Method and system for detecting urate crystal deposition and phagocytosis in synovial organ tissue

Abstract

The invention belongs to the technical field of medical detection, and particularly relates to a method and a system for detecting urate crystal deposition and phagocytosis in synovial organ tissues. The system comprises a synovial organ culture and verification module used for obtaining a human synovial sample, performing synovial tissue culture and forming different synovial organs; the stimulated Raman scattering microscope imaging module is used for imaging the cultured synovial organs; specifically, a specific pump-Stokes wavelength combination, a spatial deposition quantitative analysis and detection module of urate crystals and a spatial phagocytosis quantitative analysis and detection module of urate crystals are adopted; according to the method, the spatial deposition conditions of urate crystals in different synovials and the phagocytosis of osteoarthritis synovial cells on the crystals can be effectively imaged, and the unit quantity density and the intensity density of deposited or phagocytosed crystals are quantitatively calculated and analyzed; therefore, guidance is provided for development of related basic research and clinical quantitative diagnosis of gout.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and specifically relates to a method and system for detecting urate crystal deposition and phagocytosis in synovial organoid tissue. Background Art

[0002] Gout is a systemic disease with an increasing incidence and disease burden. The main pathological mechanism of gout is the deposition of urate crystals in joints and soft tissues, leading to joint destruction. Rapid, label-free, real-time quantitative detection of urate crystal deposition is conducive to accurate diagnosis of the disease and judgment of the severity of the disease, thereby guiding the implementation of treatment plans. Traditionally, the gold standard for diagnosing gout is to detect urate crystals by compensated polarized light microscopy (CPLM). However, CPLM detection by relevant personnel without professional training will increase the error rate of identifying urate crystals, resulting in certain false negatives and false positives. In recent years, advanced imaging technologies have been continuously updated in the diagnosis of gout, especially ultrasound and dual-energy computed tomography (DECT), which have become indispensable methods for clinical detection of urate crystals. However, ultrasound scanning takes a long time and has low specificity, and successful ultrasound diagnosis requires professional technicians. Although DECT can accurately detect the location of urate crystals and measure the volume of urate, it has ionizing radiation, is relatively expensive, and has low sensitivity in the early stages of gout.

[0003] Stimulated Raman scattering microscopy has become an advanced chemical imaging method in the biomedical field due to its fast imaging speed, strong chemical specificity, high spectral resolution, and strong three-dimensional imaging capabilities. In the field of gout, we have previously demonstrated that stimulated Raman scattering microscopy can detect urate crystals in tissues without labels, observe the phagocytosis of urate crystals by cells, and reflect the inflammatory infiltration of tissues based on the unique fingerprint Raman spectrum. However, the analysis and quantitative detection of spatial deposition and phagocytosis of urate crystals in tissues have not yet been achieved. Therefore, exploring a method for rapid and specific detection of spatial deposition and phagocytosis of urate crystals is an urgent need to optimize treatment strategies. Summary of the invention

[0004] The purpose of the present invention is to provide a method and system for detecting urate crystal deposition and phagocytosis in synovial organoid tissue with fast imaging speed and strong specificity.

[0005] The present invention provides a method for detecting urate crystal deposition and phagocytosis in synovial organoid tissues, using chemically specific, label-free stimulated Raman scattering microscopy technology and a preclinical model, synovial organoids, to perform in situ imaging and quantitative detection of urate crystal spatial deposition and phagocytosis; the specific steps are:

[0006] The first step is to cultivate and verify synovial organoids: Human synovial samples were obtained from the Department of Sports Medicine of a hospital in Shanghai from non-osteoarthritis and osteoarthritis patients undergoing knee arthroscopy (after ethical review). The synovial tissue was digested and cultured in DMEM medium containing 10% fetal bovine serum. Primary synovial cells from different sources were cultured in ice-cold Matrigel matrix to form different synovial organoids, which were verified by histochemical staining;

[0007] Step 2: stimulated Raman scattering microscopy imaging: using a stimulated Raman scattering microscopy system, the cultured synovial organoids were imaged using a specific pump-Stokes wavelength combination, specifically, the pump light power was 70-90 mW, and the Stokes beam power was 150-170 mW;

[0008] The third step is to quantitatively analyze and detect the spatial deposition of urate crystals. Specifically, the open source software ImageJ is used to process stimulated Raman scattering image data. First, the background of each image of the three chemical substances, lipids, proteins, and urate crystals, is removed by subtracting the corresponding non-resonant images. Then, the mask of the organoid tissue area is determined, and the organoid area is manually selected. For further processing, the selected area is converted into a binary mask. Then, after using the "threshold" to select urate crystals, the "analysis particles" is used to obtain an index indicating urate, including the number and intensity of single urate crystals. Next, the total number or total intensity of urate crystals in the tissue is divided by the tissue space volume to obtain the urate crystal deposition index in the tissue, as shown in formulas (1) and (2):

[0009]

[0010] The fourth step is to quantitatively analyze and detect the spatial engulfment of urate crystals. Specifically, the stimulated Raman scattering image data is processed using the open source software ImageJ. First, the background of each image of the three chemical substances, lipids, proteins, and urate crystals, is removed by subtracting the corresponding non-resonant images. Then, the mask of the organoid cell area is determined. The "Find Edges" function is used to distinguish the cell area based on the background and signal of the lipid channel. For further processing, the extracted area is converted into a binary mask. Then, after selecting urate crystals using "Threshold", "Analyze Particles" is used to obtain an index indicating urate, including the number and intensity of individual urate crystals. Next, the total number or total intensity of urate crystals in the cell is divided by the total number or total intensity of crystals to obtain the urate crystal engulfment rate, as shown in formulas (3) and (4):

[0011]

[0012]

[0013] Corresponding to the above-mentioned method for detecting urate crystal deposition and phagocytosis in synovial organoid tissue, the present invention also provides a detection system for urate crystal deposition and phagocytosis in synovial organoid tissue, specifically comprising: a synovial organoid cultivation and verification module, a stimulated Raman scattering microscope imaging module, a spatial deposition quantitative analysis detection module of urate crystals, and a spatial phagocytosis quantitative analysis detection module of urate crystals; wherein:

[0014] The synovial organoid cultivation and verification module is used to obtain human synovial samples from non-osteoarthritis and osteoarthritis patients during knee arthroscopic surgery, culture synovial tissue, and form different synovial organoids; and then verify them by histochemical staining method; wherein, the synovial tissue culture adopts DMEM culture medium containing 10% fetal bovine serum; the different synovial organoids are formed by culturing primary synovial cells from different sources in ice-cold Matrigel matrix;

[0015] The stimulated Raman scattering microscope imaging module is used to image the cultured synovial organoids; specifically, a specific pump-Stokes wavelength combination is used, wherein the power of the pump light is 70-90 mW and the power of the Stokes beam is 150-170 mW;

[0016] The spatial deposition quantitative analysis detection module of urate crystals is used for quantitative analysis and detection of spatial deposition of urate crystals. Specifically, the stimulated Raman scattering image data is processed using the open source software ImageJ. First, the background of each image of three chemical substances, namely lipids, proteins and urate crystals, is removed by subtracting the corresponding non-resonant image. Then, the mask of the organoid tissue area is determined, and the organoid area is manually selected. For further processing, the selected area is converted into a binary mask. Then, after selecting urate crystals using the "threshold", the "analysis particle" is used to obtain an index indicating urate, including the number and intensity of single urate crystals. Next, the total number or total intensity of urate crystals in the tissue is divided by the tissue space volume to obtain the urate crystal deposition index in the tissue, which is shown in formulas (1) and (2):

[0017]

[0018] The spatial engulfment quantitative analysis detection module of urate crystals is used for quantitative analysis and detection of spatial engulfment of urate crystals; specifically, the stimulated Raman scattering image data is processed using the open source software ImageJ; first, the background of each image of the three chemicals is removed by subtracting the corresponding non-resonant image; then the mask of the organoid cell area is determined; the "find edge" function is used to distinguish the cell area based on the background and signal of the lipid channel. For further processing, the extracted area is converted into a binary mask. Then, after selecting urate crystals using the "threshold", the "analyze particles" is used to obtain an index indicating urate, including the number and intensity of single urate crystals. Next, the urate crystal engulfment rate is obtained by dividing the total number or total intensity of urate crystals in the cell by the total number or total intensity of crystals, as shown in formulas (3) and (4):

[0019]

[0020] The technical features of the present invention mainly include:

[0021] The present invention can clearly image the spatial deposition of urate crystals in different synovial tissues and the phagocytosis of crystals by synovial cells, and perform quantitative calculation and analysis on the unit number density and intensity density of deposited or phagocytic crystals, thereby providing guidance for conducting relevant basic research and clinical quantitative diagnosis of gout. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The flowchart of the method for detecting urate crystal deposition and phagocytosis in synovial organoid tissue of the present invention is shown in Figure 1. A is the culture and verification of organoids; B is the component diagram of stimulated Raman scattering microscope; C is the reconstruction image of 3D synovial organoids using stimulated Raman scattering microscope; and D is the observation of spatial deposition and phagocytosis of urate crystals using stimulated Raman scattering microscope.

[0023] Figure 2 Schematic diagram of the culture process of synovial organoids.

[0024] Figure 3 Validation of normal and osteoarthritic synovial organoids.

[0025] Figure 4 Stimulated Raman scattering spectra of normal and osteoarthritis synovial organoids.

[0026] Figure 5 Spatial imaging of normal and osteoarthritic synovial organoids by stimulated Raman scattering.

[0027] Figure 6 To examine the spatial deposition of urate crystals in normal and osteoarthritic synovial organoids.

[0028] Figure 7To investigate the spatial phagocytosis of urate crystals in three-dimensional cells of normal and osteoarthritic synovium.

[0029] Figure 8 To detect spatial phagocytosis of urate crystals in normal and osteoarthritic synovial 2D cells. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of embodiments in conjunction with the accompanying drawings.

[0031] In this example, stimulated Raman scattering microscopy was used to perform real-time label-free detection and quantitative analysis of urate crystal spatial deposition and phagocytosis in normal and osteoarthritis synovial organoids. The specific experimental steps are as follows:

[0032] (1) Organoid culture and validation: After ethical review, human synovial samples from different sources were obtained from the Sports Medicine Department of a hospital in Shanghai. The synovial tissue was digested and cultured in DMEM medium containing 10% fetal bovine serum. Primary synovial cells from different sources were cultured in Matrigel matrix to form a variety of synovial organoids.

[0033] Figure 2 The synovial organoids culture process is shown. Histological examination of the cultured synovial organoids revealed that the synovial cells in the inner layer of the synovium spontaneously accumulated and compacted, which is very similar to the human synovium in terms of anatomy. Compared with normal synovial organoids, osteoarthritis synovial organoids are thickened and disordered ( Figure 3 A). In synovial tissue, synovial cells autonomously generate extracellular matrix components, such as reticular fibers, which are similar to the reticular fibers in human synovium. Gomori reticular cellulose staining showed that the reticular fibers in osteoarthritis synovial tissue were more disorganized than those in normal synovial tissue ( Figure 3 B). Lubricin is abundant in synovial fluid and is produced by synovial lining cells. Reduced lubricin expression has been reported to be associated with cartilage degradation / damage in animal models of acute joint injury or osteoarthritis. Less immunoreactive lubricin has been detected in the lining layer of osteoarthritic synovial organ tissue ( Figure 3 C and F). To further explore the expression of inflammatory factors, immunohistochemical staining of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) was performed ( Figure 3 D and E). The results showed that the expression levels of these two inflammatory factors in osteoarthritis synovial tissue were significantly higher than those in normal synovial tissue ( Figure 3F). Overall, normal and osteoarthritis synovial organoids were successfully established, and osteoarthritis synovial organoids showed more organizational disorder, less lubrication proteins, and more inflammatory cytokine expression, similar to human osteoarthritis synovium. Urate crystals were then added to the organoid tissue in vitro to observe the spatial deposition and phagocytosis of the crystals in different tissues.

[0034] (2) Spatial imaging of organoids by stimulated Raman scattering: Spontaneous Raman spectroscopy of standard chemicals in the range of 550-3,100 cm-1 was performed to determine the spectral characteristics of the experimental samples. The spectral characteristics of the standard chemicals in this range were reproduced in the average SRS spectrum. The Raman spectrum showed several obvious peaks, including prominent peaks at 625, 2920, and 2845 cm-1, which originated from the purine ring vibration of urate, the CH3 stretching vibration of proteins, and the CH2 stretching vibration of lipids, respectively ( Figure 4 A). The spectra of viable synovial organoids and fresh synovial tissue are consistent, indicating that the organoids are well cultured ( Figure 4 B). Figure 5 A and 5B show representative side and top views of normal and OA synovial organoids 8 h after addition of urate crystals.

[0035] (3) Stimulated Raman scattering detection of spatial deposition of urate crystals in tissues: The spatial deposition of urate crystals in organ tissues was obtained by quantitative analysis and calculation using ImageJ (Equations (1), (2)). Figure 6 A is a three-dimensional top view of normal and osteoarthritis synovial organoids after urate crystals were added for 8 hours. Figure 6 B shows representative cross-sectional images. Figure 6 C and D show the quantitative statistical analysis results of the number density and intensity density of urate crystals in tissues.

[0036]

[0037] (4) Stimulated Raman scattering detection of urate crystal phagocytosis in tissues: Then, ImageJ was used to quantitatively analyze the spatial phagocytosis of urate crystals in organ tissues (Equations (3), (4)). In order to better observe the phagocytosis of urate crystals by synovial cells, we selected the three-dimensional stimulated Raman scattering stereoscopic side views and representative magnified images of cells in normal and osteoarthritis synovial organoids after 8 hours of adding urate crystals ( Figure 7 A and B). Figure 7 C and D show the quantitative statistical analysis results of urate crystal phagocytosis rate. Monolayer cell experiments of normal and osteoarthritis synoviocytes stimulated with urate crystals showed similar results ( Figure 8 A), Figure 8B and C show the results of quantitative statistical analysis of urate crystal phagocytosis rate in 2D cells.

[0038]

Claims

1. A method for detecting urate crystal deposition and phagocytosis in synovial organoid tissue, characterized in that: In situ imaging and quantitative detection of phagocytosis of spatial urate crystal deposition were performed using chemically specific, label-free stimulated Raman scattering microscopy and a preclinical model, synovial organoids. The specific steps were: The first step is to cultivate and validate synovial organoids: obtain human synovial samples from non-osteoarthritis and osteoarthritis patients undergoing knee arthroscopic surgery; digest the synovial tissue and culture it in DMEM medium containing 10% fetal bovine serum; culture primary synovial cells from different sources in ice-cold Matrigel matrix to form different synovial organoids, which are then validated using histochemical staining methods; Step 2: Stimulated Raman Scattering Microscope Imaging: Using a stimulated Raman scattering microscope system, the cultured synovial organoids were imaged using a specific pump-Stokes wavelength combination, specifically, the pump light power was 70-90 mW and the Stokes beam power was 150-170 mW; The third step is to quantitatively analyze and detect the spatial deposition of urate crystals. Specifically, the open source software ImageJ is used to process stimulated Raman scattering image data. First, the background of each image of the three chemical substances, lipids, proteins, and urate crystals, is removed by subtracting the corresponding non-resonant images. Then, the mask of the organoid tissue area is determined, and the organoid area is manually selected. For further processing, the selected area is converted into a binary mask. Then, after using "threshold" to select urate crystals, "analyze particles" is used to obtain an index indicating urate, including the number and intensity of single urate crystals. Next, the total number or total intensity of urate crystals in the tissue is divided by the tissue space volume to obtain the urate crystal deposition index in the tissue, as shown in formulas (1) and (2): The fourth step is to quantitatively analyze and detect the spatial engulfment of urate crystals. Specifically, the stimulated Raman scattering image data was processed using the open source software ImageJ. First, the background of each image of the three chemical substances, lipids, proteins, and urate crystals, was removed by subtracting the corresponding non-resonant images. Then, the mask of the organoid cell area was determined. The "Find Edges" function was used to distinguish the cell area based on the background and signal of the lipid channel. For further processing, the extracted area was converted into a binary mask. Then, after selecting urate crystals using "Threshold", the "Analyze Particles" was used to obtain an index indicating urate, including the number and intensity of single urate crystals. Next, the total number or total intensity of urate crystals in the cell was divided by the total number or total intensity of crystals to obtain the urate crystal engulfment rate, as shown in formulas (3) and (4):

2. A detection system for urate crystal deposition and phagocytosis in synovial organoid tissue, characterized in that: Specifically include: Synovial organoid cultivation and verification module, stimulated Raman scattering microscope imaging module, spatial deposition quantitative analysis and detection module of urate crystals, spatial phagocytosis quantitative analysis and detection module of urate crystals; among them: The synovial organoid cultivation and verification module is used to obtain human synovial samples from non-osteoarthritis and osteoarthritis patients during knee arthroscopic surgery, culture synovial tissue, and form different synovial organoids; and then verify them by histochemical staining method; wherein, the synovial tissue culture adopts DMEM culture medium containing 10% fetal bovine serum; the different synovial organoids are formed by culturing primary synovial cells from different sources in ice-cold Matrigel matrix; The stimulated Raman scattering microscope imaging module is used to image the cultured synovial organoids; specifically, a specific pump-Stokes wavelength combination is used, wherein the power of the pump light is 70-90 mW and the power of the Stokes beam is 150-170 mW; The spatial deposition quantitative analysis detection module of urate crystals is used for quantitative analysis and detection of spatial deposition of urate crystals; specifically, the stimulated Raman scattering image data is processed using the open source software ImageJ; the background of each image of three chemical substances, namely lipids, proteins and urate crystals, is removed by subtracting the corresponding non-resonant images; the mask of the organoid tissue area is determined, the organoid area is manually selected, and the selected area is converted into a binary mask; the urate crystals are selected using the "threshold value" and the "analysis particles" are used to obtain an index indicating urate, including the number and intensity of single urate crystals; the total number or total intensity of urate crystals in the tissue is divided by the spatial volume of the tissue to obtain the urate crystal deposition index in the tissue, which is shown in formulas (1) and (2): The spatial engulfment quantitative analysis detection module of urate crystals is used for quantitative analysis and detection of spatial engulfment of urate crystals; specifically, the stimulated Raman scattering image data is processed using the open source software ImageJ; the background of each image of the three chemical substances is removed by subtracting the corresponding non-resonant image; the mask of the organoid cell area is determined; the "find edge" function is used to distinguish the cell area according to the background and signal of the lipid channel; the extracted area is converted into a binary mask; the "threshold" is used to select urate crystals, and the "analysis particle" is used to obtain an index indicating urate, including the number and intensity of single urate crystals; the total number or total intensity of urate crystals in the cell is divided by the total number or total intensity of crystals to obtain the urate crystal engulfment rate, which is shown in formulas (3) and (4):

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