Method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis

Abstract

The invention discloses a method for detecting mechanical properties of articular cartilages at different positions based on osteoarthritis, and relates to the field of pathological analysis. Magnetic resonance imaging (MRI) and image analysis; according to mechanical indentation mechanical measurement and statistical analysis, the mechanical characteristics of articular cartilage (AC) of different stages and multiple areas of osteoarthritis (OA) are measured through a depth sensing indentation technology system, and specifically, the mechanical characteristics comprise the inner side far-end femoral condyle, the outer side far-end femoral condyle, the inner side rear femoral condyle and the outer side rear femoral condyle. The result shows that the AC mechanics of the four areas presents a consistent trend along with the progress of OA. It is worthy of notice that the AC elastic modulus of the OA3 stage is minimum in the last three OA stages, and the inflection point lays a solid foundation for subsequent understanding of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of pathological analysis, and particularly to a method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis. Background Art

[0002] The prevalence of osteoarthritis (OA) is quite high, and osteoarthritis is a progressive, irreversible and incurable disease. Therefore, a comprehensive understanding of the prevalence, incidence and modifiable risk factors of OA is crucial for effective prevention and early intervention. From a structural perspective, the degeneration of articular cartilage (AC) is a defining feature of osteoarthritis, because articular cartilage plays a key role in joint protection, being able to distribute the applied load, reduce potential stress concentrations, and provide a low-friction surface to ensure smooth joint movement.

[0003] Articular cartilage (AC) evolves into a remarkable and non-homogeneous tissue, characterized by a heterogeneous composition, which is closely related to its biological function. This structural tissue is mainly composed of water (60 - 85%), a solid matrix of collagen (15 - 22%), proteoglycans (PG, 4 - 7%) and chondrocytes (3 - 5%). Collagen and water together constitute the largest component of AC, followed by proteoglycans. Each component has unique mechanical properties and performs different biological functions. For example, the collagen network serves as the structural framework of articular cartilage, providing the main tensile and shear strength, while proteoglycans, due to their significant net negative charge, attract water into the tissue, thus enhancing the compressive strength of articular cartilage. The synergistic effect between these components endows articular cartilage with excellent mechanical properties. On the contrary, any change in any component of this complex system may lead to damage to articular cartilage, thus impairing its normal function in the body. In addition, changes in the composition of articular cartilage may further alter its mechanical properties.

[0004] To further explore the goal of using the mechanical changes of articular cartilage as the basis for early diagnosis of osteoarthritis, the present invention uses a depth-sensing indentation technology system to measure the mechanical properties of articular cartilage at different stages of osteoarthritis and multiple regions. Summary of the Invention

[0005] The present invention aims to provide a method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, so as to solve the problem that the prior art cannot accurately obtain the mechanical properties of articular cartilage.

[0006] To achieve the above object, the present invention provides the following technical solutions: A method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, comprising the following steps: S1. Obtain a knee joint sample. For the first part of the knee joint sample, perform slicing and pathological grade classification, and use hematoxylin-eosin staining to obtain histological images. For the second part of the knee joint sample, which includes articular cartilage and subchondral bone, store it in an anti-degradation solution and dissolve it in a buffer solution. S2. Obtain magnetic resonance images of the articular cartilage. Then, outline the regions of interest (ROIs) of different articular cartilages on the histological images and magnetic resonance images to make the ROIs of the histological images match those of the magnetic resonance images. S3. Extract articular cartilage samples from the medial distal femoral condyle, lateral distal femoral condyle, medial posterior femoral condyle, and lateral posterior femoral condyle of the second part of the knee joint sample. Place the articular cartilage samples in a sealed chamber filled with a buffer solution and perform mechanical indentation measurements within the ROIs respectively. S4. Obtain the elastic modulus of the articular cartilage of osteoarthritis with different pathological grades through mechanical indentation measurements, and obtain the mechanical properties of the articular cartilage at different positions.

[0007] For the method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, in step S1, the condyle samples in the first part of the knee joint sample are fixed in 3 - 6% formaldehyde and decalcified with a rapid bone decalcifying agent for 36 - 48 hours, and then embedded in paraffin. The section thickness of the fixed sample is 6 - 10 µm.

[0008] For the method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, in step S1, the anti-degradation solution includes 1 - 2 mM ethylenediaminetetraacetic acid, 3 - 6 mM benzamidine hydrochloride, 8 - 12 mM N-ethylmaleimide, 1 - 3 mM phenylmethylsulfonyl fluoride, and penicillin; alternatively, the penicillin is replaced with streptomycin.

[0009] For the method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, in step S1, perform histopathological evaluation on the sliced first part of the knee joint sample based on the standardized Osteoarthritis Research Society International cartilage assessment system and the Kellgren-Lawrence grading system to complete the pathological grade classification.

[0010] For the method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, in step S2, the step of obtaining magnetic resonance images of the articular cartilage specifically includes: obtaining three sagittal magnetic resonance images, including a fat-suppressed proton density weighted imaging sequence, a T2 mapping sequence, and a T1ρ mapping sequence.

[0011] The method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, wherein in step S2, the step of outlining the regions of interest of different articular cartilages on the histological image and the magnetic resonance image includes: In the histological image, taking the c-line of the condyle as the reference line, draw an R-line parallel to the c-line, and the distance between the c-line and the R-line is represented by the p-distance; Taking the c-line of the condyle as the reference line, draw an R-line in the magnetic resonance image according to the p-distance.

[0012] The method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, wherein in step S3, at the start of mechanical indentation measurement, the tip of the indenter approaches the sample surface at a speed of 18 - 25 nm / s. When the instrument detects a value reaching 2 - 4 μN, the instrument starts to load at a speed of 15 - 58 nm / s. When the maximum penetration depth reaches 2800 - 3000 nm, the load on the indenter remains unchanged for 60 seconds. Finally, the indenter unloads at a speed of 280 - 300 nm / s, and then a low-load cycle is performed to evaluate the temperature drift effect.

[0013] The method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, wherein in step S3, at least 100 points are randomly selected on each sample for mechanical indentation measurement, and the distance between the indentation points is 45 - 60 microns.

[0014] A method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, wherein in step S4, the elastic modulus equation of the articular cartilage of different pathological grades of osteoarthritis is:

[0015] Wherein, E sample is the elastic modulus, λ sample is the Poisson's ratio of the test sample, E r is the reduced modulus between the indenter and the sample, and its relationship with the contact stiffness follows the equation:

[0016] Wherein, S is the contact stiffness, A C is the contact area between the indenter and the sample, the projected contact area A C is used as the contact depth h c as a function of can be described by the following equation:

[0017] wherein hc is the contact indentation depth, and the coefficients C0 , C1 ,…, C8 are determined by fitting experimental data.

[0018] Advantageous effects: The present invention provides a method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, including the steps of: obtaining a knee joint sample, wherein the first part of the knee joint sample is sectioned and pathologically graded, and hematoxylin-eosin staining is used to obtain a histological image; the second part of the knee joint sample includes articular cartilage and its underlying bone, which is stored in an anti-degradation solution and dissolved in a buffer solution; obtaining a magnetic resonance image of the articular cartilage, and then outlining regions of interest of different articular cartilages on the histological image and the magnetic resonance image to make the regions of interest of the histological image match those of the magnetic resonance image; thawing the second part of the knee joint sample, respectively extracting articular cartilage samples at the medial distal femoral condyle, lateral distal femoral condyle, medial posterior femoral condyle and lateral posterior femoral condyle, placing the articular cartilage samples in a closed chamber filled with a buffer solution, and respectively performing mechanical indentation measurements; obtaining the elastic modulus of the articular cartilage of osteoarthritis with different pathological grades through mechanical indentation measurements, and obtaining the mechanical properties of articular cartilage at different positions. The present invention uses a depth-sensing indentation technology system to measure the mechanical properties of AC at different OA stages and multiple regions, specifically including the medial distal femoral condyle, lateral distal femoral condyle, medial posterior femoral condyle and lateral posterior femoral condyle. The results show that the AC mechanics in these four regions show a consistent trend with the progression of OA, and the AC elastic modulus at the OA3 stage is the smallest among the last three OA stages, and this inflection point lays a solid foundation for the subsequent understanding of osteoarthritis. Description of the drawings

[0019] Figure 1 is a typical load-displacement curve for nanoindentation measurement of articular cartilage, and the upper left inset is the loading plan adopted; Figure 2 shows the changes of cartilage from OARSI grade I to OARSI grade IV (in panel c). The scale bar in panel c represents 500 T2 mapping images (panels a and b) and the corresponding histopathological microns; Figure 3Relationship between elastic modulus and osteoarthritis grade on the surface of knee joint cartilage in different regions: medial distal femoral condyle (LDC, N = 17) (A), lateral distal femoral condyle (MDC, N = 16) (B), lateral posterior femoral condyle (LPC, N = 17) (C), and medial posterior femoral condyle (MPC, N = 17) (D). The letters (a, b, and c) above the bar graphs indicate that the distributions are significantly different (different letters) or not significantly different (same letters) at the p < 0.05 level.

[0020] Figure 4 Elastic modulus of AC in different regions (LDC, MDC, LPC, and MPC) under grade II (A) and grade III (B) of OA. The letters (a and b) above the bar graphs indicate that the distributions are significantly different (different letters) or not significantly different (same letters) at the p < 0.05 level.

[0021] Figure 5 Schematic diagram of indentation measurement of articular cartilage on the upper joint at Osteoarthritis Research Society International (OARSI) grades 1 - 4; Detailed implementation mode The present invention will be further described in detail below in conjunction with the accompanying drawings and the implementation mode. The specific implementation process is as follows: A method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, comprising the following steps: S1. Obtain knee joint samples through total knee arthroplasty. For the first part of the knee joint samples, sectioning is performed and pathological grading is carried out. Using hematoxylin - eosin (HE) staining, histological images are obtained; for the second part of the knee joint samples, which include articular cartilage and the underlying bone, they are preserved in an anti - degradation solution containing 2 mM ethylenediaminetetraacetic acid, 5 mM benzamidine hydrochloride, 10 mM N - ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, and penicillin / streptomycin, dissolved in 1 liter of phosphate - buffered saline (PBS). This preserved part is then stored at - 80 °C for subsequent mechanical testing.

[0022] It should be clear that the articular cartilage samples used in the present invention are sourced from surgical waste generated during total knee arthroplasty. A total of 21 volunteers participated, and all of them provided consent through signed informed consent forms. The inclusion criteria included meeting the osteoarthritis diagnostic criteria established by the American College of Rheumatology, being over 50 years old, and showing a Kellgren - Lawrence grade of 3 - 4 in radiological assessment. The exclusion criteria included: (1) having an autoimmune disease; (2) being diagnosed with rheumatoid arthritis; (3) having a history of knee infection; (4) participants who were considered clinically unsuitable for inclusion in the study.

[0023] HE staining and histopathological evaluation Preferably, the obtained condyle samples were fixed in 4% formaldehyde and decalcified using a rapid bone decalcifying agent (AmericanMasterTech Inc., Lodi, CA, USA) for 48 hours, and then embedded in paraffin. The sections of the fixed samples were approximately 8 µm thick and stained with hematoxylin and eosin (HE). Then, at least two experienced pathologists independently performed a rigorous histopathological evaluation of each tissue section, following the standardized Osteoarthritis Research Society International (OARSI) cartilage assessment system and the recognized Kellgren-Lawrence grading system. The evaluation results were subsequently reviewed and compared to ensure consensus among the evaluators. If there were differences in the evaluation of a specific sample, the sample was re-evaluated by another pathologist, and finally, a final consensus on the pathological grade was reached through sufficient discussion.

[0024] S2. Obtain magnetic resonance images of the articular cartilage before surgery, and then outline the regions of interest (ROIs) of different articular cartilages on the histological images (HE staining) and magnetic resonance images to match the ROIs of the histological images with those of the magnetic resonance images; Preoperative MRI images were obtained using a commercial 3.0T MRI scanner (Philips Healthcare, Eindhoven, Netherlands). Three sagittal MR images were obtained, including fat-suppressed (FS) proton density-weighted imaging (PDWI) sequence, T2 mapping sequence, and T1ρ mapping sequence. All sagittal images were obtained without angulation and were parallel to the magnetic static field (B0). The acquisition parameters of the sagittal T2 mapping sequence were as follows: repetition time = 1000 ms, echo time = 0 ms, optional lock time = 9.5 / 19.1 / 28.6 / 38.1 / 47.6 / 57.2 / 66.7 / 76.2 ms, slice thickness = 4 mm, slice gap = 0.5 mm, number of slices = 20, field of view = 160 mm, matrix size = 320×256, number of excitations = 1, scan time = 8 minutes and 46 seconds.

[0025] Regions of interest (ROIs) of different cartilages were outlined. The condyle was divided into ROI parts to match the histological images (HE staining). During the operation, the c-line (Whiteside line) of the condyle was marked for surgical consideration. The histological ROI was selected by drawing an R-line parallel to the c-line. The p-distance was applied to determine the interval between the c-line and the R-line, and this ROI was identified and marked by the surgeon during the operation. Then, the Whiteside line was drawn in the MRI image and set as the reference line, and by applying the p-distance, the tissue section of interest could be marked in the MRI image. By this method, the tissue section was matched with the ROI of the MRI.

[0026] S3. Thaw the second part of the knee joint samples, and separately extract the articular cartilage samples at the medial distal femoral condyle (MDC), lateral distal femoral condyle (LDC), medial posterior femoral condyle (MPC), and lateral posterior femoral condyle (LPC). Place the samples in a sealed chamber filled with phosphate buffered saline (PBS) solution, and perform mechanical indentation measurements within the region of interest respectively; Preferably, to thaw the frozen articular cartilage samples, a sequential thawing process is employed. This process includes incubating at -20°C for 10 hours, then incubating at 4°C for another 10 hours, and finally incubating at room temperature for 10 hours. After thawing, AC samples for mechanical measurements are extracted using a circular stamping tool with a diameter of 10 mm. Then, the prepared samples are firmly fixed on the mechanical measurement bracket using 3M rapid drying glue (AD119, 3M, USA) to ensure good adhesion at the interface between the sample and the bracket. After fixation, the samples are immediately subjected to mechanical measurements.

[0027] The micro-mechanical properties of articular cartilage are evaluated by depth-sensing indentation technique. For this purpose, measurements are carried out using a NanoIndenter G200 (Keysight Technologies, Inc., Santa Rosa, California, USA) equipped with a standard Berkovich indenter. The elastic modulus of this indenter is 1140 GPa and the Poisson's ratio is 0.07. The bracket carrying the prepared AC samples is safely placed in a custom-made sealed chamber filled with phosphate buffered saline (PBS) solution. This setup ensures that both the AC sample and the indenter tip are immersed in the PBS solution, thus simulating the mechanical properties of AC in its natural state in vivo.

[0028] Preferably, at the start of the indentation test, the indenter tip approaches the sample surface at a speed of 20 nm / s. The contact point is determined by setting a setpoint of 2 μN, that is, when the instrument detects that the value of 2 μN is reached, it is considered that contact with the sample surface has occurred. Then, the instrument starts loading at a speed of 50 nm / s. When the maximum penetration depth reaches 3000 nm, the load on the indenter remains constant for 60 seconds. To minimize the influence of viscosity on the calculation of elastic modulus, the indenter is unloaded at a higher speed of 300 nm / s, followed by a low-load cycle to evaluate the temperature drift effect. A typical load-displacement curve is as Figure 1 shown, and the designed loading and unloading schemes are illustrated in the inset panel. To ensure the reproducibility of the results, more than 100 points are randomly selected on each sample for indentation measurements. The distance between indentation points is 50 microns to prevent potential strain field interactions.

[0029] S4. Obtain the elastic modulus of the articular cartilage of osteoarthritis with different pathological grades through mechanical indentation measurements, and obtain the mechanical properties of the articular cartilage at different positions.

[0030] To obtain a more realistic elastic modulus of the sample, the contact zero point of the load-displacement curve was examined and corrected if necessary. The unloading portion included 50% - 95% of each load-displacement curve and was analyzed using the Oliver-Pharr model. The reduced modulus (E r ) between the indenter and the sample and the relationship with the contact stiffness (S) obtained from the unloading curve ( Figure 1 ) follow the following equation:

[0031] where A C is the contact area between the indenter and the sample. The shape of a Berkovich indenter is typically described as a three-sided pyramid with a face angle of 65.3°, and its shape is represented by an area function. The projected contact area AC as a function of the contact depth h c can be described by the following equation:

[0032] where h c is the contact indentation depth, and the coefficients C0, C1, …, C8 are determined by fitting experimental data. For an ideal Berkovich indenter, C0 is 24.5 and the other coefficients are 0.

[0033] The elastic modulus of the sample can be described by the following equation:

[0034] where λ sample and λ indenter are the Poisson's ratios of the test sample and the indenter, respectively. Considering that the elastic modulus of the indenter (E indenter , 1140 GPa) is much larger than that of the sample (E sample ), the above equation can be simplified to:

[0035] According to existing research considerations, the Poisson's ratio (λ C ) of A sample is considered to be 0.5.

[0036] Statistical analysis Data are presented as mean ± standard deviation (SD). To calculate significance, one-way ANOVA was used followed by Tukey's post hoc test. Statistical significance was defined as p < 0.05. All analyses were performed using Graphpad Prism (version: 5.01, GraphPad Software Inc, California, USA).

[0037] Results Anthropometric information of osteoarthritis patients Table 1 shows the anthropometric characteristics of the patients involved in the present invention. Evaluation of knee X-rays using the Osteoarthritis Research Society International criteria and the Kellgren-Lawrence grading system revealed that none of the patients were rated grade 0. However, it should be noted that not all four articular cartilage regions (MDC, LDC, MPC, and LPC) exhibited all four stages of OA. This difference can be attributed to the samples being derived from discarded materials generated after total knee arthroplasty.

[0038] Table 1 Anthropometric information of osteoarthritis patients.

[0039]

[0040] T2 mapping images To visually understand the progression of OA and the compositional changes of AC, magnetic resonance imaging (MRI) was used to obtain images of the samples. Figure 2 Shown are the T2 mapping images of AC at different OA grades and their corresponding histopathological analyses. The main findings include: (1) In grade I OA, initial depletion of proteoglycans was observed, accompanied by the onset of collagen matrix degradation; (2) As OA progressed to grade II, proteoglycans were severely depleted and collagen fibers were broken into shorter fragments; (3) In grade III OA, the content of proteoglycans and collagen matrix decreased significantly, resulting in the formation of a softened layer; (4) Finally, in grade IV OA, the cartilage was almost completely depleted, reflecting the late stage of the degenerative process.

[0041] Elastic modulus of AC at different OA stages MRI results indicated that as OA progressed, the structure and morphology of AC changed. Therefore, it is reasonable to expect that the mechanical properties of AC would also change with the development of OA. To study these changes, a depth-sensing indentation technique was used to measure the elastic modulus of AC at different OA stages. Figure 3 Shown are the elastic moduli of AC in four different regions at different OA stages. The research results showed that the elastic modulus of AC in the hydrated state ranged from 1 to 100 MPa.

[0042] The elastic moduli of AC in all four regions showed a consistent trend of change during the OA process ( Figure 3 ). Specifically, the elastic modulus of AC at OA stage 2 was higher than that at OA stage 1 (as shown by C and D in Figure 3 ). As OA progressed, the elastic modulus of AC reached its lowest point at OA stage 3, compared to stages 2 and 4 (as shown in Figure 3A, B, D) in it. In addition, the AC elastic modulus in OA stage 4 is the highest. However, it is worth noting that when OA progresses to stage 3, a significant inflection point in the elastic modulus of AC occurs, which may provide valuable insights for further understanding the pathology of OA.

[0043] To quantitatively evaluate the mechanical properties of AC in the four regions, their elastic moduli at different OA stages were compared (as Figure 4 shown). The results showed that there were statistically significant differences in the elastic moduli of AC in the four regions at grade III stage (p < 0.05), while there were no significant differences at grade II stage.

[0044] According to the results of depth-sensing indentation tests, it is obvious that the elastic modulus of AC is significantly affected by the progression of OA. In addition, it is worth noting that the measured elastic moduli of the four regions of AC show a consistent trend during the progression of OA, although the levels of mechanical loads they bear are different. However, there are indeed differences in the absolute values of the measured elastic moduli among different regions, which can be attributed to the uneven progression of OA in these regions and thus different bearing loads on AC.

[0045] Changes in the content or interaction of AC components are expected to alter its mechanical properties. Therefore, in order to reveal the reasons for the mechanical changes of AC during the progression of OA, it is crucial to further study the components of AC and the process of OA. Although the etiology of OA has not been fully clarified, it is generally believed that there are differences in the degradation composition of AC at different OA stages. For example, in the early stage of OA, the main change observed is the loss of glycosaminoglycans, followed by changes in the collagen structure. In addition, the orientation of collagen fibers in the superficial AC also changes significantly in early OA, while a significant decrease in collagen content mainly occurs in the late stage of OA (as Figure 5 shown). Since the degradation of components is different at different stages of OA, the changes in the mechanical properties of AC during the progression of OA are also different. For example, the collagen network, as the structural framework of AC, is the main source of tensile and shear strength, while proteoglycans endow AC with considerable compressive strength due to their significant net negative charge. Therefore, the compositional changes occurring at different stages of OA lead to significant differences in the mechanical behavior of AC.

[0046] In addition, OA also changes the arrangement of components in AC. In healthy AC, three structurally different regions can be distinguished according to the main orientation of collagen fibers (as Figure 5As shown in the figure: (i) The superficial zone (10 - 20% of the total thickness), where the fibers are arranged parallel to the joint surface; (ii) The transition zone (40 - 60%), where the fibers are randomly oriented; (iii) The radial zone (30%), where the fibers are almost perpendicular to the AC. With the progression of OA, an irregular arrangement of collagen fibers is observed in the superficial and deep zones of the AC. Therefore, the second possible reason for the change in the mechanical properties of the AC during the development of OA is as follows (as shown in Figure 4): (1) In grade I OA, the degradation of the intact structure of the proteoglycan and collagen matrix in the AC leads to direct contact between the collagen fibers and the indenter; (2) In grade II OA, the proteoglycans are severely depleted and the collagen fibers are broken into segments. Therefore, more collagen segments are under the indenter, resulting in a higher elastic modulus at this grade compared to grade I; (3) With the further development of OA (grade III), the soft layer formed by the degradation of the proteoglycans and collagen matrix leads to a decrease in the elastic modulus; (4) In grade IV OA, since the cartilage is almost completely depleted, the obtained elastic modulus may represent that of the subchondral bone, which is higher than that of the AC.

[0047] The research results show that although there are significant differences in the mechanical properties of the four AC regions at specific OA stages, they exhibit similar trends during the progression of OA. Therefore, it is necessary to study the changes in the mechanical properties of each AC component at different OA stages and establish the complex relationship between OA progression and damaged AC components.

[0048] With the progression of OA, the elastic modulus of the AC shows more obvious discreteness, which may be related to the heterogeneity of AC damage at different OA stages. As a biological tissue, the biomechanical function of the AC is closely related to its composition, including the water content, the concentrations of collagen, proteoglycans, and hyaluronic acid, as well as the complex interactions between these components. With the development of OA, the integrity of the AC is damaged, and even at the same anatomical location and OA stage, the degree of damage varies because of the different mechanical forces acting on different regions of the knee joint. Therefore, the biomechanical properties of the damaged AC show higher discreteness within the same OA grade, as Figure 2 shown.

[0049] The mechanical properties of articular cartilage (AC) are jointly influenced by its composition and morphology, so these two factors must be considered simultaneously. This invention systematically explored the relationship between the mechanical properties of the overall AC (divided into four different regions: medial distal femoral condyle (MDC), lateral distal femoral condyle (LDC), medial posterior femoral condyle (MPC), and lateral posterior femoral condyle (LPC)) and the progression of osteoarthritis (OA). The results showed that the mechanical properties of AC in all four regions exhibited a consistent trend with the development of OA. Notably, the elastic modulus of AC at OA stage 3 was the lowest among stages 2 to 4. This result emphasizes that the mechanical properties of AC during OA depend on its degree of damage, which is closely related to the compositional changes of AC at different OA stages.

[0050] The observed regional differences in the mechanical properties of AC in OA highlight the importance of this invention. The identification of the inflection point emphasizes the necessity of studying the relationship between AC mechanics and OA based on specific AC locations. In addition, more detailed segmentation may yield more accurate results.

[0051] The findings of this invention provide a new perspective for the understanding of OA, pave a potential path for future therapeutic interventions, and emphasize the crucial role of location in studying AC mechanics. By examining the mechanical properties of AC in different regions, this invention provides more detailed information than previous studies. Although the basic mechanisms of OA still need to be further explored, studying the mechanical properties of AC at the micro level will help significantly uncover its complexity.

[0052] The above are only embodiments of the present invention, and common general knowledge such as specific technical solutions and / or characteristics known in the solution are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting the mechanical properties of articular cartilage at different positions based on osteoarthritis, characterized in that: The steps include: S1. Obtain knee joint samples, wherein the first part of the knee joint samples is sliced ​​and pathologically graded, and stained with hematoxylin-eosin to obtain a histological image; the second part of the knee joint samples includes articular cartilage and underlying bone, which are stored in an anti-degradation solution and dissolved in a buffer solution; S2, acquiring a magnetic resonance image of the articular cartilage, and then outlining regions of interest of different articular cartilages on the histological image and the magnetic resonance image, so that the regions of interest of the histological image match the regions of interest of the magnetic resonance image; S3, extracting articular cartilage samples at the medial distal femoral condyle, the lateral distal femoral condyle, the medial posterior femoral condyle and the lateral posterior femoral condyle from the second part of the knee joint samples, placing the articular cartilage samples in a closed chamber filled with a buffer solution, and performing mechanical indentation measurements in the regions of interest respectively; S4. The elastic modulus of articular cartilage of osteoarthritis of different pathological grades is measured by mechanical indentation, and the mechanical properties of articular cartilage in different positions are obtained.

2. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S1, the condylar samples in the first part of the knee joint sample are fixed in 3-6% formaldehyde and decalcified using a rapid bone decalcifier for 36-48 hours, and then embedded in paraffin. The slice thickness of the fixed sample is 6-10 µm.

3. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S1, the anti-degradation solution includes 1-2 mM ethylenediaminetetraacetic acid, 3-6 mM benzylbutyric acid chloride, 8-12 mM ethylmaleimide, 1-3 mM benzylthiourea fluoride and penicillin; or, the penicillin is replaced by streptomycin.

4. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S1, the first part of the knee joint sample after sectioning is subjected to histopathological evaluation based on the standardized Osteoarthritis Research Society cartilage assessment system and Kellgren-Lawrence grading system to complete the pathological grade classification.

5. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S2, the step of acquiring the magnetic resonance image of the articular cartilage is specifically: acquiring three sagittal magnetic resonance images, including a fat-suppressed proton density weighted imaging sequence, a T2 mapping sequence, and a T1ρ mapping sequence.

6. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S2, the step of outlining the regions of interest of different articular cartilages on the histological image and the magnetic resonance image comprises: In the histological image, the c-line of the condyle is used as a reference line, and an R-line parallel to the c-line is drawn, and the interval between the c-line and the R-line is represented by a distance p; Taking the c-line of the condyle as a reference line, an R-line is drawn in the magnetic resonance image according to the p-distance.

7. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S3, at the beginning of the mechanical indentation measurement, the tip of the indenter approaches the sample surface at a speed of 18-25 nm / s. When the instrument detects that a value of 2-4 μN is reached, the instrument starts loading at a speed of 15-58 nm / s. When the maximum penetration depth reaches 2800-3000 nm, the load on the indenter remains unchanged for 60 seconds. Finally, the indenter is unloaded at a speed of 280-300 nm / s, followed by a low-load cycle to evaluate the temperature drift effect.

8. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S3, at least 100 points are randomly selected on each sample for mechanical indentation measurement, and the distance between the indentation points is 45-60 μm.

9. The method for detecting mechanical properties of articular cartilage at different positions based on osteoarthritis according to claim 1, characterized in that: In step S4, the elastic modulus equation of the elastic modulus of the articular cartilage of osteoarthritis of different pathological grades is: in, E sample is the elastic modulus, λ sample is the Poisson’s ratio of the test sample, E r is the reduced modulus between the indenter and the sample, and its relationship with the contact stiffness follows the equation: in, S is the contact stiffness, A C is the contact area between the indenter and the sample, the projected contact area A C As contact depth h c The function can be described by the following equation: in hc is the contact indentation depth, the coefficient C0 , C1 ,…, C8 Determined by fitting experimental data.

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