of the present invention. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, illustrating only the basic structure of the invention, and therefore only show the components relevant to the invention.

[0047] refer to Figures 1-2 A method for preparing a gradient-degrading skeletal muscle repair cryogel includes: S1, preparing a host-side endmember precursor solution H and a defect-center-side endmember precursor solution C, both containing methacrylated fucoidan, methacrylated gelatin, and a free radical initiation system, such that the mass ratio of methacrylated fucoidan to methacrylated gelatin and the molar concentration of methacrylated double bonds in the host-side endmember precursor solution H are less than the corresponding values ​​in the defect-center-side endmember precursor solution C, to obtain an endmember precursor solution group; S2, outputting the endmember precursor solution group at complementary flow rates, and after static mixing, filling the mold along the thickness direction, so that the volume fraction of the host-side endmember precursor solution H increases from the host side to the defect-center side. S3. The freezing front is continuously reduced to form a composition field; S4. The time for the freezing front to cross the composition field gradient region is less than the diffusion homogenization characteristic time. The freezing front is initiated and adjusted from the host side to obtain a frozen gradient body; S5. Free radical low-temperature cross-linking is performed in the frozen concentrated microphase of the frozen gradient body. After thawing and washing, a cold gel without discrete layer interfaces is obtained. The effective cross-linking density and the interconnected pore size increase continuously from the host side to the defect center side; S6. The enzymatic mass loss order of the cold gel along the thickness direction is verified. With the mass loss occurring on the host side before the defect center side as a constraint, the end-member precursor solution formulation and freezing procedure are combined to obtain a gradient degradation skeletal muscle repair cold gel.

[0048] In this specific embodiment, S1 includes:

[0049] This specific embodiment uses the preparation of a gradient cold gel with a thickness of 8 mm and a planar size of 40 mm × 20 mm as the same process scenario. The same batch of methacrylamide fucoidan, methacrylamide gelatin, phosphate buffer with pH 7.4 and ionic strength of 0.15 mol / L, and redox initiation system composed of ammonium persulfate and tetramethylethylenediamine were selected. The batch number of raw materials, solid content, moisture content, degree of substitution test results and retest date were all recorded in the formula control record. All solution preparation and sampling containers were sterilized and operated at 8 ℃ in the dark.

[0050] Two methacrylamide polymers were measured three times in parallel using nuclear magnetic resonance hydrogen spectroscopy. The degree of substitution was calculated by the ratio of the proton integral area of ​​the methacrylamide olefin to the characteristic proton integral area of ​​each polymer that was not affected by the modification. The amount of substitutable group per unit mass obtained from batch inspection was used as the conversion benchmark. When the relative standard deviation of the three measurements was not greater than 5%, the arithmetic mean was taken. If it exceeded 5%, the sample was re-dissolved and re-tested. If the re-test still exceeded the limit, the batch of raw material was not included in the preparation of the end-member precursor solution.

[0051] For any end-member precursor liquid, according to Calculate the mass ratio of methacrylated fucoidan to methacrylated gelatin. The symbol represents the mass ratio of the end-member precursor solution and is a dimensionless quantity. The symbol indicates the feed mass of methacrylamide fucoidan in this endmember, and the unit is g. The symbol indicates the mass of methacrylamide gelatin added to this endmember, and the unit is g. The symbol represents the endmember category and is taken as either host-side endmember H or defect-center-side endmember C;

[0052] Based on the degree of substitution and the quality of feed, Calculate the molar concentration of the methacrylated double bond. The symbol indicates the molar concentration of methacrylated double bonds in the end-member precursor solution, and the unit is mol / L. The symbol represents the amount of substituted groups per unit mass of methacrylamide fucoidan, and the unit is mol / g. The symbol represents the degree of substitution of methacrylamide fucoidan and is a dimensionless quantity. The symbol represents the amount of amino substance that can be substituted per unit mass of methacrylamide gelatin, and the unit is mol / g. The symbol represents the degree of substitution of methacrylamide gelatin and is a dimensionless quantity. The symbol represents the constant volume of the corresponding end-member precursor solution and the unit is L. Both terms in the formula are the amount of substance of the double bond and are closed by dividing by the constant volume to get mol / L.

[0053] In this batch, the concentrations of methacrylated fucoidan and methacrylated gelatin in the host-side endmember precursor solution H were set at 25 mg / mL and 75 mg / mL, respectively, and the corresponding concentrations in the defect-center endmember precursor solution C were set at 45 mg / mL and 55 mg / mL, respectively, so that the total polymer concentration of both endmembers was 100 mg / mL and the mass ratio of the host side was less than that of the defect-center side. After calculating the molar concentration of the double bond based on the measured value of the degree of substitution in this batch, the formulation was released only when the value of the host side was less than that of the defect-center side.

[0054] Add the same batch of buffer solution to both end units to ensure that the final concentrations of ammonium persulfate and tetramethylethylenediamine are both 5 mol / m³. Ensure that the amount of buffer salt per unit volume is the same by adding sodium chloride and phosphate at constant volume. After preparation, sterilize by passing through a 0.22 μm low-protein adsorption filter membrane and degas at 8 °C for 20 min. Measure pH, total polymer concentration, initiator concentration, and osmotic pressure. If the relative difference between the end units for any corresponding item exceeds 5%, the sample is not included in the metering unit. For each batch, take three additional samples of the same formulation and perform small-amplitude oscillatory rheology at 8 °C, measuring complex viscosity and storage modulus every 5 min. Determine the earlier of the time when the complex viscosity increases by 10% relative to the initial coexistence of the two initiating components and the time when the storage modulus first reaches the loss modulus as the applicable period for that batch of initiation system. The cumulative time from the addition of the last initiating component to all five locations in the mold reaching their respective freezing initiation temperatures should not exceed half of this applicable period. On the production line, every 5... The viscosity increase is verified by the bypass microsample; if the cumulative time exceeds the limit, the viscosity increase reaches 10%, or visible gel particles appear, the precursor liquid and the current sample are immediately isolated and re-numbered from step S1. The applicable period, cumulative time, viscosity increase and disposal results are written into the formulation control record.

[0055] To determine the end-member combination, three candidate levels were set around the mass ratio of the host side and the defect center side, respectively. Three double bond molar concentration levels were formed by adjusting the degree of substitution of the same batch of polymers or the feeding ratio of the two polymers. Nine orthogonal combinations were used to prepare candidate end-member samples with a diameter of 10 mm and a thickness of 2 mm, with no less than five parallel samples in each group. The candidate level, measured double bond molar concentration, crosslinking time and sample number were written into the candidate end-member screening table.

[0056] After the candidate end-member samples reached equilibrium swelling data in phosphate buffer at 37 °C and achieved two consecutive weighing changes of less than 1%, the reciprocal of the volume swelling ratio was used as the polymer volume fraction and then... Determine the effective crosslinking density. The symbol represents the effective crosslinking density and the unit is mol / m³. The symbol represents the polymer volume fraction under equilibrium swelling conditions, and is a dimensionless quantity that satisfies the following conditions: , The symbols represent the Flory interaction parameters between the same batch of polymer and the buffer solution, and are dimensionless quantities obtained by fitting from equilibrium swelling calibration experiments. The symbol represents the equivalent molar volume of the solvent in the buffer solution, and the unit is m³ / mol. In this batch, the density of the same formulation buffer solution was measured three times at 37 °C using a 10 mL specific gravity bottle calibrated with pure water. The average density was 0.993 g / mL, which was converted by dividing the molar mass of water (18.015 g / mol) by this density. The value is 0.00001814 m³ / mol, and the temperature, tertiary density, instrument number, and conversion value are written into the equilibrium swelling calibration record;

[0057] The measurement object is clearly defined as each numbered candidate end-member sample. The equilibrium swelling measurement uses wet mass, dry mass and the volume swelling ratio converted from the density of the two materials. The enzymatic measurement uses the mass loss ratio converted from the freeze-dried mass before and after culture. Each candidate level has at least five parallel samples and the instrument is calibrated with standard weights and standard volume blocks. Only when the effective crosslinking density and degradation time on the host side are both less than the corresponding center side value is the combination determined to meet the directionality requirement.

[0058] The candidate set will be constructed based on the directional requirements of the formulations, and then... Calculate the time difference of endmember degradation. The symbol represents the time difference between the defect center and the host side reaching 30% mass loss, and the unit is d. The symbol indicates the time it takes for the sample at the center of the defect to reach a 30% mass loss, and the unit is days (d). The symbol represents the time when the host-side end-member sample reaches 30% mass loss and the unit is d. The time difference must be greater than 0 and the larger the value, the more sufficient the degradation order margin.

[0059] First, delete any combination whose concentration, pH or osmotic pressure exceeds the raw material inspection range. Then, sort the candidates by end-member degradation time difference from largest to smallest. If the order is the same, select the combination with the smallest deviation in total polymer concentration between the two end-members. Write the selected combination number, order value and reason for deletion into the formulation control record.

[0060] The formulation joint calibration status record includes the mass ratio of the two end members, the molar concentration of double bonds, the degradation sequence parameters that reach the same proportion at each sampling location, the effective crosslinking density, the median of the equivalent interconnected pore size, and the cyclic compression results. The executable actions are limited to adjusting the polymer feed mass on the host side or the defect center side, replacing the batch with the tested degree of substitution, and sending a temperature control curve correction request to step S3. The goal is to make the degradation sequence parameters monotonically increase from the host side to the defect center side and to ensure that all mechanical and pore structure constraints are passed simultaneously.

[0061] The results of cyclic compression, equilibrium swelling and three-dimensional pore structure tests obtained before the verification of the enzymatic mass loss order, together with the temperature recording field, are used as read-only feedback input. If the same proportion cannot be achieved at all positions within the range of 20% to 40% within the verification time limit, the verification time limit is extended for retesting. If the retest still fails or the monotonic increase constraint is not met, the reason for the abnormality is recorded and the process is rolled back to this step to prepare again.

[0062] During the rollback period, both metering units remain in a stopped state. The formula that fails to pass is not written into the release area and the corresponding sample is isolated and stored. Only when the new combination completes the end-member screening again and is subsequently compressed for no less than one thousand cycles with the upper limit of the physiological shrinkage strain measurement range as the set shrinkage strain, and the compression modulus retention rate is not less than 80%, the cold gel has no through cracks and the degradation sequence is passed, will the formula version be updated and the safety status be released.

[0063] The formula input record is constructed sequentially according to the raw material batch number, end-member category, polymer feed mass, degree of substitution, amount of substitutable group per unit mass, fixed volume, calculated mass ratio, calculated double bond molar concentration, amount of initiator, amount of buffer salt, and version status. If any required field is empty, the units are inconsistent, or the two gradient indicators on the host side are not less than the corresponding values ​​on the center side, the generation program will refuse to issue the record and keep the metering unit stopped.

[0064] Candidate end-member samples are placed in the same mixed enzyme system as in step S5 to obtain enzymatic mass loss curves. Formula combinations in which the effective crosslinking density on the host side is less than the effective crosslinking density on the defect center side and the time for the host side to reach 30% mass loss is less than the corresponding time on the defect center side are selected. The final feed mass, degree of substitution, mass ratio, double bond molar concentration, total polymer concentration, initiator concentration, and buffer salt concentration are written into the formula control record that can be read by the two metering units and the end-member precursor solution group is output.

[0065] In this specific embodiment, S2 includes:

[0066] The first metering unit is connected to the host-side end-member precursor solution H, and the second metering unit is connected to the defect center-side end-member precursor solution C. The two lines use low-temperature resistant injection pump lines with the same inner diameter and merge into a static mixer with six built-in spiral mixing units and a measured retention volume of 0.24 mL. The outlet of the static mixer is close to the host-side inlet of the mold. The effective volume of the mold is 6.4 mL and the thickness direction coordinate points from 0 mm on the host side to 8 mm on the defect center side.

[0067] The constant total flow rate was set to 1.20 mL / min. The ratio of the actual cumulative volume entering the mold to the effective volume of the mold was used as the normalized filled volume, and then... Generate host-side endmember volume fraction function. The symbol represents the volume fraction of the target host-side endmember at the static mixer outlet, and is a dimensionless quantity with a value ranging from 0 to 1. The symbol represents the normalized filled volume, which is dimensionless and ranges from 0 to 1; for any given time... , The symbol represents the time calculated from the start of the two metering units, with the unit being min and the start time being 0. The controller uses the two pumps from start time 0 to time 1. The sum of the cumulative output volumes of the feedback circuits minus the cumulative sampling volume of the bypass circuits during the same period yields the actual cumulative volume entering the mold. This volume is then divided by 6.4 mL to obtain the final volume. , The value is 0 when the start time is earlier than the start time, and 1 when the actual cumulative volume reaches or exceeds 6.4 mL. The function is equal to 1 at the start of the filling process and equal to 0 at the end of the filling process, and continuously and monotonically decreases within the interval.

[0068] Based on the static mixer retention volume Determine the time shift amount of the flow command. The symbol represents the static mixer residence time, and the unit is min. The symbol represents the static mixer retention volume calibrated by the weighing method, and the unit is mL. The symbol represents the constant total flow rate of the two metering units and the unit is mL / min. The compensation time for this batch is 0.20 min.

[0069] The controller reads the target volume fraction one residence compensation time in advance at the moment of mold filling, and then... and Output complementary flow, Symbols represent time. The instantaneous flow rate of the host-side endmember precursor solution H, in mL / min. Symbols represent time. The instantaneous flow rate of the precursor solution C at the defect center end cell, in mL / min. The symbol represents the time since the metering unit started, and the unit is minutes. The symbol represents the normalized feedforward prediction of the filled modulus volume at the compensation time and is dimensionless; the controller only uses the time. The actual cumulative volume already obtained within the mold, plus the constant total flow rate, is used to calculate the volume. The expected output volume is subtracted from the bypass sampling volume scheduled in the control table that falls within the compensation interval. The resulting volume is divided by 6.4 mL and truncated to the range of 0 to 1. Future pump feedback is not read. The outlet weighing value is recalculated to the predicted value according to the residence compensation time. The comparison is performed every 100 ms. When the absolute volume deviation exceeds 0.02 mL for three consecutive cycles, the mold filling is paused and the uncrosslinked sample is discarded. This ensures that the sum of the two flow rates is equal to the constant total flow rate at any time and that the feedforward relationship can be executed in real time.

[0070] Before formal mold filling, the two pumps were calibrated by weighing at three flow rates of 0.10, 0.60, and 1.10 mL / min, with a single-point flow rate relative error not exceeding 2%. Subsequently, the dead volume of the pipeline was replaced with end-member precursor solution twice to remove air bubbles; the flow rate was then set to the corresponding... The host-side end-members are pre-filled with a retention volume of no less than twice that of the static mixer and outlet pipeline, and all of them are directed to the waste liquid. When the local mass fraction of three consecutive 20 μL microsamples at the outlet deviates from the measured value of the host-side end-members by no more than 2%, the waste liquid branch is closed, the mold inlet is opened, and this switching moment is defined as the start of formal mold filling. The controller records the two flow commands, pump feedback flow, two cumulative output volumes, bypass cumulative sampling volume, actual cumulative volume in the mold after deducting the sampling, the calculated normalized filled volume, and the corresponding thickness position at a period of 100 ms. When the flow rate deviates from the command by more than 3% for three consecutive cycles, the mold filling is paused and the uncrosslinked sample is discarded.

[0071] The statically mixed precursor liquid is continuously advanced along the thickness direction through a slit inlet parallel to the mold plane. The mold is maintained at 8 °C to limit diffusion during filling. Whenever the actual cumulative volume inside the mold, after deducting previous sampling volumes, reaches the next integer multiple of 0.8 mL, a 20 μL verification sample is taken from the bypass, for a total of eight times, with a total sampling volume of 0.16 mL. After each sampling, the 20 μL is immediately added to the cumulative sampling volume in the bypass. Both pumps continue to output until the cumulative output volume reaches 6.56 mL, confirming that the actual cumulative volume inside the mold reaches 6.4 mL, to compensate for diversion and avoid underfilling at the end. When the actual cumulative volume inside the mold reaches 6.4 mL, the mold inlet is immediately closed, and the remaining tailings in the static mixer and outlet pipes are switched to the waste liquid branch. The tailings must not flow back or continue to be forced into the mold. The outlet corresponds to... The endpoint boundary was confirmed to be valid when the deviation of the measured mass fraction of three consecutive 20 μL microsamples from the defect center end element was no greater than 2%. The local mass fraction was converted using the sulfur content calibration curve established with the same batch of methacryloylfucose. The calibration curve recorded the sulfur content, polymer mass fraction, blank value, recovery rate and batch version. The calibration curve was re-established when the recovery rate was not between 95% and 105%.

[0072] The mass fractions of locally methacrylated fucoidan in the eight verification samples are sorted according to their corresponding thickness positions. Each measured value is required to be between the measured values ​​of the two endmembers, with no reverse increase between adjacent positions, and the absolute deviation from the target composition function conversion value is no greater than 8% of the endmember difference. When the conditions are met, the metered flow rate, cumulative mold volume, thickness position, local mass fraction, and calibration curve version are written into the composition field record, and the continuous composition field in the mold is transferred to step S3 at 8 ℃.

[0073] In this specific embodiment, S3 includes:

[0074] Before production molding begins, use the same batch of end-member precursor solution, or use an equivalent calibration batch whose total polymer concentration, the mass ratio of the two polymers, the molar concentration of double bonds, the complex viscosity, and the ionic strength all deviate from the production batch by no more than 5%. At the same 8 ℃ and 0.15 mol / L ionic strength as the molding batch, form an initial gradient consistent with the mold in an 8 mm diffusion cell. Every 5 minutes, take equal volumes of microsamples from both the host side and the defect center side, and measure the mass fraction of methacrylated fucoidan and the molar concentration of double bonds, respectively. Calculate two gradient decay curves using the absolute value of the difference between the two measured values. Production molding can only proceed after the calibration results have been verified and a valid version has been issued. Production teams must not remain at the site waiting for diffusion calibration.

[0075] The time required for the mass fraction difference of fucoidan to decay to one-third of its initial value was recorded as a candidate time, and the time required for the molar concentration difference of double bonds to decay to one-third of its initial value was recorded as another candidate time. Determine the diffusion homogenization characteristic time. The symbol represents the diffusion homogenization characteristic time and the unit is min. The symbol represents the time required for the mass fraction difference to decay to one-third of its initial value, and the unit is min. The symbol represents the time required for the molar concentration difference of the double bond to decay to one-third of its initial value, and the unit is min. Both candidate times are obtained by linear interpolation between adjacent sampling points.

[0076] T-type micro thermocouples, calibrated at both the freezing point and 0 °C, were placed at positions of 0, 2, 4, 6, and 8 mm along the mold thickness direction. The sampling period was 1 s. The freezing initiation temperature of the precursor liquid at that position, measured by differential scanning calorimetry, was taken as the arrival time of the freezing front. The time it takes for the frozen front to cross the gradient region is obtained. The symbol represents the time taken for the freezing front to cross an 8 mm gradient zone, and the unit is min. The symbol indicates the arrival time of the freezing front at the side measuring point of the defect center, and the unit is min. The symbol indicates the arrival time of the freezing front at the host-side metric point, and the unit is min. The subscript... This indicates that the arrival time chain is frozen;

[0077] Five independent temperature control zones are set at the bottom of the mold and numbered from the host side to the defect center side. The host side temperature control zone first cools down to -20 ℃ at 4 ℃ / min and then continues to cool down to -35 ℃. The remaining temperature control zones are started sequentially according to the arrival time of the adjacent measurement points and the cooling rate decreases from 3.2 ℃ / min to 1.6 ℃ / min. The controller only releases the temperature program when the freezing front crossing time is less than 0.8 times the diffusion homogenization characteristic time, thus leaving a safety margin for temperature measurement and fitting errors.

[0078] During the freezing procedure calibration, calibration samples with the same formulation were prepared using four constant freezing front advance speeds of 0.05, 0.10, 0.20, and 0.30 mm / min. Three parallel samples were prepared for each speed. The actual speed was calculated based on the distance between adjacent temperature measurement points and the time difference of arrival. The median of the equivalent connected aperture along the freezing direction of each sample was obtained by micron-CT. After removing abnormal measurements that deviated from the median of the three measurements by more than 15%, a monotonic calibration curve was established with the freezing front advance speed as input and the median of the equivalent connected aperture as output using piecewise linear interpolation.

[0079] The median of the initial target equivalent connectivity aperture for five nodes from the host side to the defect center side was set to 80, 110, 140, 170, and 200 μm. The target aperture for each adjacent interval was uniquely taken as the arithmetic mean of the target apertures of the upstream and downstream nodes within that interval, resulting in four interval target apertures of 95, 125, 155, and 185 μm. Then, the advance velocity of the target freezing front in each of the four intervals was calculated from the same calibration curve. Calculate the measured propulsion speed between adjacent measuring points. The symbol represents the first The measured freezing front advance velocity between adjacent measuring points, in millimeters per minute. The symbol represents the first The distance between adjacent measuring points, in mm. The symbol indicates the arrival time of the freezing front at the downstream measuring point in this interval, and the unit is min. The symbols indicate the arrival time of the freezing front at the upstream measuring point in this interval, and the unit is min; the five measuring points are numbered 0, 1, 2, 3, and 4 sequentially from the host side to the defect center side, corresponding to 0, 2, 4, 6, and 8 mm respectively. , , , and , The symbol indicates that the adjacent interval indices are frozen and only 0, 1, 2, and 3 are used; the subscript... Throughout this step, the object being measured for temperature or the area being frozen is always indicated.

[0080] For each adjacent interval, Calculate speed deviation, The symbol represents the first The speed deviation of each freezing zone is expressed in millimeters per minute. The symbol represents the advance velocity of the target freezing front, which is derived from the target aperture in this interval via a calibration curve, and the unit is millimeters per minute. and The symbols are defined in the previous section and Using only 0, 1, 2, and 3, the allowable deviation is obtained by recalculating the interval velocity based on the arrival time variation corresponding to the thermocouple temperature expansion uncertainty and the 0.1 mm measuring point positioning error according to the most unfavorable combination;

[0081] If the velocity deviation in any interval is greater than the allowable deviation, the freezing front crossing time is not less than the diffusion homogenization characteristic time, or the defect center side reaches the freezing initiation temperature before the host side, the controller triggers the freezing abnormality interlock, maintains the current temperature, saves all temperature timings, and prevents the batch from entering step S4; the abnormally frozen sample is immediately isolated and must not be thawed and then used as a production sample to execute step S3, the start time or cooling slope of the corresponding temperature control zone is corrected according to the deviation direction, and it is first verified with the existing calibration sample. After the verification is passed, it is renumbered from step S1 and a new candidate batch is completely prepared, and then steps S2 to S5 are executed in sequence;

[0082] The temperature control zone program table stores the starting temperature, start delay, cooling slope, termination temperature and holding time for each zone. The controller compares the measured temperature with the program table every second. The cooling rate per unit time in the host-side temperature control zone is significantly greater than that in the defect-center-side temperature control zone. If the deviation exceeds 1°C for 10 consecutive seconds, the current command is frozen and the abnormal interlock is entered without continuing to advance the freezing front.

[0083] Each node recorded in the calibration curve simultaneously includes the freezing front advance speed, the median of the cubic aperture, the temperature measurement batch, the micron CT segmentation threshold, and the expiration date. When calculating the target speed, linear interpolation is performed between the two nodes surrounding the target aperture. If the target aperture exceeds the calibration range, instead of extrapolating, the candidate profile is marked as unexecutable and the process returns to the aperture profile setting stage.

[0084] Before verifying the enzymatic mass loss sequence, step S5 generates the test results of cyclic compression, equilibrium swelling, and three-dimensional pore structure. The freeze feedback interface only reads the degradation sequence parameters, compression modulus retention rate, and through crack status at each location. Only when the upper limit of the physiological shrinkage strain measurement range is used as the set shrinkage strain for no less than one thousand cyclic compressions, and the compression modulus retention rate after cyclic compression is not less than 80% and there are no through cracks in the cold gel, is it allowed to correct the pore profile based on the degradation deviation.

[0085] The freeze feedback record uses the batch number and sampling location number as a joint key, and includes the same mass loss ratio, the measured time for each location to reach that ratio, the target degradation order parameters for the five locations, the batch from which the target value comes, the target version, the measured pore size and target pore size for the five nodes, the target pore size for the four intervals obtained by the arithmetic mean of the target pore sizes of adjacent nodes, the target velocity for the corresponding interval, the number of compression cycles, the compression modulus retention rate, and the crack state. When establishing the target for the first time, the arrival time of the host side and the defect center side endmembers of the same candidate formulation under the selected same mass loss ratio in step S1 is read. The time difference between the two ends is divided into four consecutive intervals and assigned to five locations of 0, 2, 4, 6, and 8 mm in sequence to form a strictly incremental initial target. The target version is frozen within the current verification cycle. Failed batches cannot be updated. Only when three consecutive batches of the same formulation appear in the same direction at the same location and the absolute value exceeds 1 can the target version be released. When the deviation of d is less than 1, the arithmetic mean of the measured times of the three batches is used to generate a candidate target. After passing the strict incremental check of adjacent positions, a new version is issued. If the field is missing, the position order is inconsistent, or the batch number is mismatched, the temperature control curve of the previous version is retained and the data of the current batch is returned for verification.

[0086] When correcting the pore size, first calculate the signed deviation of the measured degradation sequence parameter relative to the target value according to the same target version in the freeze feedback record. A positive deviation indicates that the degradation at this position is too slow and the target pore size is increased by 5 μm every 1 day. A negative deviation indicates that the degradation at this position is too fast and the target pore size is decreased by 5 μm every 1 day. If the monotonically increasing relationship from the host side to the defect center is disrupted after correction, then adjust from this position to both sides until the adjacent target pore sizes differ by at least 10 μm.

[0087] The new aperture profile is used to reverse-engineer the temperature control program within the calibration range of 80 to 220 μm. The controller first verifies the start-up time and cooling slope of each temperature control zone with an empty model thermal response test, and then verifies the freezing front crossing time, velocity deviation and aperture profile with a calibration sample. Only after all three items pass can the new curve be marked as a candidate production version; otherwise, the previous version is restored and the reason for failure is saved.

[0088] When all sections pass through, maintain at -35 ℃ for 30 min to allow the components within the mold to freeze completely and form a freezing gradient. Then, following the procedure determined by the empty mold thermal response test and the post-freezing pore size maintenance test, simultaneously and controlledly raise the temperature of the five temperature control zones to -12 ℃ at a rate of 1 ℃ / min, with a total transition time not exceeding 25 min. The temperature difference between any two probes should not exceed 2 ℃. Only after all probes have reached -12 ℃ ± 0.5 ℃ and remained stable for 10 min will the transition status be output. If any of the following exceeds the limits: heating slope, transition time, probe temperature difference, or stability condition, the batch will be isolated and discarded. Temperature records, diffusion homogenization characteristic time, freezing front crossing time, measured speed, target speed, allowable deviation, transition thermal history, and calibration curve version will be written into the freezing procedure record. If step S5 feedback indicates that the degradation sequence parameter at a certain sampling location is higher than the target, the target pore size at that node will be increased by 5 μm for every 1 day higher, and limited to 80 to 220 μm. Within μm, the arithmetic mean of the target apertures of adjacent nodes is used to generate the interval target aperture. The new target velocity is then calculated by reverse calculation using the same calibration curve. After verification in step S5, the corrected profile is written into the target aperture process parameters.

[0089] In this specific embodiment, S4 includes:

[0090] After reading the freezing procedure record in step S3 and confirming that the controlled heating slope, total transition time, probe temperature difference, and -12 ℃ stability conditions are all passed, the freezing gradient body is kept at -12 ℃. A redox initiation system composed of persulfate and tertiary amine is used to induce free radical crosslinking of the methacrylamide double bonds in the frozen concentrated microphase while the precursor solution is kept frozen. Specifically, ammonium persulfate and tetramethylethylenediamine of equal molar concentration in the end-member precursor solution are used. This batch is first crosslinked for 4 hours, and parallel samples of the same batch are taken every 30 minutes to determine the dry weight after the removal of soluble matter.

[0091] according to Calculate the first gel fraction of each parallel sample The symbol represents the first The gel fraction of each sample, expressed as a percentage. The symbol indicates the dry mass of the insoluble gel after thorough washing and vacuum drying of the sample, and the unit is mg. The symbol indicates the theoretical dry mass of the polymer contained in the sample before crosslinking, and the unit is mg. The symbols represent the serial numbers of parallel samples at the same crosslinking time, and only 1, 2, and 3 are used;

[0092] The plateau region is defined as the gel fraction increment of two consecutive 30 min intervals not exceeding 2 percentage points and the relative standard deviation of the gel fraction of three parallel samples not exceeding 5%. The time when the condition is first met and its average gel fraction are used as the crosslinking endpoint. If the plateau region is not reached after 4 h, the test is extended by 4 h and then repeated. If the crosslinking endpoint is still not reached after the retest, the batch is discarded and crosslinking failure is recorded.

[0093] After reaching the crosslinking endpoint, the temperature was increased from 2 °C to 4 °C per minute to complete the thawing. After thawing, the gel was washed with buffer solution, and the cold gel was transferred to sterile phosphate buffer solution with a volume of 50 times its volume. The gel was washed at 60 rpm for 12 h, and the washing solution was replaced. The concentration of unreacted persulfate in the washing solution was determined by UV absorption calibration curve established with standard solutions, and the concentration of tetramethylethylenediamine was determined by headspace gas chromatography-flame ionization detection. The concentration, response value, blank value, recovery rate, limit of quantitation and version number were recorded for the two calibration curves, respectively. The recovery rate must be between 95% and 105%, and the limit of quantitation must be less than one-tenth of the corresponding safety threshold.

[0094] The upper limits of the concentrations corresponding to a decrease in cell viability of myoblasts relative to the blank control of no more than 10% in the 24-hour extract pre-test were used as the safety thresholds for persulfate and tetramethylethylenediamine in biological materials. In this batch, dose-response curves were obtained for the two substances at five concentration groups (0, 0.01, 0.02, 0.05, and 0.10 mol / m³) and a blank control. The safety thresholds for persulfate and tetramethylethylenediamine were determined to be 0.02 mol / m³ and 0.05 mol / m³, respectively. The cell viability decrease was verified to be no more than 10% when both substances were simultaneously at their respective thresholds.

[0095] Washing and release and implement, The symbol indicates the measured concentration of unreacted persulfate in the current eluent, and the unit is mol / m³. The symbol represents the persulfate safety threshold and the unit is mol / m³. The symbol indicates the measured concentration of tetramethylethylenediamine in the current eluent, and the unit is mol / m³. The symbol represents the safety threshold of tetramethylethylenediamine and the unit is mol / m³. The batch is released only if both inequalities are satisfied. If neither inequality is satisfied, the washing cycle is extended by 12 h and both inequalities are retested. If either inequality is still not satisfied on the second test, the batch is discarded.

[0096] Equilibrium swelling samples were obtained at positions of 0, 2, 4, 6 and 8 mm along the thickness direction. The effective crosslinking density was determined according to the equilibrium swelling conversion and Flory relationship defined in step S1. The three-dimensional pore structure was obtained using a micron-scale CT with a voxel size of no more than 10 μm. The original volume data were first subjected to three-dimensional median filtering for noise reduction and beam hardening correction. Then, background voxels connected to the outer boundary of the sample were removed, and the pore connectivity domain was skeletonized and the maximum inscribed sphere was analyzed. The median diameter of the pore channel at each sampling position was taken as the median equivalent pore diameter.

[0097] The continuity test used a 0.4 mm scanning step size to perform Raman surface scanning on the entire 8 mm gradient region. The mass fraction conversion relationship of local methacrylamide fucoidan was established using the same batch of sulfur element calibration samples from step S2. At the same time, the initial local double bond molar concentration was calculated based on the end-member double bond molar concentration and local end-member volume fraction from step S1. It was required that the two indicators at all locations were between their respective end-member values ​​and changed monotonically from the host side to the defect center.

[0098] Before crosslinking begins, read the freezing procedure record in step S3, and confirm item by item that the freezing front crossing time is less than the diffusion homogenization characteristic time, all measurement points arrive at valid times, and the velocity deviation formed by the absolute value of the difference between the measured freezing front advance velocity and the target freezing front advance velocity is not greater than the allowable deviation. If any field is missing or exceeds the limit, maintain the frozen state and return to step S3. Do not use the extension of crosslinking time to replace the freezing procedure correction.

[0099] During the crosslinking process at -12 ℃, three temperature probes at the center and both ends of the mold are used for continuous monitoring. If any probe exceeds the measured freezing initiation temperature by 0.5 ℃ for 30 seconds, the timing is stopped and the batch is isolated. Only when all three probes remain frozen and the temperature difference does not exceed 2 ℃ is the current crosslinking time included in the gel fraction platform determination.

[0100] For each gel fraction determination, the parallel samples were first washed with the same batch of buffer solution until the change in conductivity of the washing solution was less than 2%, and then vacuum dried until the change in two consecutive weighings was less than 1%. The platform judgment program checked two consecutive increments in chronological order and output the first pass time. If the relative standard deviation of the parallel sample exceeded 5%, the time point was invalid and three parallel samples were added.

[0101] The washing process is recorded with washing solution batch number, volume, temperature, start and end time, unreacted persulfate concentration, tetramethylethylenediamine concentration, two calibration curve versions and number of solution changes. The washing status is only output when the actual concentrations of the two measured values ​​are lower than or equal to the safety threshold of their respective biological materials. If either value is higher than the threshold, only one additional cycle of equal duration is allowed and the test is repeated. If either value is still higher than the limit on the second test, the waste status is output and the sample number is locked.

[0102] Before calculating the effective crosslinking density, check whether the change in equilibrium wet mass at each position is less than 1% for two consecutive times. When this stable condition is met, calculate according to the defined relationship in step S1 and write it into the position record. If it is not met, extend the swelling time by 12 hours and retest. If the second time is still not balanced, mark the position as invalid and discard the batch.

[0103] The hole structure processing program requires that the proportion of effective connected hole voxels at each location is not less than 70% of the total hole voxels. When this requirement is met, the voxels are sorted by channel diameter from smallest to largest and the median value is taken. If the proportion is less than 70%, the segmentation threshold determined by the blank and solid samples in the same batch is readjusted. If the requirement is still not met after recalculation, the hole structure is output as unqualified.

[0104] The scanning procedure compares the mass fraction and local double bond molar concentration of adjacent sections cross-section by cross-section. If any index changes in the opposite direction or exceeds the closed interval of the two ends, the composition is output as unqualified. After all sections pass, the continuous abrupt change zone of the cross-section is checked. Only when both levels of judgment pass is the state of no discrete layer interface output and the process is allowed to proceed to step S5.

[0105] When the local mass fraction change of adjacent scanning sections is greater than 15% of the endmember difference, a continuous abrupt change band appears across the cross section of the cold gel, or the effective cross-linking density and median of the equivalent connected pore size at any position disrupt the monotonically increasing relationship from the host side to the defect center side, the batch is judged to have a discrete layer interface or gradient distortion and is discarded. Otherwise, the gel fraction, residual initiator concentration, effective cross-linking density, median of the equivalent connected pore size, composition scanning results, and sample orientation are written into the gelation inspection record to obtain a cold gel without a discrete layer interface that proceeds to step S5.

[0106] In this specific embodiment, S5 includes:

[0107] In this specific embodiment, a method for preparing a gradient degradation skeletal muscle repair cryogel is described. In step S5, the enzymatic mass loss sequence of the cryogel along the thickness direction is verified. For the same batch of cryogel that passed the test in step S4, five equally spaced sampling positions are set at 0 mm, 2 mm, 4 mm, and 6 mm on the host side and 8 mm on the defect center side. At seven predetermined weighing time points of 0, 4, 7, 10, 14, 21, and 28 days, five independent endpoint parallel samples with a diameter of 6 mm and a thickness of 1.5 mm are prepared for each position and each time point. Equal independent samples are reserved for four extended time points of 35, 42, 49, and 56 days. Each sample is taken out, freeze-dried, and weighed only once at its respective time point. The sample number includes the batch number, position number, endpoint time point, and duplication number.

[0108] Before verifying the order of enzymatic quality loss, at least five enzyme-free wet parallel samples with a diameter of 6 mm and a thickness of 1.5 mm, and bearing batch number, position number, and duplicate number, were prepared at five positions: 0, 2, 4, 6, and 8 mm. Cyclic compression test, equilibrium swelling test, and three-dimensional pore structure test were performed on each sample. For the five positions, a uniform procedure was used, with a strain range of 0 to 15%, a compression rate of 1 mm per minute, the upper limit of the physiological shrinkage strain measurement range as the set shrinkage strain, a frequency of 1 Hz, and the checkpoint at exactly the 1000th cycle. The effective crosslinking density, median equivalent connected pore size, initial compressive modulus, compressive modulus after cycling, compressive modulus retention rate, and through crack status were recorded for each position. If a single sample was invalid due to incorrect numbering, size, or loading record, a parallel sample reserved at the same position was used to supplement the test, ensuring that there were at least five valid samples at each position. If the compressive modulus retention rate at any position was lower than 80% or through cracks appeared, the current batch was isolated and backed up according to the joint calibration rules. Enzymatic samples were only allowed to enter mixed enzyme culture when these pre-fields were complete and all positions passed.

[0109] The gelation test record received in this step also proves that when the precursor solution was kept frozen, a redox initiation system composed of persulfate and tertiary amine was used to cause free radical crosslinking of the methacrylamide double bonds in the frozen concentrated microphase, and that it was washed with buffer after thawing until the residual initiator was no higher than the safety threshold. If any crosslinking or washing field fails, the degradation sequence verification will not be carried out.

[0110] The gelation test record also includes the composition distribution obtained by continuous cross-sectional imaging with a scanning step size of no more than one-twentieth of the gradient region thickness. Only when the local mass fraction of methacrylamide fucoidan and the local molar concentration of double bonds are both located between their respective endmember values ​​and change monotonically, and there is no compositional abrupt interface across the cross section of the cold gel, is the sample state set to no discrete layer interface and the test continues.

[0111] The mixed enzyme system used a pH 7.4 phosphate buffer containing 100 U / mL collagenase and 20 U / mL fucoidan-degrading enzyme. The enzyme concentration was determined by the number of enzyme activity units per milliliter of enzyme solution. 10 mL of mixed enzyme solution was added to each sample with mass loss. The samples were incubated at 37 °C and 60 r / min, and the same volume of fresh enzyme solution was replaced every 48 h. The blank control used enzyme-free buffer, and all samples had their surface liquid removed by the same contact time with absorbent paper before weighing.

[0112] according to Calculate the mass loss ratio. The symbol represents the first Degradation sampling locations at the endpoint The The mass loss ratio of each independent parallel sample, expressed as a percentage. The symbol indicates the initial dry mass of the independent sample before rehydration and enzymatic culture, and the unit is mg. The symbol indicates the time when the independent sample was incubated to its respective endpoint. The dry weight after freeze-drying according to a uniform procedure is expressed in mg; subscript This represents the computational chain for enzymatic degradation. The symbols represent the degradation sampling location number and only take the values ​​0, 1, 2, 3, and 4, corresponding to 0, 2, 4, 6, and 8 mm respectively. The symbols represent the serial numbers of parallel samples at the same location and the same endpoint, and only 1, 2, 3, 4, and 5 are used. The symbol represents the enzymatic culture time in days and is taken from a preset set of 0, 4, 7, 10, 14, 21, 28, 35, 42, 49 and 56 days.

[0113] Set 28 days as the verification time limit determined based on the target repair cycle. Among the five candidate mass loss ratios of 20%, 25%, 30%, 35%, and 40%, select the first ratio that the average mass loss curve of all sampling locations can reach within 28 days, from high to low, as the same ratio. For each location and time point, take the arithmetic mean of the mass loss ratios of five valid independent endpoint samples to form a location average curve. Samples with mismatched numbers, missing freeze-drying weight loss records, or failed weighing quality control are deemed invalid and re-sampled at the same location, time point, and new duplicate number to restore the number of valid samples entering the average value to five. If there are fewer than five valid samples, the time point cannot be used for interpolation. When there is no qualified ratio, use the reserved 35, 42, 49, and 56 day endpoint samples to extend the culture to 56 days. If there is still no qualified ratio after extension, record the unreached location, curve, and enzyme activity verification results, adjust the endmember mass ratio or double bond molar concentration, and return to step S1.

[0114] For each sampling position, first determine the sampling times of two adjacent valid endpoints that hold the same mass loss ratio, and then perform linear interpolation using the following formula;

[0115] according to Degradation order parameters were obtained. The symbol represents the first The time it takes for all degradation sampling locations to reach the same proportion is expressed in days (d). The symbols are the same as those defined earlier and only 0, 1, 2, 3, and 4 are used. The symbol represents the effective endpoint sampling time before the location-average mass loss ratio first reaches the same ratio, and the unit is d. The symbol indicates the sampling time of the adjacent valid endpoint, and the unit is d. The symbol represents the percentage of the same quality loss selected from the candidate range of 20% to 40%, and the unit is %. and They represent the first Each position has five valid positions at the corresponding time. The arithmetic mean mass loss percentage, expressed in terms of %; each position is represented by five parallel values. and Each sample was resampled 1000 times with replacement and interpolated repeatedly to obtain the results. The standard deviation of the distribution is used as the standard uncertainty of the degradation order parameter at that location;

[0116] according to Determine the minimum interval for the degradation order. The symbol represents the minimum difference in degradation order parameters between adjacent degradation sampling locations, and the unit is d. Symbols and The symbols use the degradation order parameters and degradation sampling position numbers defined in this step, and the minimum value operation only iterates through the data. Take four adjacent pairs of positions: 0, 1, 2, and 3; during the calculation, simultaneously retain the pair of positions that produce the smallest difference, and then... The square root of the sum of the squares of the standard uncertainties is used to obtain the combined standard uncertainty of the time difference. When the minimum difference is found, the combined standard uncertainty with the largest value at each of the parallel positions is used as the most unfavorable threshold. Only when the minimum interval is greater than 0 and greater than this threshold is it determined that the difference from the host side to the defect center side increases monotonically.

[0117] If the monotonic increase condition is not met, first check the enzyme activity, initial dry mass and sample orientation. If the test is still not met after eliminating abnormalities, record the deviation direction. If the host side degradation is too slow, reduce the host side mass ratio or double bond molar concentration. If the center side degradation is too fast, increase the corresponding value on the center side. Then, correct the temperature change curve of the relevant temperature control zone according to the pore size feedback rule in step S3 and return to step S1.

[0118] Before enzyme-catalyzed verification, compression tests were performed using enzyme-free wet parallel samples. The peak contractile stress measured before implantation of the target defect site was compared with the upper limit of the measurement range of physiological contractile strain of surviving muscle tissue. Determine the support modulus threshold. The symbol represents the support modulus threshold and the unit is kPa. The symbol represents the peak shrinkage stress at the target defect location, and the unit is kPa. The symbol represents the upper limit of the physiological contraction strain measurement range and is a dimensionless quantity. The peak contraction stress measured in this batch was 12 kPa and the upper limit of strain was 0.15, corresponding to a support modulus threshold of 80 kPa.

[0119] Five parallel wet samples without enzyme were sampled at five locations. The initial compressive modulus was measured at a compression rate of 1 mm / min within the 0-15% strain range. Cyclic compression was then performed using the upper limit of the physiological shrinkage strain measurement range as the set shrinkage strain and a frequency of 1 Hz. Loading was paused after exactly 1000 cycles, and the compressive modulus at that checkpoint was measured. Further cycles could then be performed without changing the checkpoint diameter in this formula. Each location was... Calculate the compression modulus retention rate. The symbol represents the compressive modulus retention rate at the 1000th cycle checkpoint at this location, and the unit is %. The symbol represents the compressive modulus at that position after exactly 1000 cycles, and the unit is kPa. The symbol represents the initial compressive modulus before the cycle at that position and the unit is kPa. At the same time, check for through cracks at each position under a stereomicroscope. The host side must also meet the requirement that the initial compressive modulus is not less than the support modulus threshold. All five positions must meet the requirement that the compressive modulus retention rate after cyclic compression is not less than 80% and that the cold gel has no through cracks. If any position fails, the batch is isolated and renumbered from step S1.

[0120] The joint calibration status record uses the mass ratio of the two end members, the molar concentration of double bonds, the measured degradation sequence parameters at five positions, the corresponding target values, the target source batch and target version, the velocity deviation formed by the absolute value of the difference between the target and measured pore size and the measured and target freezing front propulsion speeds, the initial compressive modulus, and the compressive modulus retention rate as state quantities, and the increase or decrease of the two end member polymer feed, switching the batch with the tested degree of substitution, correcting the target pore size, and correcting the start time or cooling slope of the temperature control zone as action quantities;

[0121] Action selection prioritizes hard constraints, first excluding actions that cause the initial compressive modulus of the host side to be lower than the support modulus threshold, the compressive modulus retention rate to be lower than 80%, the appearance of through cracks, the freezing rate deviation to exceed the limit, or the composition to change abruptly. Then, from the remaining actions, select the action that minimizes the difference between adjacent degradation sequence parameters. When no action satisfies all hard constraints, retain the previously validated formulation and temperature control curve, isolate the current batch, and roll back to step S1.

[0122] The rollback triggering conditions are divided into five categories: no same proportion after the verification time limit is extended, non-monotonic degradation sequence, mechanical conditions are not passed, non-monotonic pore structure, and freezing speed deviation exceeds the limit. When any rollback condition is triggered, the triggering position and value are recorded and only the corresponding adjustment action is performed. The adjusted candidate batch must be renumbered from step S1 and the molding, freezing, crosslinking and verification must be completely repeated. Only when all the conditions are met again can the isolation be lifted and the previous candidate version be replaced.

[0123] The joint calibration is only passed when the initial compressive modulus on the host side is not less than the support modulus threshold, the compressive modulus retention rate is not less than 80%, the cold gel has no through cracks, and the effective crosslinking density and the median of the equivalent connected pore size obtained in step S4 both increase monotonically from the host side to the defect center side and the degradation order parameter increases strictly in the same direction. If any condition is not met, it shall not be compensated by other indicators and shall be backed to the corresponding formulation or freezing procedure based on the failure indicator.

[0124] The batch verification record saves the following data: Step S1: the feed mass, degree of substitution, mass ratio, and double bond molar concentration of the two end-member precursor solutions; Step S2: the flow rate sequence, cumulative output volume of both channels, cumulative sampling volume of the bypass channel, actual cumulative volume in the mold, and composition verification data; Step S3: the moment when each temperature measuring point first reaches the freezing initiation temperature, the freezing front advance speed, the target degradation sequence parameters at five locations and their sources and versions, the target pore size profile; and Step S4: the measured values ​​of the two types of residues and the number, initial dry mass, endpoint dry mass, pore structure, equilibrium swelling, cyclic compression, enzymatic mass loss, cause of abnormality, number of backtrackings, and final release conclusion of each independent endpoint sample in this step.

[0125] After all joint calibration conditions are met, a V-shaped notch with a depth of 2 mm and an opening width of 2 mm is punched into the host-side edge of the cryogel, while keeping the edge of the defect center straight. The shape is coded as host-side V-shape No. 2, and the host-side orientation, punching time, and operator are written into the batch verification record. During packaging, the V-shaped notch is aligned with the host-side arrow on the label, thereby forming a directional implantation error prevention mark and completing a method for preparing a gradient degradation skeletal muscle repair cryogel.

[0126] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0127] The control and joint calibration algorithm combination employed in this invention does not process the end-member formulation, mold filling flow rate, freezing procedure, and degradation results independently. Instead, it establishes a correlation between the parameters according to the formation process of the cryogel. The algorithm combination first determines the end-member differences based on the mass ratio of the two end-member precursor solutions and the molar concentration of the methacrylated double bond. Through complementary flow functions under constant total flow rate and time-shift compensation for the static mixer retention volume, the end-member volume fraction corresponds to the thickness position within the mold. Then, it uses the time for the freezing front to traverse the gradient region being less than the diffusion homogenization characteristic time as the locking condition for the composition gradient, and adjusts the temperature change curves of each temperature control zone based on the calibration relationship between the freezing front propagation speed and the median of the equivalent interconnected aperture. Finally, it uses the degradation sequence parameter, which is the time required for each sampling position to reach the same mass loss ratio, as feedback to jointly calibrate the end-member precursor solution formulation and freezing procedure. Therefore, the algorithm combination can connect end-member formulation differences, spatial composition gradients, effective crosslinking density gradients, interconnected pore size gradients, and enzymatic degradation order, reducing the interference of diffusion homogenization and pore size mass transfer effects on the preset degradation direction, so that the host side degrades before the defect center side, while the defect center side maintains the necessary structural support in the early stage of repair, thereby taking into account both the mechanical stability in the early stage of repair and the tissue ingrowth and material clearance requirements in the later stage of repair.

[0128] To address the problem in existing technologies where adjusting only a single factor such as polymer composition, crosslinking degree, or pore size makes it difficult to stably obtain a directional degradation front, this invention improves the traditional open-loop process parameter setting into a closed-loop structure of "gradient generation—time sequence locking—pore size reverse calculation—actual verification—feedback back." Complementary flow control and retention volume compensation improve the accuracy of the correspondence between compositional gradients and spatial positions; diffusion homogenization characteristic time criteria and freezing rate deviation criteria identify the risk of distortion in compositional or pore size gradients before crosslinking; and degradation sequence monotonicity criteria verify the actual degradation results formed by the combined effects of chemical crosslinking and pore size mass transfer after gelation. When any criterion is not met, the algorithm structure prevents the current batch from entering subsequent processes, or corrects the end-member precursor liquid formulation, target pore size profile, and temperature change curve of the temperature control zone, and re-executes the corresponding steps. The aforementioned structural improvements allow the process parameters to be optimized in a coordinated manner, rather than focusing on individual material indicators. Instead, they are adjusted in a coordinated manner to achieve the goal of "preferential degradation on the host side and continuous support on the defect center side." This is beneficial for improving the stability and batch repeatability of continuous gradient molding and for more reliably producing gradient-degradable skeletal muscle repair cryogels without discrete layer interfaces.

Claims

1. A method for preparing a gradient-degrading skeletal muscle repair cryogel, characterized in that, include: S1. Prepare host-side endmember precursor solution H and defect-center-side endmember precursor solution C, both containing methacrylated fucoidan, methacrylated gelatin, and a free radical initiation system. The mass ratio of methacrylated fucoidan to methacrylated gelatin and the molar concentration of methacrylated double bonds in host-side endmember precursor solution H are lower than their corresponding values ​​in defect-center-side endmember precursor solution C, thus obtaining the endmember precursor solution group. S2. Output the endmember precursor solution group at complementary flow rates. After static mixing, fill the mold along the thickness direction, causing the volume fraction of host-side endmember precursor solution H to continuously decrease from the host side to the defect-center side, forming a composition field. S3. Make the time for the freezing front to cross the composition field gradient region less than the diffusion homogenization characteristic time, start and adjust the freezing front from the host side to obtain a frozen gradient body; S4. Perform free radical low-temperature cross-linking in the frozen concentrated microphase of the frozen gradient body, thaw and wash to obtain a cold gel without discrete layer interfaces, whose effective cross-linking density and interconnected pore size continuously increase from the host side to the defect center side; S5. Verify the enzymatic mass loss order of the cold gel along the thickness direction, with the host side experiencing mass loss before the defect center side as a constraint, and combine the calibration end-member precursor solution formulation and freezing program to obtain a gradient degradation skeletal muscle repair cold gel.

2. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 1, characterized in that, S1 includes: determining the degree of substitution of methacrylamide fucoidan and methacrylamide gelatin respectively, and calculating the molar concentration of methacrylamide double bonds in each end-member precursor solution based on the degree of substitution and the feed mass; preparing the host-side end-member precursor solution H and the defect-center-side end-member precursor solution C using the same batch of buffer solution and free radical initiation system, such that the total polymer mass, initiator amount, and buffer salt amount per unit volume of the two end-member precursor solutions are the same; calculating the mass ratio of methacrylamide fucoidan to methacrylamide gelatin based on the feed mass, and using the mass ratio and the molar concentration of methacrylamide double bonds as the formulation input parameters for the two metering units.

3. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 1, characterized in that, S2 includes: A first metering unit and a second metering unit are respectively connected to the host-side end-member precursor liquid H and the defect-center-side end-member precursor liquid C. The sum of the instantaneous flow rates of the first metering unit and the second metering unit is set as the constant total flow rate. The ratio of the instantaneous flow rate of the host-side end-member precursor liquid H to the constant total flow rate is set as a flow function that continuously decreases from 1 to 0 as the normalized filled mold volume decreases. The instantaneous flow rate of the defect-center-side end-member precursor liquid C is set as the difference between the constant total flow rate and the instantaneous flow rate of the host-side end-member precursor liquid H. The flow function is time-shifted and compensated according to the stagnation volume of the static mixer so that the end-member volume fraction at the outlet of the static mixer corresponds to the thickness position in the mold. The mass fraction of locally methacrylated fucoidan collected along the thickness direction of the mold is used as the verification data of the composition field, and the metered flow rate, cumulative filled mold volume, and corresponding thickness position are recorded.

4. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 1, characterized in that, S3 includes: under the same temperature and ionic strength as the molding process, measuring the difference between the upper and lower bounds of the mass fraction of methacrylated fucoidan and the upper and lower bounds of the molar concentration of methacrylated double bonds in the gradient zone, respectively, and taking the time required for each difference to decay to one-third of its initial value as a candidate characteristic time, and determining the first candidate characteristic time after arranging the two candidate characteristic times in ascending order of numerical value as the diffusion homogenization characteristic time; recording the moment when each measuring point first reaches the freezing temperature of the precursor liquid by setting temperature measuring points along the thickness direction of the mold, and determining the time difference between the arrival time of the host-side measuring point and the defect center-side measuring point as the time for the freezing front to cross the gradient zone; setting the temperature change curve of each temperature control zone of the mold according to the comparison results of the two times, so that the time for the freezing front to cross the gradient zone is less than the diffusion homogenization characteristic time, and the unit time cooling amplitude of the host-side temperature control zone is greater than the unit time cooling amplitude of the defect center-side temperature control zone.

5. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 1, characterized in that, S4 includes: using a redox initiation system composed of persulfate and tertiary amine, to induce free radical crosslinking of the methacrylamide double bonds in the frozen concentrated microphase while the precursor solution remains frozen; determining the gel fraction at the plateau region of the gel fraction versus crosslinking time curve as the crosslinking endpoint; washing with buffer after thawing; determining the upper limit of the concentration of unreacted initiator that does not produce a decrease in cell viability in the pre-cell compatibility test as the biomaterial safety threshold; extending the treatment time by the same duration as the original crosslinking or washing if the gel fraction does not reach the crosslinking endpoint or the concentration of unreacted initiator in the wash solution exceeds the biomaterial safety threshold; and extending the treatment time by the same duration as the original crosslinking or washing time if the gel fraction does not reach the crosslinking endpoint or the concentration of unreacted initiator in the wash solution exceeds the biomaterial safety threshold. The batch is periodically retested, and if the retest still fails to meet the standard, the batch is discarded and does not proceed to step S5. The effective crosslinking density at each sampling position along the thickness direction is determined based on the equilibrium swelling data. The median of the equivalent connected pore diameter at each sampling position is determined based on the three-dimensional pore structure image. The composition distribution along the thickness direction is checked by continuous cross-sectional imaging with a scanning step size no greater than one-twentieth of the gradient region thickness. When the mass fraction of locally methacrylated fucoidan and the molar concentration of locally methacrylated double bonds are both located between their respective endmember values ​​and change monotonically, and there is no compositional abrupt interface extending along the cross-section of the cold gel, it is determined that the cold gel has no discrete layer interface.

6. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 1, characterized in that, S5 includes: setting multiple sampling positions along the thickness direction of the cryogel at equal intervals, including host-side sampling positions and defect center-side sampling positions; using a mixed enzyme system containing collagenase and fucoidan-degrading enzyme; using the number of enzyme activity units per unit volume of enzyme solution as the enzyme concentration; and acquiring data on the change in mass loss over time at each sampling position under the same enzyme concentration, temperature, and oscillation conditions; determining the verification time limit as the in vitro enzymatic culture period preset according to the target repair cycle; selecting the same proportion that all sampling positions can achieve within the verification time limit from a mass loss ratio range of 20% to 40%; and ensuring that each sampling position reaches the target mass loss ratio. The same proportion of time is used as the degradation sequence parameter, and the degradation sequence parameter is verified to increase monotonically from the host side to the defect center side. If the same proportion cannot be selected within the verification time limit, the verification time limit is extended and the test is repeated. If it still cannot be selected after the retest, the reason for the selection failure is recorded, the mass ratio of the end-member precursor liquid or the molar concentration of the methacrylamide double bond is adjusted, and the process is reversed to step S1 to prepare and verify again. If the verification result does not meet the monotonically increasing constraint, the reason for the abnormality is recorded, the mass ratio of the end-member precursor liquid or the molar concentration of the methacrylamide double bond and the temperature change curve of the corresponding temperature control zone are adjusted, and the process is reversed to step S1 to prepare and verify again.

7. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 6, characterized in that, Step S1 further includes: setting at least three candidate levels for the mass ratio and the molar concentration of the methacrylamide double bond, preparing candidate end-member samples and measuring the equilibrium swelling ratio and enzymatic mass loss curve, determining the effective crosslinking density of each candidate end-member sample based on the equilibrium swelling ratio; selecting a formulation combination from the candidate end-member samples where the effective crosslinking density of the host-side end-member sample is less than that of the defective center-side end-member sample, and the time taken for the host-side end-member sample to reach the same ratio is less than the corresponding time taken for the defective center-side end-member sample, and writing the mass ratio and molar concentration of the methacrylamide double bond in the formulation combination into the formulation control record.

8. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 4, characterized in that, Step S3 further includes: preparing calibration samples using multiple constant freezing front advance velocities; calculating the freezing front advance velocity based on the arrival time of temperature measurement points on each calibration sample; determining the corresponding median equivalent connected aperture; establishing a calibration curve with the freezing front advance velocity as input and the median equivalent connected aperture as output; using the candidate median equivalent connected aperture profile that monotonically increases from the host side to the defect center side as the initial target profile; calculating the target freezing front advance velocity between adjacent temperature measurement points from the calibration curve based on the initial target profile; and comparing the measured freezing front advance velocity with the target... The absolute value of the difference between the freezing front advance speeds is taken as the speed deviation. When the speed deviation is greater than the allowable deviation determined by the temperature measurement uncertainty and the distance between measuring points, the freezing anomaly interlock is triggered, the batch is stopped from entering step S4, and the arrival time of each temperature measuring point is saved. After the temperature change curve of the corresponding temperature control zone is corrected, step S3 is re-executed. When the degradation sequence verification in step S5 fails, the median profile of the candidate equivalent connected aperture is corrected according to the degradation sequence parameter deviation of each sampling position, and the corrected profile that has passed the verification in step S5 is written into the target aperture process parameters.

9. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 6, characterized in that, Step S5 further includes: before verifying the enzymatic mass loss sequence, performing cyclic compression tests, equilibrium swelling tests, and three-dimensional pore structure tests on each sampling location; determining the effective crosslinking density of each sampling location based on the equilibrium swelling test results; determining the median of the equivalent connected pore diameter of each sampling location based on the three-dimensional pore structure test results; determining the ratio of the peak contractile stress measured before implantation of the target defect to the upper limit of the physiological contractile strain measurement range of the surviving muscle tissue of the target defect as the support modulus threshold, and performing no less than one thousand cyclic compressions using the upper limit of the physiological contractile strain measurement range as the set contractile strain; setting the following items as the pass conditions for joint calibration: the initial host-side sampling location The initial compressive modulus is not less than the supporting modulus threshold; the compressive modulus retention rate after cyclic compression is not less than 80% and the cold gel has no penetrating cracks; the effective crosslinking density and the median of the equivalent connected pore size both increase monotonically from the host side to the defect center side; the degradation sequence parameter increases monotonically from the host side to the defect center side; the feed mass and degree of substitution of the two end-member precursor liquids in step S1 are used as formulation record fields, and the moment when each temperature measuring point first reaches the freezing temperature of the precursor liquid in step S3 is used as temperature record fields. The formulation record fields, temperature record fields, pore structure test results, cyclic compression test results, enzymatic mass loss data, and abnormal reasons are written into the batch verification record.

10. The method for preparing the gradient-degrading skeletal muscle repair cryogel according to claim 9, characterized in that, Step S5 further includes: after meeting the pass conditions of the joint calibration, forming a notch or flange on the host side edge of the cryogel that is asymmetrical with the shape of the defect center side edge, and writing the shape code of the notch or flange and the correspondence with the host side orientation into the batch verification record to form a directional implantation error prevention mark.

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