A method and system for printing a hydrogel scaffold

Abstract

The application provides a printing method of a hydrogel support, uses a three-dimensional imaging module to obtain a three-dimensional image of a single filament of the hydrogel, calculates a printability score according to the width-height ratio and layer thickness standard deviation information of the three-dimensional image, and adjusts a printing temperature, carries out layer-by-layer printing, obtains top layer height information through the three-dimensional imaging module, and generates a z-axis height of the next layer of printing, in the printing process, detects a printing defect in real time, locates a defect coordinate, feeds back to a computer to generate a corresponding repair code for repair. The printing system for the above method comprises a high-precision three-axis mechanical arm, a three-dimensional imaging module, a gas pressure control system, a temperature control system and a computer. The application has the advantages that it is suitable for the three-dimensional printing of hydrogels and similar temperature-sensitive materials and materials with poor mechanical properties, can realize segmented detection, timely feedback and accurate repair, and can improve the quality and stability of the printed model.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of extrusion type biological 3D printing technology, and particularly relates to a printing method and system of a hydrogel scaffold. BACKGROUND

[0002] Extrusion type biological 3D printing technology is a commonly used technology for constructing living tissues / tissue models / tissue engineering scaffolds, and has the advantages of wide range of materials that can be selected, moderate printing flux and printing resolution. Hydrogels are often used as materials for constructing living tissues / scaffolds by extrusion type biological 3D printing due to their good water absorption and biocompatibility, however, the mechanical properties of hydrogels are poor, which greatly challenges the printability and printing stability of hydrogel materials.

[0003] The reconstruction of tissue structure and function is a spatiotemporal dynamic process, and the printed tissue may undergo new morphogenesis as the culture time increases, which requires re-printing; in-situ printing on complex tissue structures may also occur, and accurate analysis of the structure before and after printing is required to control the subsequent printing events, improve the printing accuracy and stability. SUMMARY

[0004] The purpose of the present application is to provide a printing method and system of a hydrogel scaffold that is suitable for stereoscopic printing, segmented detection, timely feedback, accurate repair and can improve the quality and stability of the printed model.

[0005] In order to achieve the above purpose, the present application realizes the technical scheme as follows:

[0006] A printing method of a hydrogel scaffold, comprising:

[0007] (1) quantitatively analyzing and determining the printing parameters including the printing temperature

[0008] A three-dimensional imaging module is used to obtain a three-dimensional image of the single filament of the hydrogel, and the filament diameter, layer thickness and other statistical information of the printing are obtained according to the three-dimensional image. The three-dimensional imaging module can be selected from OCT technology, laser confocal technology, two-photon imaging technology, light sheet microscopy technology, laser three-dimensional scanning technology, etc.

[0009] The single filament is segmented, and two-dimensional cross-sectional information is obtained from each segment. The filament diameter and layer thickness of each cross section in the segment are calculated through the cross-sectional information, and the average filament diameter, average layer thickness, width-height ratio and layer thickness standard deviation of the segment are statistically calculated. The calculation formula is as follows:

[0010]

[0011] Wherein, m is the selected segment length, n is the number of cross-sectional images of the three-dimensional imaging module in the segment, i is the i-th cross-section, FS is the filament diameter of the single filament in the segment, LT is the layer thickness of the single filament in the segment, AR is the aspect ratio of the single filament in the segment, and STL is the layer thickness standard deviation of the single filament in the segment.

[0012] By quantitatively analyzing the aspect ratio AR and the layer thickness standard deviation STL of the single filament, the printing nozzle is controlled at a suitable printing temperature.

[0013] The printability judgment method is as follows:

[0014] The aspect ratio and the layer thickness standard deviation show the gel state of the hydrogel at the current temperature. The single filament printed by the under-gel hydrogel is in a liquid state, the aspect ratio will be greater than that in the normal and over-aggregation states, and will usually be greater than 2. The single filament printed by the over-aggregation hydrogel is rough and uneven in surface, and the layer thickness standard deviation will be greater than 0.023. The single filament printed by the normal aggregation hydrogel is smooth in surface and stable in shape, and both indicators will be lower than the above two values. In order to characterize the printability of the single filament, a printability score Pr score is used to indicate the printability of the hydrogel single filament, and the calculation formula is as follows:

[0015]

[0016] In the formula, the value of k1 is 5, the value of k2 is 500, and the aspect ratio and the layer thickness standard deviation of the single filament are substituted to obtain Pr score .

[0017] When the hydrogel is in an under-gel state, the value of Pr score will be close to 1; when it is in a normal gel state, the value of Pr score will be around 0; and when it is in an over-gel state, the value of Pr score will be close to -1.

[0018] Therefore, when the value of Pr score is close to -1, it can be determined that the current printing temperature is too low and is not suitable for printing; when the value of Pr score is close to 1, it can be determined that the current printing temperature is too high and is not suitable for printing. The detection results are fed back to the computer, the computer makes a judgment, and the temperature control system is controlled to raise or lower the temperature of the printing nozzle, so that the temperature of the printing nozzle is maintained at a suitable printing temperature, and the value of Pr score is maintained around 0.

[0019] (2) Defect positioning and repair printing

[0020] The length of the segment is selected according to the actual situation, for example, for small uneven problems or broken wire problems, the length of the segment can be set to be short, and through the statistical data, the subtle defects can be found; for example, when the overall shape of a single wire is studied, the length of the segment can be set to be long or a whole wire is intercepted for analysis.

[0021] The wire diameter FS or layer thickness LT obtained by the segment detection can obtain the current segment situation; if the wire breaking situation occurs, the value of FS or LT in the segment is 0. m or LT m of the segment is 0.

[0022] Taking the printing starting point as the coordinate origin, the distance between the cross-sectional axis of the single wire and the origin coordinate is calculated, the x-axis coordinate of the single wire is recorded, and thus the x-axis coordinate of the defect is determined;

[0023] Using the segment method of the formula l = m * 10 / 1024, the y-axis coordinate of the defect is determined as y = lp, l is the actual distance of the unit segment wire, p is the pth segment where the defect occurs, and the length of the defect is obtained by subtracting the y coordinate of the first defect from the y coordinate of the last defect;

[0024] Corresponding the printing starting point to the coordinate origin in the Gcode, after obtaining the defect coordinate, a segment of the repaired Gcode is automatically generated and fed back to the printer for repair.

[0025] (3) Hydrogel printing z-axis offset quantification method

[0026] Before printing, the position of the printing platform on the z-axis is determined through the three-dimensional imaging module; after printing one or more layers of the support, only the top layer height information of the printed structure needs to be obtained when the three-dimensional imaging module collects the image of the printed structure;

[0027] The formula of the actual height of the current three-dimensional support is as follows:

[0028]

[0029] Wherein, Height(n) is the actual height of the nth layer support, base is the position of the printing platform on the z-axis at the initial moment, material n is the position of the nth layer material surface in the coordinate system, △Rz is the resolution of the three-dimensional image on the z-axis, and the last term in the formula is the summation of the z-axis offset of the first n-1 layers;

[0030] According to the obtained current support top layer height information, the z-axis height of the next layer printing is generated. The z-axis height of the next layer printing is the current support top layer height plus the z-axis offset, and the z-axis offset is generally a fixed value.

[0031] Before quantifying the filament diameter and layer height of a single filament, the collected three-dimensional image is corrected, the actual position of the filament on the printing platform is obtained after correction by compensation algorithm, and the corrected image is binarized, wherein the calculation of the refractive index is expressed as the ratio of the actual thickness h1 of the filament to the optical path length (h1+h2), and h2 is the optical path length; the product of the number of pixels at both ends of the bottom of the filament cross-section binary image and the resolution in the x or y direction is defined as the actual filament diameter value, and the product of the number of pixels between the top of the filament and the printing platform and the resolution in the z direction is defined as the actual layer height value.

[0032] When obtaining the aperture of the multi-layer printed, a plurality of XY sections between every two layers are intercepted, the section images are processed and the holes are extracted, the product of the number of pixels and the pixel size of the hole part is calculated as the hole area, and the aperture value is obtained after the square root of the hole area; the aperture value of each two layers of hydrogel is the average value of the holes at the corresponding positions of the plurality of XY sections.

[0033] For multi-layer scaffolds or in-situ printing on complex structures, according to the obtained scaffold top layer height information, the z-axis offset of the next layer printing is generated to ensure that the filament position of the printing nozzle and the scaffold top layer always maintain a constant height difference.

[0034] For the spatiotemporal iterative printing of multi-layer scaffolds or complex tissues, according to the analysis of the tissue morphogenesis of the obtained three-dimensional image of the current structure, the key morphological parameters of the three-dimensional image of the current structure include the channels, branches, cavitation, boundaries and volume of the multi-layer scaffold or complex tissue, the centroid of the scaffold channel boundary or the centroid of the complex tissue cavitation is located to determine the origin position of the spatiotemporal iterative printing, and the relationship between the average feeding speed and the printing temperature, extrusion pressure, nozzle inner diameter is established as follows:

[0035] Q=v x p d 2 / 4; wherein Q is the capacity of the printing required for quantitative extrusion, v is the average feeding speed, and d is the inner diameter of the printing nozzle.

[0036] For another way of spatiotemporal iterative printing of multi-layer scaffolds or complex tissues, the three-dimensional image of the current structure is displayed in the coordinate system interface of the printing software, the position of the iterative printing component is visually designed in the microcavity array of the current structure, and then the G code for printing is generated. The hydrogel solution loaded with organoids / growth factors / organ elements / new cells is loaded into a new extrusion printing nozzle, the temperature of the printing nozzle and the printing platform is set, and according to the pre-designed G code path, the printing nozzle distributes the hydrogel in the selected microcavity of the current structure / tissue morphology for a short time, then rises to the highest z-axis offset obtained, and moves to the next position. During the iterative printing process, the intermittent air pressure, extrusion time (recommended as 500-1000 ms), minimum extrusion volume (recommended <0.1 pl), printing speed and other printing parameters of the new printing nozzle are controlled.

[0037] A printing system of a hydrogel scaffold is integrated on a biological 3D printer, comprising a high-precision three-axis mechanical arm, a three-dimensional imaging module, a gas pressure control system, a temperature control system and a computer, the three-dimensional imaging module collects image information of the biological scaffold, generates a Gcode repair file or a printing instruction after computer image processing, and then sends the printing instruction to a printer motion control mechanism comprising a three-axis mechanical arm, a gas pressure control system and a temperature control system to complete corresponding repair or printing operation.

[0038] Compared with the prior art, the present application has the following advantages:

[0039] The printing method and system of the hydrogel scaffold of the present application are suitable for the stereoscopic printing of hydrogels and similar materials, and can improve the quality and stability of the printed model through segmented detection, timely feedback and accurate repair. Specifically, since the mechanical properties of hydrogels are weak and easily affected by temperature, the silk may collapse or swell during the printing of the three-dimensional scaffold. The present application uses a three-dimensional imaging module as a monitoring system of the printer to obtain data information of the current printing layer, evaluates the printability of the hydrogel by segmented detection, feeds back the control of the extrusion gas pressure and speed parameters, so that the width and height of the printed single silk material are within the designed error range, and adjusts the temperature of the printing nozzle and platform to ensure that the printed three-dimensional hydrogel scaffold is under the ideal pore size. In the segmented detection, the silk diameter or layer thickness can obtain the current segment condition, if the silk is broken, the average silk diameter or average layer thickness value in the segment is 0, the three-dimensional imaging module is used to calculate the distance between the cross-sectional axis of each silk and the printing origin coordinate, to quickly locate the position of the defect, and to automatically generate a segment of repaired Gcode feedback to the printer for repair.

[0040] The three-dimensional imaging module can realize three-dimensional imaging of the printed scaffold or tissue, obtain key morphological parameters such as the channel, pore size or complex tissue morphology of the scaffold, and determine the required quantitative extrusion capacity according to the cavitation / morphology volume. At the same time, the three-dimensional imaging module can compare the printed scaffold or tissue, the cultured scaffold or tissue, the scaffold and tissue after spatiotemporal iterative printing with the collected three-dimensional image, analyze the growth trend and activity distribution of cells in the scaffold or tissue, and based on the spatiotemporal iterative printing, can positionally guide / induce the proliferation or differentiation of cells in the scaffold or tissue. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A1 is a storage modulus and loss modulus change graph of 5% Gel + 1% Alg at a temperature from 5℃ to 40℃.

[0042] Figure 1A2 is the relationship between the viscosity of 5% Gel + 1% Alg hydrogel and the shear rate at 27℃.

[0043] Figure 1 B is the needle tip wire drawing diagram, single wire material diagram and OCT reconstruction diagram at different temperatures.

[0044] Figure 1 C1 is the AR value distribution diagram of the needle tip of three different specifications at three groups of printing temperatures.

[0045] Figure 1 C2 is the LST value distribution diagram of the needle tip of three different specifications at three groups of printing temperatures.

[0046] Figure 1 D1 is the change of width-height ratio of single wire material when continuously printing for 30 min at three groups of temperatures.

[0047] Figure 1 D2 is the change of layer thickness standard deviation of single wire material when continuously printing for 30 min at three groups of temperatures.

[0048] Figure 1 E1 is the change diagram of three-dimensional fitting wire diameter and speed pressure when the wire diameter range is 0.2 to 0.3.

[0049] Figure 1 E2 is the change diagram of three-dimensional fitting layer thickness and speed pressure when the wire diameter range is 0.15 to 0.2.

[0050] Figure 2 A is the OCT image (XZ section) of single wire material, refractive index correction image and wire diameter layer thickness schematic diagram.

[0051] Figure 2 B is the schematic diagram of segmented calculation of wire diameter, layer thickness, width-height ratio and layer thickness standard deviation.

[0052] Figure 3 A, B, C are the pore size change diagrams corresponding to the number of layers in the A, B, C three groups of experiments.

[0053] Figure 4 A, B, C are the multi-layer printing material diagrams in the A, B, C three groups of experiments. DETAILED DESCRIPTION

[0054] The embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0055] For extrusion type biological 3D printing, the quality of the printed structure is mainly controlled by four process parameters: wire drawing pressure, shaft moving speed, temperature and z-axis height. Among them, the z-axis height has an important influence on whether the three-dimensional scaffold can be formed.

[0056] The temperature will directly affect the gel state of the hydrogel, and the inappropriate gel state will affect the printability, so the temperature needs to be determined before the filament pressure and the shaft moving speed are determined. When the temperature is low, the hydrogel is in the over-gel state, and the hydrogel is difficult to be extruded due to the small caliber of the printing nozzle needle tip, and the filament will be in an uneven state when extruded at a large pressure; if the hydrogel contains cells, the cells will die due to the large shear stress. When the temperature is high, the hydrogel is in the under-gel state, and the extruded hydrogel is in a liquid state, so that the filaments are fused when a small scaffold is printed, and the layers are also fused, so that it is difficult to form a hole. When the hydrogel is in the ideal gel state, the extruded filament is uniform, and the filaments and layers can maintain an ideal state. The three-dimensional penetration characteristics of the OCT system are used in this embodiment, and the printability of the single filament is determined according to the width-height ratio AR of the single filament and the layer thickness standard deviation SLT.

[0057] The prepared gelatin sodium alginate hydrogel was taken, and a needle with a specification of 23G, 25G or 27G (the hole diameter is 0.34 mm, 0.26 mm or 0.21 mm, respectively) was used for printing. In order to distinguish the gel state of the gelatin sodium alginate hydrogel, the temperature of the printing nozzle was set to 22℃, 27℃ or 32℃. The temperature of the printing platform was set to 10℃, so that the hydrogel could quickly become a gel state when it was printed onto the printing platform, and the state could be maintained stably. When the single filament was printed, the Z offset The z-axis offset was 0.9 times the diameter of the needle hole, and too high would cause the extruded filament to gather around the needle and not adhere to the platform, and too low would cause the needle to extrude and result in a high width-height ratio, causing deviation in the experimental results. In this embodiment, two groups of air pressure and speed were applied to the printing of the single filament at each temperature, and the specific values were determined according to the gel state of the filament. A larger extrusion air pressure was applied when the filament was in the over-gel state, and a smaller extrusion air pressure was applied when the filament was in the under-gel state, so that the single filament could be maintained within a suitable filament diameter range.

[0058] The printed filament was scanned and reconstructed using OCT, and a segmented detection method was used, including the following steps: (1) segmenting the single filament and obtaining a two-dimensional cross section from each segment; (2) representing three-dimensional data information through the cross section information; and (3) calculating the filament diameter and layer thickness of each cross section in the segment, and calculating the average filament diameter, average layer thickness, width-height ratio and layer thickness standard deviation information of the segment, and the calculation method is as follows:

[0059]

[0060] The length of the segment can be selected according to different situations, such as small-scale unevenness or filament breakage, and the segment length can be set to be short, and the statistical data can find subtle defects; when the overall shape of the single filament is studied, the segment length can be set to be long or the entire filament can be analyzed.

[0061] The rheological properties of the hydrogel used in this embodiment from 5℃ to 40℃ are as follows Figure 1 A1 shows that at 32℃, the hydrogel has melted, and at 27℃, the storage modulus of the hydrogel has decreased significantly. Due to the shear thinning property, as shown in Figure 1 A2 shows that at this temperature, the hydrogel can maintain a good shape in the extruded state. Under the action of low temperature of the printing platform, the shape of the extruded filament can be well maintained on the platform, as shown in Figure 1 B shows that at 22℃, the hydrogel is extruded from the needle by high pressure, and from the physical object and the OCT reconstructed image, it can be seen that the shape of the filament at this time presents a wrinkled state; at 27℃, the hydrogel is extruded by moderate air pressure, and the filament deposited on the printing platform is flat and smooth; at 32℃, even if the minimum air pressure of the printer is used for extrusion, the hydrogel will form a water droplet-like shape at the needle tip, and the single filament deposited on the printing platform is flat and smooth, but due to the action of gravity, the hydrogel will spread to both sides, and the filament width and layer thickness cannot meet the expectations.

[0062] In this embodiment, the value of m is the whole filament, and the width-height ratio AR and layer thickness standard deviation SLT of the whole single filament at different temperatures are obtained, as shown in Figure 1 C1 shows the width-height ratio AR, and as shown in Figure 1 C2 shows the layer thickness standard deviation SLT. No matter which size needle is used for printing, the layer thickness standard deviation STL of the single filament printed at 22℃ in the over-gel state is high, exceeding 0.023; the layer thickness standard deviation STL of the single filament printed at 27℃ in the normal gel state and at 32℃ in the over-gel state is maintained at about 0.01. The width-height ratio AR of the single filament printed at 32℃ in the under-gel state is greater than 2.0, higher than that in the over-gel state and the normal gel state; the width-height ratio AR of the single filament printed at 27℃ is larger than that printed at 22℃, and the overall comparison is close and maintained at a low level.

[0063] Based on the width-height ratio and layer thickness standard deviation data, Pr score is calculated, and the box plot obtained is as shown in Figure 1 D1 shows that the data differentiation is obvious in the three states, and the box length is relatively short, and the data is relatively concentrated. Even if there are a few abnormal values in the data, the value of the abnormal value is still within the corresponding state range, which fully illustrates that Pr score can well distinguish the printability of the hydrogel, and has high accuracy, and is suitable for computer to directly judge the printability, thereby guiding the adjustment of the process parameters of the printer.

[0064] At each temperature, a 27G needle is used to continuously print on the platform for 30min, and single filament image data is collected every 5min, Pr score is calculated, and the data obtained is as shown in Figure 1 D1 shows that at 27℃, the Prscore Maintaining a temperature close to 0°C, compared to the other two temperature groups, the silk printed at 27°C is smooth and flat, and less prone to collapse.

[0065] After determining the appropriate printing temperature and ensuring the printability of the hydrogel, the printing parameters required for the appropriate filament width and layer thickness were obtained under normal gel conditions. The print head movement speed was controlled within the range of 4–11 mm / s, with intervals of 1 mm / s. Due to the slight delay between the application of air pressure by the air pump and the extrusion of the filament, excessively fast printing speeds can cause gaps at the beginning, while excessively slow printing speeds can cause buildup at bends. First, the minimum air pressure for continuous filament extrusion at 27°C was determined. Then, with the filament diameter not exceeding 70% of the pinhole diameter as a standard, the maximum air pressure for printing at 11 mm / s was determined, resulting in an air pressure range of 0.14 MPa–0.21 MPa, with intervals of 0.01 MPa. Eight sets of air pressure parameters were combined with eight sets of speed parameters, and 64 sets of data were printed. The results obtained using second-order surface fitting are shown below. Figure 1 As shown in E1 and 1E2. Selecting a wire diameter range of 0.2–0.3 mm and a layer thickness range of 0.15–0.2 mm, the resulting parameter ranges are as follows: Figure 1 The yellow areas are shown in E1 and 1E2.

[0066] Before quantifying the diameter and layer height of a single filament, the acquired images need to be calibrated. For example... Figure 2 Figure A shows a two-dimensional OCT cross-sectional image of a single filament. Due to the different refractive indices of air, hydrogel, and the printing platform, the resulting OCT image is distorted. Therefore, refractive index correction is necessary before obtaining information such as filament diameter and layer thickness. The refractive index is calculated as the ratio of the actual material thickness h1 to the optical path length (h1+h2). After correcting the refractive index using a compensation algorithm, the true position of the single filament on the printing platform can be obtained. The corrected image is binarized, and the actual filament diameter is defined as the product of the number of pixels at the bottom two ends of the binary image of the single filament cross-section and the resolution along the x-axis or y-axis. The actual layer height is defined as the product of the number of pixels between the top of the single filament and the base plate and the resolution along the z-axis.

[0067] When obtaining the aperture of multi-layer printing, 30 XY cross-sections are taken from the middle section between every two layers. The images are processed to extract the holes. The product of the number of pixels in the hole area and the pixel size is calculated to obtain the hole area at the corresponding location. The square root of the hole area is then used to obtain the hole diameter value. In this embodiment, the hole diameter value of the two layers of hydrogel is the average value of the holes at the corresponding locations in the 30 cross-sections.

[0068] The mechanical stability of three-dimensional scaffolds is affected by surface area and pore size, and appropriate pore size is essential for tissue formation. The design of the pore size in the 2D structure of the scaffold is more considered. The hydrogel scaffold with larger pore size has an advantage in nutrient transport, but its structural stability is easily damaged, and the cell adhesion rate is lower. The scaffold with smaller pore size has better pore connectivity and structural stability, but it limits the cell growth space and tissue formation ability. The pore size in the range of 200-400 μm is the best choice for nutrient and waste flow, cell migration. In this embodiment, the printing parameters of the 27G (0.21mm) needle are used to obtain the appropriate wire diameter and layer thickness of the single wire material, the center line of the printing wire material is taken as the printing path, the path is drawn in IGcodeTool, the pore size is designed to be 400 μm, and Gcode is generated.

[0069] In order to verify the effectiveness of the layer height feedback in multi-layer printing, three groups of experiments A, B and C were set. The three groups of experiments were printed using a 27G needle under the conditions of nozzle temperature 27℃, platform temperature 10℃, air pressure 0.15Mpa, and printing nozzle moving speed 7mm / s. According to the single wire material detection of the appropriate wire diameter and layer thickness, it can be known that the theoretical wire diameter of the printing wire material under the parameters is 0.3mm, and the theoretical layer thickness is 0.19, and the z-axis compensation is 0.9 times the diameter of the needle hole.

[0070] The three groups of experiments are designed as follows: group A does not use layer height feedback, and the z-axis compensation (Z offset ) of each layer is set to the theoretical layer thickness value 0.19; group B also does not use layer height feedback, and the Z offset of each layer is set to the theoretical layer thickness value + standard deviation, which is 0.2; group C uses layer height feedback, and the initial setting is to take Z offset 0.19, and then adjust Z offset according to the feedback layer height, and the specific adjustment value is the feedback layer height value plus the initial setting value.

[0071] The three groups of experiments are scanned once by OCT every two layers, and the difference between the actual pore size and the designed pore size after each scan is compared, and the results are shown in Figure 3 . Figure 3 The pore size of the three groups of experiments corresponding to the layer number is shown, specifically the average pore size value and the number of pores with the column chart increasing with the layer number; for example, A-4 represents the pore size of the fourth layer of group A. As shown in Figure 4 , the multi-layer printing of groups A, B and C is shown in the figure, wherein I is the printing process diagram, II is the enlarged diagram of the red box area of I, III is the OCT reconstruction result diagram after printing, and IV is the enlarged diagram of the red box area of III.

[0072] The pore size of the middle section of each layer in the three groups can be seen. In the first 8 layers, the average pore size of the three groups is stable at about 400 μm, but the error in the z-axis of groups A and B has begun to accumulate. At the 10th layer, the defects of group A begin to appear, and the average pore size and the number of pores begin to decrease, until the 18th layer, the scaffold has no pores. From Figure 4 As can be seen from A-I, when printing to a high layer, the needle of group A has penetrated into the inside of the scaffold when printing at a high layer, at this time the extruded filament accumulates on the original filament, causing the pores to be blocked, destroying the original structure of the scaffold, and the structure obtained is a non-porous scaffold (such as Figure 4 A-III). Group B begins to become unstable in pore size and number of pores from the 14th layer, the main reason is that as the number of layers accumulates, a small collapse occurs at the bottom of the scaffold, and the z-axis error accumulates, as shown in Figure 4 B-I, during the printing process at a high layer, the filament extruded by the needle does not immediately adhere to the printed scaffold, but accumulates to a certain height at the needle before depositing on the scaffold, at this time the printed filament is mostly broken, which has no meaning for repairing broken filaments, and finally a chaotic and distorted printed structure is obtained (such as Figure 4 B-III). In the C group experiment, until the 20th layer is printed, the pore size and the number of pores remain very stable, as shown in Figure 4 C, the pores do not disappear as in group A or become irregular as in group B, due to the effect of z-axis compensation correction every two layers, the filament extruded by the needle is deposited very stably on the scaffold (such as Figure 4 C-I), a porous stable scaffold is obtained (such as Figure 4 C-III).

[0073] The printing system of the present embodiment uses an independently developed integrated OCT extrusion type biological 3D printing system, which integrates a high-precision three-axis mechanical arm, a swept frequency OCT module, a gas pressure control system, and a temperature control system.

[0074] The mechanical arm movement error of the printer is ±10 μm, and the high-precision displacement can ensure that there is no large deviation between the actual printing path and the designed printing path when printing a small scaffold. The gas pressure control system can provide an extrusion gas pressure of 0.8 Mpa. The temperature control system can control the temperature of the biological ink and the printing platform respectively.

[0075] The OCT used as a monitoring system uses a swept frequency laser light source with a center wavelength of 1310 nm. The A-Scan scanning rate is 50 KHz, and the focal length is 36 mm. In air, the axial resolution is 7.2 μm, the lateral resolution is 15 μm, the maximum imaging depth is 6 mm, and the lateral field of view is 10 mm (x) * 10 mm (y).

[0076] In the system, the OCT serves as a three-dimensional imaging module, is responsible for collecting image information of the printed object, generates a repaired Gcode file after computer image processing and calculation, and sends the Gcode file to a motion control system of the printer, so that a corresponding repair operation is completed.

[0077] The above merely describes preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the concept of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A printing method of hydrogel scaffold, characterized in that: a three-dimensional imaging module is used to obtain a three-dimensional image of a single filament of the hydrogel, and information of average filament diameter, average layer thickness, aspect ratio and layer thickness standard deviation is calculated according to the three-dimensional image; the printability score is calculated by quantitatively analyzing the aspect ratio and layer thickness standard deviation of the single filament, so as to regulate the printing temperature and other printing parameters of the printing head; before printing, the position of the printing platform on the z-axis is determined by the three-dimensional imaging module; after printing one or more layers of the scaffold, the height information of the top layer of the scaffold is obtained by the three-dimensional imaging module, so as to generate the z-axis height of the next layer of printing; during printing, the printing defects are detected and located in real time, and the computer generates corresponding repair code to feed back to the printer for repair; in the quantitative analysis, the single filament is segmented, and two-dimensional cross-sectional information is obtained from each segment; the filament diameter and layer thickness of each cross section in the segment are calculated according to the cross-sectional information, and the information of average filament diameter, average layer thickness, aspect ratio and layer thickness standard deviation of the segment is calculated; when the measured layer thickness standard deviation is greater than 0.023, the printability score is close to -1, and it can be determined that the current printing temperature is too low and is not suitable for printing; when the measured aspect ratio is greater than 2.0, the printability score is close to 1, and it can be determined that the current printing temperature is too high and is not suitable for printing; when the obtained printability score is near 0, it can be determined that the current printing temperature is suitable for the printing of the hydrogel. 2.The printing method of the hydrogel scaffold according to claim 1, characterized in that: in the positioning of the defect coordinates, the printing starting point is taken as the coordinate origin, the distance between the cross-sectional axis of the single filament and the coordinate origin is calculated as the x-axis coordinate of the single filament, so as to determine the x-axis coordinate of the defect; the formula l=m*10 / 1024 is used to determine the y-axis coordinate of the defect, y=lp, l is the actual distance of the unit segment filament, m is the selected segment length, and p is the pth segment in which the defect occurs; the length of the defect is obtained by subtracting the y-coordinate of the first occurrence of the defect from the y-coordinate of the last occurrence of the defect; if a broken filament occurs, the value of the filament diameter or layer thickness of the broken filament defect in the segment is 0. 3.The printing method of the hydrogel scaffold according to claim 1, characterized in that: before quantifying the filament diameter and layer height of the single filament, the collected three-dimensional image is corrected, the actual position of the filament on the printing platform is obtained after correction by a compensation algorithm, and the corrected image is binarized; the actual filament diameter value is defined as the product of the pixel number at both ends of the bottom of the filament cross-sectional binary image and the resolution in the x-axis or y-axis direction, and the actual layer height value is defined as the product of the pixel number between the top of the filament and the printing platform and the resolution in the z-axis direction. 4.The printing method of the hydrogel scaffold according to claim 1, characterized in that: when the pore diameter of the multi-layer printing is obtained, multiple XY cross sections between every two layers are intercepted, the cross-sectional images are processed and the holes are extracted, the product of the pixel number of the hole part and the pixel size is calculated as the hole area, and the pore diameter value is obtained by taking the square root of the hole area. The pore size value of each two layers of hydrogel is the average value of the pores at the corresponding positions of multiple XY cross sections.

5. The printing method of the hydrogel scaffold according to claim 1, characterized in that: For the spatiotemporal iterative printing of the multi-layer scaffold or the complex tissue, the origin position and the average feeding speed of the spatiotemporal iterative printing are determined according to the analysis of the tissue morphogenesis based on the three-dimensional image of the current structure, and the relationship between the printing average feeding speed and the printing temperature, the extrusion pressure and the inner diameter of the nozzle is established.

6. The printing method of the hydrogel scaffold according to claim 5, characterized in that: The key morphological parameters of the three-dimensional image of the current structure include the channel, the branch, the cavitation, the boundary and the volume of the multi-layer scaffold or the complex tissue, and the origin position of the spatiotemporal iterative printing is located according to the centroid of the scaffold channel boundary or the centroid of the complex tissue cavitation.

7. The printing method of the hydrogel scaffold according to claim 1, characterized in that: For the spatiotemporal iterative printing of the multi-layer scaffold or the complex tissue, the three-dimensional image of the current structure is displayed in the printing software coordinate system interface, the position of the iterative printing is visually designed in the microcavity array of the current structure, and the printing code is generated; The printing parameters of the printing nozzle and the temperature of the printing platform are set, and the iterative printing is performed according to the generated printing code.

8. A printing system of hydrogel scaffolds integrated on a biological 3D printer, characterized in that: The system comprises a high-precision three-axis mechanical arm, a three-dimensional imaging module, a gas pressure control system, a temperature control system and a computer, the three-dimensional imaging module collects image information of the biological scaffold by using the method of any one of claims 1 to 7, generates a repair file or a printing instruction after computer image processing, and sends the repair file or the printing instruction to a printer motion control mechanism comprising a three-axis mechanical arm, a gas pressure control system and a temperature control system to complete corresponding repair or printing operations.

Images