Method and system for establishing corneal defect animal model
By screening healthy rabbits and analyzing differences in corneal structure, a standardized corneal defect model was established, which solved the problems of inconsistent results and animal welfare in the evaluation of new ophthalmic materials and realized a low-cost and efficient evaluation method.
Patent Information
- Application Number
- CN202411583191.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-01-10
AI Technical Summary
The lack of standardized animal models for corneal defects in current technologies leads to inconsistent animal test results during the research and development of new ophthalmic materials, increases costs and neglects animal welfare, and also lacks effective evaluation methods.
Healthy experimental rabbits were screened using slit-lamp and ophthalmic ultrasound imaging. The structural differences between rabbit and human corneas were analyzed, and the parameters of the corneal defect model were adjusted. The size and location of the defect were controlled using a microscope to establish a standardized animal model of corneal defect.
It improves the reliability of animal experimental results and the credibility of extrapolating them to clinical applications, provides a low-cost and efficient evaluation method, and ensures the consistency and reproducibility of experimental results.
Smart Images
Figure CN119296806A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of eyeball models for evaluating the safety and effectiveness of medical devices, and in particular relates to a method and system for establishing a corneal defect animal model. Background Art
[0002] With the rapid development of ophthalmology, the number of ophthalmic medical devices has gradually increased. The evaluation of such devices usually requires a good and stable corneal defect animal model. In the past, the instruments used in corneal treatment, such as artificial corneas made of polymer materials such as glass and PMMA, were far from the normal human cornea. With the development of modern corneal treatment surgical methods and the rapid update of surgical instruments, various new biomaterials or medical devices have gradually been used in the treatment of corneal diseases. A low-cost and universal eyeball model is needed to evaluate various new biological ophthalmic materials.
[0003] At the same time, corneal disease is one of the most important reversible and blinding eye diseases in the world today. According to statistics from the World Health Organization: corneal disease is the second largest blinding eye disease in the world after cataracts. Corneal disease is an important cause of vision loss. It causes grayish-white turbidity in the transparent cornea, which can cause blurred vision, decreased vision, and even blindness. In the early stages of corneal disease, if it is treated promptly and accurately, it can be cured. However, if the lesions are severe or recurring, thick scars will be left on the cornea. At this time, the only treatment method is corneal transplantation to remove the turbid cornea and replace it with a transparent cornea, so that the patient can regain vision. In addition, ocular trauma and corneal ulcers also play an important role in corneal blindness. Lamellar keratoplasty is an important means of treating corneal diseases. In order to make up for the shortage of donated corneas and solve the current situation that corneal transplantation requires a long wait for donated corneas, many medical device companies have positioned their products as substitutes for human corneas or materials, and have carried out research and development of such products. Similarly, drugs and new treatment technologies used to treat such diseases are also constantly improving, and standardized animal models are urgently needed to evaluate products and technologies.
[0004] With the development of modern corneal transplantation methods and the rapid update of surgical instruments, corneal transplantation has become one of the conventional surgical procedures for treating corneal blindness, and has achieved good surgical results. Tens of thousands of patients have gained sight. At present, before new materials are applied to humans, animal tests must be conducted to determine whether the new materials can effectively alleviate or treat corneal diseases. However, excessive application on animals during the research and development stage will cause animal ethics issues, such as whether a test is required at each stage or process, which will lead to neglect of animal welfare and increase the corresponding costs. This has brought huge difficulties to the development of some small and medium-sized enterprises. At the same time, since there are no specific standards for preclinical animal experiments, the level of operators in each institution that can carry out animal experiments is different, and the understanding of the test is different, which will result in a situation where the test results of a sample conducted in two institutions are biased. Summary of the invention
[0005] In view of the above shortcomings, the present invention hopes to establish a standardized corneal defect eyeball model for evaluating the safety and effectiveness of various new ophthalmic materials. At the same time, combined with the clinical experience of experienced operators, it can bring better experience to various new ophthalmic workers and better study the effects of surgery.
[0006] To achieve the above-mentioned purpose, the present invention provides a method and system for establishing a corneal defect animal model. Among them, a method for establishing a corneal defect animal model comprises:
[0007] Slit lamp was used to preliminarily screen the experimental rabbits to exclude visible eye diseases or defects. Ophthalmic ultrasound imaging was also used to further identify and screen hidden eye abnormalities and obtain experimental rabbits with normal eye conditions.
[0008] Based on the differences in anatomical structure between rabbit cornea and human eyeball, the thickness and hierarchical structure parameters of rabbit cornea and human cornea were obtained, and the degree of difference between the two was determined through comparative analysis, and the preparation parameters of corneal defect model were adjusted accordingly;
[0009] By adopting standardized operation procedures and parameter settings and using a microscope to control the size, depth and position of the corneal defect, an eyeball model with corneal defect was constructed to obtain a structurally uniform and repeatable corneal defect animal model.
[0010] Preferably, the process of obtaining an experimental rabbit with normal eye condition comprises:
[0011] According to the pre-established imaging characteristic model of normal eye condition of experimental rabbits, the experimental rabbits are preliminarily screened using slit lamps, and the slit lamp images are compared with the imaging characteristic model of normal eyes to determine whether the experimental rabbits have visible eye diseases or defects. If so, the experimental rabbits are excluded;
[0012] For the experimental rabbits that have been initially screened, ophthalmic ultrasound equipment is used to obtain ultrasound images of eye tissues. The ultrasound images are enhanced and segmented through image processing technology to extract key characteristic parameters of eye tissues.
[0013] The extracted key characteristic parameters of the eye tissue are input into a pre-trained latent eye abnormality recognition model, and the probability value of whether the experimental rabbit has a latent abnormality in the eye is obtained through the model calculation;
[0014] According to the preset hidden eye abnormality threshold, it is determined whether the probability value exceeds the threshold, if it exceeds the threshold, the experimental rabbit is excluded, otherwise it is determined that the eye condition of the experimental rabbit is normal;
[0015] For experimental rabbits with normal eye conditions, basic information was entered and they were marked as qualified individuals and included in the candidate object library for subsequent experiments.
[0016] Preferably, the process of adjusting the preparation parameters of the corneal defect model includes:
[0017] Obtain anatomical data of rabbit cornea and human cornea, including corneal thickness and layer structure parameters;
[0018] Compare and analyze the anatomical structure data of rabbit cornea and human cornea, and calculate the difference in thickness and layer structure between the two.
[0019] Determining the degree of difference in anatomical structure between the rabbit cornea and the human cornea according to the difference value;
[0020] If the degree of difference exceeds a preset threshold, the preparation parameters of the corneal defect model are adjusted according to the degree of difference, including but not limited to the resection depth and resection diameter;
[0021] A corneal defect model was prepared on the rabbit cornea according to the adjusted preparation parameters;
[0022] The anatomical structure data of the prepared rabbit corneal defect model is obtained, compared with the anatomical structure data of the human corneal defect, and the similarity is calculated; if the similarity does not reach the preset threshold, the preparation parameters are continued to be adjusted until the similarity meets the requirements, thereby obtaining a rabbit corneal defect model with high similarity and comparability with the human corneal defect.
[0023] Preferably, the process of comparing and analyzing the anatomical structure data of the rabbit cornea and the human cornea and calculating the difference values between the two in thickness and hierarchical structure comprises:
[0024] Obtain anatomical images of rabbit and human corneas and perform segmentation to extract structural features of each layer of the cornea;
[0025] According to the segmentation results, the thickness data of rabbit cornea and human cornea at different levels are measured;
[0026] Statistical methods were used to calculate the mean and variance of the thickness of rabbit and human corneas at each level;
[0027] The hierarchical structures of the rabbit cornea and the human cornea were compared, and the similarity of the hierarchical structures of the two was calculated using an image similarity algorithm. The differences in the anatomical structures of the rabbit cornea and the human cornea were comprehensively analyzed based on the thickness difference value and the hierarchical structure similarity, and quantitative difference analysis results were obtained.
[0028] Preferably, the process of obtaining a structurally uniform and repeatable corneal defect animal model comprises:
[0029] According to the preset standardized operation process and parameter settings, the cornea is observed and located using a microscope to obtain the position coordinates of the target area of corneal defect;
[0030] Based on the position coordinates of the corneal defect target area, the size and shape of the defect area are measured by an image processing algorithm to obtain a quantitative defect size parameter;
[0031] Compare the defect size parameters with a preset standard defect model, and determine whether the current defect meets the standard model requirements through a similarity matching algorithm. If not, adjust the defect parameters and re-prepare the defect;
[0032] According to the depth parameters of the standard defect model, the cornea is cut layer by layer in real time, and the defect structure image of the corneal defect area after cutting is obtained through microscopic imaging technology;
[0033] A batch preparation method was adopted to perform parallel operations on several animal individuals to obtain corneal defect models in batches.
[0034] Preferably, the method further comprises: obtaining data on tissue compatibility, degradation absorption and induced repair response of rabbit cornea and human cornea to the material based on evaluating the effect of the biomaterial on corneal defect repair, judging the difference in the effect of the material in the two corneas through comparative analysis, and optimizing the design and preparation process of the material accordingly.
[0035] Preferably, the process of optimizing the design and preparation process of the material comprises:
[0036] According to the preset standards, the physical and chemical properties and preparation process parameters of candidate biomaterials are obtained, and the materials are classified by clustering algorithms;
[0037] Using tissue engineering technology, rabbit cornea and human cornea defect models were constructed, and different types of biomaterials were implanted into the defect sites.
[0038] At different time points after implantation, histological sections and immunohistochemical staining images of corneal tissue were obtained, and quantitative parameters of material degradation, absorption, and cell infiltration were extracted using image segmentation algorithms;
[0039] Gene expression profile data of corneal tissue were obtained, and principal component analysis was used to compare the differences in expression of genes related to tissue repair induced by different materials;
[0040] The tissue compatibility, material degradation and absorption, and induced repair effect parameters of rabbit and human corneas were normalized, and a prediction model of material properties and corneal repair effects was established using a support vector machine algorithm.
[0041] The key performance parameters that affect the corneal repair effect of the material were determined based on the prediction model, and the response surface optimization method was used to optimize the material composition and preparation process;
[0042] The optimized materials were implanted into rabbit cornea and human corneal defect models to evaluate the repair effect.
[0043] Preferably, the corneal defect animal model comprises five layers, namely: epithelial cell layer, anterior Descemet's layer, parenchymal layer, posterior Descemet's layer, and endothelial cell layer.
[0044] Preferably, the corneal defect animal model is made of PVC, the defect depth of the eyeball model is 0.15 mm, and the defect diameter is 5-6 mm.
[0045] The present invention also provides a system for establishing a corneal defect animal model, comprising:
[0046] The experimental rabbit screening module is used to conduct preliminary screening of experimental rabbits using a slit lamp to exclude visible eye diseases or defects, and to further identify and screen hidden eye abnormalities through ophthalmic ultrasound imaging examinations to obtain experimental rabbits with normal eye conditions;
[0047] Corneal structure comparison module, based on the differences in anatomical structure between rabbit cornea and human eyeball, obtains the thickness and hierarchical structure parameters of rabbit cornea and human cornea, and determines the degree of difference between the two through comparative analysis;
[0048] The corneal defect model establishment module is used to adjust the preparation parameters of the corneal defect model according to the degree of difference, adopt standardized operating procedures and parameter settings based on the adjusted preparation parameters, and use a microscope to control the size, depth and position of the corneal defect to construct a corneal defect eyeball model, so as to obtain a structurally uniform and repeatable corneal defect animal model.
[0049] Compared with the prior art, the present invention has the following advantages and technical effects:
[0050] The present invention first screens out normal-eyed experimental rabbits through slit lamp and imaging examinations, and compares the differences in structural parameters between rabbit cornea and human cornea, and then establishes a standardized corneal defect model to control the size, depth and position of the defect. The present invention effectively improves the reliability of extrapolating animal experimental results to clinical practice, and provides an important basis for the development and application of corneal defect repair materials.
[0051] The standardized eyeball model established by the present invention can eliminate interference from other external factors and provide relatively consistent data results for reference when used in scientific research. The present invention uses easily available materials to establish a human eyeball model, which is low in cost but high in market benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:
[0053] Figure 1 The figure is a schematic diagram of a method flow of an embodiment of the present invention. DETAILED DESCRIPTION
[0054] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0055] It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.
[0056] like Figure 1 As shown, this embodiment provides a method for establishing a corneal defect animal model, comprising:
[0057] Slit lamp was used to preliminarily screen the experimental rabbits to exclude visible eye diseases or defects. Ophthalmic ultrasound imaging was also used to further identify and screen hidden eye abnormalities and obtain experimental rabbits with normal eye conditions.
[0058] Based on the differences in anatomical structure between rabbit cornea and human eyeball, the thickness and hierarchical structure parameters of rabbit cornea and human cornea were obtained, and the degree of difference between the two was determined through comparative analysis, and the preparation parameters of corneal defect model were adjusted accordingly;
[0059] By adopting standardized operation procedures and parameter settings and using a microscope to control the size, depth and position of the corneal defect, an eyeball model with corneal defect was constructed to obtain a structurally uniform and repeatable corneal defect animal model.
[0060] Furthermore, the process of obtaining experimental rabbits with normal eye conditions includes:
[0061] According to the pre-established imaging characteristic model of normal eye condition of experimental rabbits, the experimental rabbits are preliminarily screened using slit lamps, and the slit lamp images are compared with the imaging characteristic model of normal eyes to determine whether the experimental rabbits have visible eye diseases or defects. If so, the experimental rabbits are excluded;
[0062] For the experimental rabbits that have been initially screened, ophthalmic ultrasound equipment is used to obtain ultrasound images of eye tissues. The ultrasound images are enhanced and segmented through image processing technology to extract key characteristic parameters of eye tissues.
[0063] The extracted key characteristic parameters of the eye tissue are input into the pre-trained latent eye abnormality recognition model, and the probability value of whether there is a latent abnormality in the experimental rabbit's eye is calculated by the model;
[0064] According to the preset hidden eye abnormality threshold, determine whether the probability value exceeds the threshold. If it exceeds the threshold, the experimental rabbit is excluded; otherwise, the experimental rabbit's eye condition is considered normal;
[0065] For experimental rabbits with normal eye conditions, basic information was entered and they were marked as qualified individuals and included in the candidate object library for subsequent experiments.
[0066] This embodiment uses a decision tree algorithm to classify the experimental rabbit eye imaging data generated during the screening process, continuously optimizes the imaging feature model of normal eyes, and improves the accuracy of subsequent screening. Regularly re-examine the eyes of rabbits in the experimental candidate library, continuously monitor their eye health, and update the experimental candidate library in a timely manner to ensure the reliability of experimental research.
[0067] Specifically, in the early stage of the experiment, by collecting slit lamp images and ultrasound images of 100 healthy experimental rabbits, a normal eye imaging feature model containing 10 key feature parameters such as corneal thickness, anterior chamber depth, and lens thickness was established. The model was used to preliminarily screen the 50 newly selected experimental rabbits, and the slit lamp images were detected and classified by the YOLOv5 algorithm. Five experimental rabbits were identified to have visible abnormalities such as cataracts and corneal ulcers in their eyes, and they were eliminated. For the remaining 45 experimental rabbits, the Resnet50 convolutional neural network was used to segment and extract features from their eye ultrasound images. The obtained feature parameters were input into the random forest classifier for hidden abnormality identification. By comparing with the threshold of 85, it was determined that three experimental rabbits had hidden abnormalities in their eyes and were excluded. Finally, 42 experimental rabbits with normal eye conditions were selected, and their strain, age, weight and other information were entered and included in the experimental candidate library. The ID3 decision tree algorithm was used to train the 2,000 eye image data from the screening process, and the feature parameters were sorted by information gain to optimize the imaging model containing only 5 key features, increasing the model accuracy from 92% to 96%. In order to ensure the quality of the experimental subjects, the eyes of the rabbits in the experimental candidate library were re-examined every month, and the candidate library was updated according to the results of slit lamp examination and ultrasound examination. Individuals with eye abnormalities were removed in time. A total of 5 abnormal individuals were removed within 3 months, maintaining the health level of the experimental object library.
[0068] Furthermore, the process of adjusting the preparation parameters of the corneal defect model includes:
[0069] Obtain anatomical data of rabbit cornea and human cornea, including corneal thickness and layer structure parameters;
[0070] Compare and analyze the anatomical structure data of rabbit cornea and human cornea, and calculate the difference in thickness and layer structure between the two.
[0071] The degree of difference in anatomical structure between rabbit cornea and human cornea was determined based on the difference value;
[0072] If the degree of difference exceeds a preset threshold, the preparation parameters of the corneal defect model are adjusted according to the degree of difference, including but not limited to the resection depth and resection diameter;
[0073] A corneal defect model was prepared on the rabbit cornea according to the adjusted preparation parameters;
[0074] The anatomical structure data of the prepared rabbit corneal defect model is obtained, compared with the anatomical structure data of the human corneal defect, and the similarity is calculated; if the similarity does not reach the preset threshold, the preparation parameters are continued to be adjusted until the similarity meets the requirements, thereby obtaining a rabbit corneal defect model with high similarity and comparability with the human corneal defect.
[0075] Specifically, in order to obtain the anatomical data of rabbit cornea and human cornea, optical coherence tomography (OCT) was used to scan and image the two corneas, and the corneal thickness and the size parameters of each hierarchical structure were extracted by image processing algorithm. The anatomical parameters of rabbit cornea and human cornea were subjected to paired t-test using statistical methods, and the difference values of the two sets of data in thickness and each hierarchical structure were calculated, and the Cohen's d value was calculated as a quantitative indicator of the degree of difference. The degree of difference was judged according to the Cohen's d value. If the d value was greater than 8, it was considered that the difference was significant, and the preparation parameters of the corneal defect model needed to be adjusted. The response surface optimization algorithm was used, with the similarity between the corneal defect model and the human corneal defect anatomical structure as the optimization target, and the resection depth and resection diameter as the independent variables, to establish a mathematical model of preparation parameters and similarity. The optimal combination of preparation parameters was obtained by model solution, and the corneal defect model was prepared on the rabbit cornea. After preparation, the rabbit corneal defect model was scanned and imaged using OCT, and its anatomical structure data was extracted and compared with the anatomical structure data of human corneal defects. The similarity of the two sets of data was calculated using the Euclidean distance method. If the similarity is less than 9, the response surface optimization step is returned to continue optimization based on the current preparation parameters until the similarity meets the requirements. Through iterative optimization, a rabbit corneal defect model with high similarity (greater than or equal to 9) and comparability with human corneal defects was finally obtained, providing a reliable animal model for subsequent corneal defect repair research.
[0076] Furthermore, the anatomical structure data of the rabbit cornea and the human cornea are compared and analyzed, and the process of calculating the difference values in thickness and hierarchical structure between the two includes:
[0077] Obtain anatomical images of rabbit and human corneas and perform segmentation to extract structural features of each layer of the cornea;
[0078] According to the segmentation results, the thickness data of rabbit cornea and human cornea at different levels are measured;
[0079] Statistical methods were used to calculate the mean and variance of the thickness of rabbit and human corneas at each level;
[0080] The hierarchical structures of the rabbit cornea and the human cornea were compared, and the similarity of the hierarchical structures of the two was calculated using an image similarity algorithm. The differences in the anatomical structures of the rabbit cornea and the human cornea were comprehensively analyzed based on the thickness difference value and the hierarchical structure similarity, and quantitative difference analysis results were obtained.
[0081] Specifically, in order to obtain the anatomical structure data of the rabbit cornea and the human cornea, the present embodiment uses a high-resolution confocal microscope to image the rabbit cornea and the human cornea to obtain a clear corneal tomographic image. Then, the corneal image is segmented using the image segmentation algorithm DeepLabV3+ based on deep learning, and the structural features of the corneal epithelium, stroma and endothelium are extracted. By analyzing the segmentation results at the pixel level, the average thickness of the rabbit corneal epithelium, stroma and endothelium are measured to be 32μm, 325μm and 1μm, respectively, and the average thickness of the corresponding layers of the human cornea is 54μm, 493μm and 2μm. Using a one-way analysis of variance, it was found that the differences in thickness of the rabbit cornea and the human cornea at each layer were statistically significant, among which the thickness difference of the stroma was the most significant, reaching 178μm. In terms of corneal hierarchical structure comparison, the present embodiment extracts the SIFT features of the corneal layered image, and uses the cosine similarity algorithm to calculate the similarity between the rabbit cornea and the human cornea hierarchical structure. The results show that the structural similarity between the two is 783. Based on the analysis results of thickness difference and structural similarity, this example believes that there are certain differences in the anatomical structure between the rabbit cornea and the human cornea. These differences need to be considered when constructing the rabbit corneal defect model, and the similarity and comparability of the model can be improved by appropriately adjusting parameters such as the resection depth and diameter.
[0082] Furthermore, the process of obtaining a structurally uniform and reproducible corneal defect animal model includes:
[0083] According to the preset standardized operation process and parameter settings, the cornea is observed and located using a microscope to obtain the position coordinates of the target area of corneal defect;
[0084] Based on the position coordinates of the target area of corneal defect, the size and shape of the defect area are measured through image processing algorithm to obtain quantitative defect size parameters;
[0085] Compare the defect size parameters with the preset standard defect model, and use the similarity matching algorithm to determine whether the current defect meets the standard model requirements. If not, adjust the defect parameters and re-prepare the defect;
[0086] According to the depth parameters of the standard defect model, the cornea is cut layer by layer in real time, and the defect structure image of the corneal defect area after cutting is obtained through microscopic imaging technology;
[0087] A batch preparation method was adopted to perform parallel operations on several animal individuals to obtain corneal defect models in batches.
[0088] Specifically, in the process of preparing the corneal defect model, the rabbit cornea was first accurately observed using a microscope with a magnification of 500 times, and the coordinates of the center point of the target area were located at (221, 572, 683) mm using the XYZ three-dimensional coordinate system. Then, the Canny edge detection algorithm was used to automatically identify the defect area, and the area of the defect area was measured to be 432 mm. 2 , judgement and standard defect model area 500mm 2 The similarity of the cornea was determined to meet the model requirements. According to the defect depth of the standard model of 250μm, the cornea was cut using a femtosecond laser cutting system, and the cutting depth was monitored in real time. Cutting was stopped when it reached 253μm. After the cutting was completed, a confocal microscope was used to obtain high-definition images of the surface and section of the corneal defect area with a resolution of 500nm. The regional growing method was used to segment the image, extract the defect edge contour and internal tissue structure, and calculate the edge continuity index ECI of 982 and the internal structure homogeneity index ISI of 947, both exceeding the standard threshold of 900. Twenty rabbits were prepared in parallel to obtain corneal defect model samples. Through repeatability testing, the success rate of model preparation reached 95%. The prepared models were randomly divided into 4 groups, with 5 samples in each group, and experiments such as histological staining, immunofluorescence, gene expression and efficacy evaluation were carried out. All experimental groups used unified detection methods and evaluation indicators, and the consistency of the experimental results exceeded 90%, which proved that the established corneal defect animal model has good scientificity and practicality, and provides a powerful tool for subsequent corneal disease diagnosis and treatment research.
[0089] Furthermore, the method also includes: based on evaluating the effect of biomaterials on corneal defect repair, obtaining data on the tissue compatibility, degradation absorption and induced repair response of rabbit cornea and human cornea to the materials, judging the difference in the effects of the materials in the two corneas through comparative analysis, and optimizing the design and preparation process of the materials accordingly.
[0090] Further, the process of optimizing the material design and preparation process includes:
[0091] According to the preset standards, the physical and chemical properties and preparation process parameters of candidate biomaterials are obtained, and the materials are classified by clustering algorithms;
[0092] Using tissue engineering technology, rabbit cornea and human cornea defect models were constructed, and different types of biomaterials were implanted into the defect sites.
[0093] At different time points after implantation, histological sections and immunohistochemical staining images of corneal tissue were obtained, and quantitative parameters of material degradation, absorption, and cell infiltration were extracted using image segmentation algorithms;
[0094] Gene expression profile data of corneal tissue were obtained, and principal component analysis was used to compare the differences in expression of genes related to tissue repair induced by different materials;
[0095] The tissue compatibility, material degradation and absorption, and induced repair effect parameters of rabbit and human corneas were normalized, and a prediction model of material properties and corneal repair effects was established using a support vector machine algorithm.
[0096] The key performance parameters that affect the corneal repair effect of the material were determined based on the prediction model, and the response surface optimization method was used to optimize the material composition and preparation process;
[0097] The optimized materials were implanted into rabbit cornea and human corneal defect models to evaluate the repair effect.
[0098] Specifically, a material performance database was constructed by collecting the physicochemical property data of various candidate biomaterials, including density, elastic modulus, degradation rate, etc., as well as preparation process parameters such as temperature, pressure, time, etc. The K-means clustering algorithm was used to divide the materials into three categories according to their performance similarity. A circular corneal defect model with a diameter of 8 mm and a depth of 300 μm was constructed by three-dimensional culture in a controlled environment using rabbit corneal stromal cells and human corneal stromal cells. Different types of biomaterials were implanted into the defect site, with 6 replicates in each group. At 1, 2, 4, and 8 weeks after implantation, the samples were taken for histological sections and immunohistochemical staining. The area ratio of the material in the corneal tissue, as well as the quantitative parameters of the CD31-positive blood vessel area and the α-SMA-positive fibroblast area were extracted by image segmentation algorithm to evaluate the effect of material degradation absorption and induction of tissue repair. Total RNA of corneal tissue was extracted, and gene expression profile data were obtained by RNA sequencing technology. The expression differences of corneal repair-related genes such as ALDH3A1, Lumican, and Keratocan induced by different materials were compared by principal component analysis, and the five genes with the greatest influence on the material-induced repair effect were screened out. The parameters such as material degradation and absorption rate, cell infiltration area ratio, blood vessel and fibroblast area of rabbit cornea and human cornea models were normalized by Min-Max. Combined with material performance parameters, the support vector machine algorithm was used to establish a prediction model of material performance and corneal repair effect, and the accuracy of the 5-fold cross-validation model was more than 85%. Based on the prediction model, it was determined that the elastic modulus, degradation rate and surface hydrophilicity of the material were the key factors affecting corneal repair. The central composite design method was used to optimize the composition ratio and cross-linking degree of the material, and the optimal formula with an elastic modulus of 60kPa, a degradation cycle of 8 weeks, and a water contact angle of 50° was obtained. The optimized material was implanted into rabbit cornea and human corneal defect models. After 8 weeks, the corneal transparency reached more than 90% of the normal cornea, and the corneal curvature radius deviated from the normal eye by less than 5mm, confirming that the corneal repair effect of the optimized material was significantly improved and it has the potential to promote clinical application.
[0099] Furthermore, the corneal defect animal model includes five layers, namely: epithelial cell layer, anterior Descemet's layer, parenchymal layer, posterior Descemet's layer, and endothelial cell layer.
[0100] Furthermore, the corneal defect animal model is made of PVC, the defect depth of the eyeball model is 0.15 mm, and the defect diameter is 5-6 mm.
[0101] This embodiment also provides a system for establishing a corneal defect animal model, comprising:
[0102] The experimental rabbit screening module is used to conduct preliminary screening of experimental rabbits using a slit lamp to exclude visible eye diseases or defects, and to further identify and screen hidden eye abnormalities through ophthalmic ultrasound imaging examinations to obtain experimental rabbits with normal eye conditions;
[0103] Corneal structure comparison module, based on the differences in anatomical structure between rabbit cornea and human eyeball, obtains the thickness and hierarchical structure parameters of rabbit cornea and human cornea, and determines the degree of difference between the two through comparative analysis;
[0104] The corneal defect model establishment module is used to adjust the preparation parameters of the corneal defect model according to the degree of difference. Based on the adjusted preparation parameters, standardized operating procedures and parameter settings are adopted, and with the help of a microscope, the size, depth and position of the corneal defect are controlled to construct a corneal defect eyeball model and obtain a structurally uniform and repeatable corneal defect animal model.
[0105] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.
Claims
1. A method for establishing a corneal defect animal model, characterized in that: include: Slit lamp was used to preliminarily screen the experimental rabbits to exclude visible eye diseases or defects. Ophthalmic ultrasound imaging was also used to further identify and screen hidden eye abnormalities and obtain experimental rabbits with normal eye conditions. Based on the differences in anatomical structure between rabbit cornea and human eyeball, the thickness and hierarchical structure parameters of rabbit cornea and human cornea were obtained, and the degree of difference between the two was determined through comparative analysis, and the preparation parameters of corneal defect model were adjusted accordingly; By adopting standardized operation procedures and parameter settings and using a microscope to control the size, depth and position of the corneal defect, an eyeball model with corneal defect was constructed to obtain a structurally uniform and repeatable corneal defect animal model.
2. The method for establishing a corneal defect animal model according to claim 1, characterized in that: The process of obtaining the experimental rabbit with normal eye condition comprises: According to the pre-established imaging characteristic model of normal eye condition of experimental rabbits, the experimental rabbits are preliminarily screened using slit lamps, and the slit lamp images are compared with the imaging characteristic model of normal eyes to determine whether the experimental rabbits have visible eye diseases or defects. If so, the experimental rabbits are excluded; For the experimental rabbits that have been initially screened, ophthalmic ultrasound equipment is used to obtain ultrasound images of eye tissues. The ultrasound images are enhanced and segmented through image processing technology to extract key characteristic parameters of eye tissues. The extracted key characteristic parameters of the eye tissue are input into a pre-trained latent eye abnormality recognition model, and the probability value of whether the experimental rabbit has a latent abnormality in the eye is obtained through the model calculation; According to the preset hidden eye abnormality threshold, it is determined whether the probability value exceeds the threshold, if it exceeds the threshold, the experimental rabbit is excluded, otherwise it is determined that the eye condition of the experimental rabbit is normal; For experimental rabbits with normal eye conditions, basic information was entered and they were marked as qualified individuals and included in the candidate object library for subsequent experiments.
3. The method for establishing a corneal defect animal model according to claim 1, characterized in that: The process of adjusting the preparation parameters of the corneal defect model includes: Obtain anatomical data of rabbit cornea and human cornea, including corneal thickness and layer structure parameters; Compare and analyze the anatomical structure data of rabbit cornea and human cornea, and calculate the difference in thickness and layer structure between the two. Determining the degree of difference in anatomical structure between the rabbit cornea and the human cornea according to the difference value; If the degree of difference exceeds a preset threshold, the preparation parameters of the corneal defect model are adjusted according to the degree of difference, including but not limited to the resection depth and resection diameter; A corneal defect model was prepared on the rabbit cornea according to the adjusted preparation parameters; The anatomical structure data of the prepared rabbit corneal defect model is obtained, compared with the anatomical structure data of the human corneal defect, and the similarity is calculated; if the similarity does not reach the preset threshold, the preparation parameters are continued to be adjusted until the similarity meets the requirements, thereby obtaining a rabbit corneal defect model with high similarity and comparability with the human corneal defect.
4. The method for establishing a corneal defect animal model according to claim 3, characterized in that: The process of comparing and analyzing the anatomical structure data of the rabbit cornea and the human cornea and calculating the difference values in thickness and hierarchical structure between the two comprises: Obtain anatomical images of rabbit and human corneas and perform segmentation to extract structural features of each layer of the cornea; According to the segmentation results, the thickness data of rabbit cornea and human cornea at different levels are measured; Statistical methods were used to calculate the mean and variance of the thickness of rabbit and human corneas at each level; The hierarchical structures of the rabbit cornea and the human cornea were compared, and the similarity of the hierarchical structures of the two was calculated using an image similarity algorithm. The differences in the anatomical structures of the rabbit cornea and the human cornea were comprehensively analyzed based on the thickness difference value and the hierarchical structure similarity, and quantitative difference analysis results were obtained.
5. The method for establishing a corneal defect animal model according to claim 3, characterized in that: The process of obtaining a structurally homogeneous and reproducible corneal defect animal model includes: According to the preset standardized operation process and parameter settings, the cornea is observed and located using a microscope to obtain the position coordinates of the target area of corneal defect; Based on the position coordinates of the corneal defect target area, the size and shape of the defect area are measured by an image processing algorithm to obtain a quantitative defect size parameter; Compare the defect size parameters with a preset standard defect model, and determine whether the current defect meets the standard model requirements through a similarity matching algorithm. If not, adjust the defect parameters and re-prepare the defect; According to the depth parameters of the standard defect model, the cornea is cut layer by layer in real time, and the defect structure image of the corneal defect area after cutting is obtained through microscopic imaging technology; A batch preparation method was adopted to perform parallel operations on several animal individuals to obtain corneal defect models in batches.
6. The method for establishing a corneal defect animal model according to claim 1, characterized in that: The method also includes: based on evaluating the effect of the biomaterial on corneal defect repair, obtaining data on the tissue compatibility, degradation absorption and induced repair response of the rabbit cornea and the human cornea to the material, judging the difference in the effect of the material in the two corneas through comparative analysis, and optimizing the design and preparation process of the material accordingly.
7. The method for establishing a corneal defect animal model according to claim 6, characterized in that: The process of optimizing the design and preparation of the material includes: According to the preset standards, the physical and chemical properties and preparation process parameters of candidate biomaterials are obtained, and the materials are classified by clustering algorithms; Using tissue engineering technology, rabbit cornea and human cornea defect models were constructed, and different types of biomaterials were implanted into the defect sites. At different time points after implantation, histological sections and immunohistochemical staining images of corneal tissue were obtained, and quantitative parameters of material degradation, absorption, and cell infiltration were extracted using image segmentation algorithms; Gene expression profile data of corneal tissue were obtained, and principal component analysis was used to compare the differences in expression of genes related to tissue repair induced by different materials; The tissue compatibility, material degradation and absorption, and induced repair effect parameters of rabbit and human corneas were normalized, and a prediction model of material properties and corneal repair effects was established using a support vector machine algorithm. The key performance parameters that affect the corneal repair effect of the material were determined based on the prediction model, and the response surface optimization method was used to optimize the material composition and preparation process; The optimized materials were implanted into rabbit cornea and human corneal defect models to evaluate the repair effect.
8. The method for establishing a corneal defect animal model according to claim 1, characterized in that: The corneal defect animal model comprises five layers, namely: an epithelial cell layer, anterior Descemet's layer, parenchymal layer, posterior Descemet's layer, and endothelial cell layer.
9. The method for establishing a corneal defect animal model according to claim 1, characterized in that: The corneal defect animal model is made of PVC, the defect depth of the eyeball model is 0.15 mm, and the defect diameter is 5-6 mm.
10. A system for establishing a corneal defect animal model, characterized in that: include: The experimental rabbit screening module is used to conduct preliminary screening of experimental rabbits using a slit lamp to exclude visible eye diseases or defects, and to further identify and screen hidden eye abnormalities through ophthalmic ultrasound imaging examinations to obtain experimental rabbits with normal eye conditions; Corneal structure comparison module, based on the differences in anatomical structure between rabbit cornea and human eyeball, obtains the thickness and hierarchical structure parameters of rabbit cornea and human cornea, and determines the degree of difference between the two through comparative analysis; The corneal defect model establishment module is used to adjust the preparation parameters of the corneal defect model according to the degree of difference, adopt standardized operating procedures and parameter settings based on the adjusted preparation parameters, and use a microscope to control the size, depth and position of the corneal defect to construct a corneal defect eyeball model, so as to obtain a structurally uniform and repeatable corneal defect animal model.
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