Method for testing biological efficiency of pure magnesium material

Abstract

The invention relates to a method for testing the biological efficiency of a pure magnesium material, which comprises the following steps of: selecting a pure magnesium plate with the thickness of 0.2-0.3 mm, preparing a magnesium patch with the thickness of 0.3 mm, a 3mm * 3mm grid and the porosity of 40%, and testing the in-vitro biocompatibility, the in-vivo biomechanics and the in-vivo biological activity of the magnesium patch. The method comprises the following steps: co-culturing a magnesium patch leaching solution and human dermal fibroblasts in vitro, evaluating cytotoxicity through CCK8, detecting cell morphology through Calcein-AM / PI double staining, and detecting cell cycle and apoptosis through a flow cytometer; in order to evaluate the in-vivo biomechanics of the magnesium patch, a rat subcutaneous implantation test is carried out, and the biodegradability of the degradable magnesium patch is explored; in order to evaluate the in-vivo biological effect of the magnesium patch, a rat abdominal wall defect model is established, the magnesium patch is used for repairing, and TMT protein quantitative detection, differential protein correlation analysis, PCR and western blot are carried out.

Description

Technical Field

[0001] The present invention belongs to the field of materials, and specifically relates to a method for testing the biological efficacy of pure magnesium materials. Background Art

[0002] In hernia surgery, it is a common phenomenon to repair abdominal wall defects with organic or inorganic patches, but there are also many deficiencies in the patches. Magnesium metal has good mechanical properties and can be degraded in the body, but there are few reports on the research of magnesium metal patches.

[0003] We prepared a new type of degradable magnesium patch, and evaluated its in vitro and in vivo degradation behaviors by in vitro immersion test, in vivo weight loss experiment, scanning electron microscopy examination and elemental mapping analysis of its degradation rate.

[0004] To evaluate the biocompatibility of the degradable magnesium patch, we co-cultured the magnesium patch extract with human dermal fibroblasts (HSF) in vitro, and evaluated cell toxicity by CCK8, detected cell morphology by Calcein-AM / PI double staining, and detected cell cycle and apoptosis by flow cytometry.

[0005] We further established a rat abdominal wall defect model, repaired it with the degradable magnesium patch, and explored the biological effects of the degradable magnesium patch by TMT protein quantification detection, differential protein correlation analysis, PCR and Western blot.

[0006] The degradation rate of the degradable magnesium patch in simulated body fluid solution (SBF) (2.62 mm / year) is faster than that in Hanks solution (1.14 mm / year), and there are more corrosion products on the surface of its samples. 60% of the residual structure remains undegraded 8 weeks after implantation in vivo, and the patch is tightly combined with the surrounding abdominal wall tissue.

[0007] After co-culturing the magnesium patch extracts with different concentrations with HSF for 24 hours, their relative proliferation rates are all greater than 60%, the safety rating is 0-1 level, their cytotoxicity is within a reasonable range of influence, and they can promote the proliferation of fibroblasts at a specific concentration (60%).

[0008] The degradable magnesium patch will not cause a strong foreign body reaction during the repair process. Compared with polypropylene patches, it can increase the expression levels of type III and type V collagen, which are related to wound healing, and promote the repair of abdominal wall defect tissues.

[0009] The degradation time of the degradable magnesium patch meets the requirements of the abdominal wall repair support time, has excellent biocompatibility with epithelial fibroblasts and abdominal wall tissues, and can promote the repair of abdominal wall defect tissues.

[0010] The abdominal wall can protect the internal organs of the body and stabilize the body posture, while resisting changes in intra-abdominal pressure caused by physiological events (such as breathing, digestion, coughing, urination, defecation), trunk movement, physical activity or other activities. The loss of abdominal wall function caused by abdominal wall defects is a common situation in surgical practice. When the defect is too large, about 2%-20% of patients with abdominal wall defects need abdominal wall surgery to reduce the occurrence of complications such as organ damage, infection, and acquired hernias, thereby improving the quality of life. Abdominal wall defect repair using different material patches is the main treatment method. Synthetic mesh patches have become the most widely used repair materials because of their sufficient tensile strength and elasticity. The most commonly used material is PP (polypropylene). However, due to its non-degradable property, it has the following complications including: fistula formation, intestinal obstruction, adhesion, infection, chronic pain / foreign body sensation, and erosion of surrounding tissues.

[0011] Currently, there are reports in the relevant literature on the clinical application of degradable patches. It mainly includes two major categories of materials: synthetic degradable materials and biogenic materials. Synthetic degradable materials usually include high-molecular materials such as polylactic acid-copolymer (PLGA), polycaprolactone (PCL), etc. These materials have good biocompatibility and mechanical strength, but their degradation rate is relatively fast, which may lead to insufficient mechanical support before the tissue is completely regenerated, thus affecting the surgical effect. For example, polyurethane materials are widely used in inguinal hernia repair surgery. Its degradation rate is moderate and its biocompatibility is good. However, if the degradation is too fast, it may not meet the mechanical requirements of high-tension hernias. Biogenic materials such as natural high-molecular materials like collagen and gelatin have good biocompatibility and tissue regeneration ability, but their mechanical strength is usually low, and they are prone to rupture or deformation during the operation. It is necessary to enhance their mechanical properties by compounding with other materials (such as cellulose or chitosan) [5]. In addition, the degradation rate and mechanical properties of these materials are difficult to precisely control, which may lead to insufficient mechanical support before the tissue is completely healed. At the same time, the hydrolysis products of some materials may cause a decrease in local pH, which may trigger an immune response or inflammation, increasing the risk of postoperative complications.

[0012] In this study, we designed a magnesium metal patch and detected its cytotoxicity, degradation time, and bioactivity in in vitro experiments. The feasibility of magnesium metal as a material for repairing abdominal wall defects in vivo was preliminarily evaluated. Summary of the Invention

[0013] Design a new type of abdominal tissue patch that can temporarily support the defective abdominal wall, promote repair and regeneration, and has complete biodegradability, which is expected to meet the current clinical material requirements for abdominal wall defect repair. In recent years, as a biodegradable and absorbable metal biomaterial, magnesium has been applied in many fields due to its good biocompatibility, appropriate degradation properties, and sufficient mechanical properties, including cardiovascular, gastrointestinal, orthopedic fields, etc. Current research shows that magnesium can improve the adhesion, proliferation, migration, and extracellular matrix remodeling ability of human gingival fibroblasts by activating PI3K to induce AKT phosphorylation. Zhen et al. co-cultured magnesium leaching solution with mouse fibroblast epithelial cells (L929) and found that the magnesium leaching solution could promote the proliferation of L929 cells through pathways such as activating the cell cycle, energy metabolism activation, and protein synthesis. The experiments of Killilea et al. proved that magnesium deficiency could lead to the accelerated senescence of human fibroblasts. However, there is still little research on the use of biodegradable magnesium materials in the repair of abdominal wall defects, and more research is needed to deeply explore its feasibility and mechanism of action in the repair of abdominal wall defects.

[0014] Materials and Methods 1. Preparation of patches and leaching solutions: 1.1 Patch preparation: Select pure magnesium plates with a thickness of 0.2 - 0.3 mm, chemically polish them in a phosphoethylene glycol solution, then ultrasonically clean them in absolute ethanol, dry and sterilize them (Suzhou Aoruiji Medical Technology Co., Ltd.), and finally obtain magnesium patches (0.3 mm thick, 3 mm × 3 mm grid, 40% porosity). Polypropylene patches (PROLENE PolypropyleneMesh) were purchased from Johnson & Johnson (Shanghai) Medical Devices Co., Ltd.

[0015] 1.2 Preparation of leaching solutions: According to the ISO10993 - 5 standard, soak the magnesium patches and polypropylene patches in the iCell primary fibroblast medium for 24 hours, collect the supernatant, and dilute it to different concentrations (20%, 40%, 60%, 80%, 100%) for storage.

[0016] 3. Immersion test: The immersion solution for the experimental group was simulated body fluid solution (SBF), and the immersion solution for the control group was Hanks solution. The experimental operations were carried out according to the ASTM G31-72 standard. The degradable magnesium patches were placed into the research group and the control group according to the ratio of surface area to liquid of 1 cm2 to 20 ml. The magnesium patches were removed on the 1st, 7th, and 14th days of the experiment. After washing and drying them successively in chromic acid solution, acetone, and alcohol, they were weighed on a balance. The corrosion rate was calculated according to the formula CR = (K × W) ÷ (A × T × D), where K is a constant, K = 8.76*104, W represents the difference in the mass of the two measured samples (g), A represents the surface area of the sample exposed to the test liquid (cm2), T represents the exposure time (h), and D represents the density of the test sample material (g·cm-3). At the same time, the magnesium patches on the 7th and 14th days were examined by scanning electron microscopy.

[0017] 4. Cell treatment: 4.1 Cell culture All the human dermal fibroblast cell lines (iCell, Cyagen Biosciences Inc.) involved in this study were cultured in a complete medium prepared with the iCell primary fibroblast culture system (this system includes 10% fetal bovine serum (FBS), double antibiotics (penicillin / streptomycin, P / S), primary fibroblast basal medium, and primary fibroblast culture additive), and placed in a constant temperature incubator with 5% CO2 at 37°C for culture.

[0018] 5. CCK8 experiment: The HSF cells in the logarithmic phase were digested and resuspended, and inoculated into 96-well plates at a density of 1000 cells / well and a content of 100 μl per well. After standing in the incubator for 24 hours, the old medium was removed, and polypropylene patch extracts (0%) and magnesium patch extracts at concentration gradients (20%, 40%, 60%, 80%, 100%) were added, with 5 concentration gradients, 100 μl per well, and three replicates for each group. After 24 h and 72 h of culture, the old medium was discarded, 10 μl of CCK-8 working solution and 90 μl of complete medium were added to each well, and then it was placed in the incubator for incubation for 2 hours, and the absorbance at 460 nm was detected. The relative proliferation rate was calculated according to the following formula: relative growth rate (RGR): relative proliferation rate = experimental group / control group × 100%.

[0019] 6. Live / dead cell double staining experiment: Operate strictly according to the instructions of the Calcein-AM / PI double staining kit from Solarbio. Digest HSF cells after culturing them for 1 day and 3 days in the presence of concentration gradient magnesium patch extracts (20%, 60%, 100%) and polypropylene extracts. Collect the cells, wash them with 1× Assay Buffer, resuspend them to a cell suspension count of 1×105-6 cells / ml, add 1-2 μl of Calcein-AM to every 1 ml and pipette to mix evenly, incubate in the dark at 37°C for 20-25 min, add 3-5 μl of the original PI solution, and incubate in the dark at room temperature for 5 min. Centrifuge at 450 g for 50 min to remove the staining solution, resuspend the cells with PBS, take 3-5 μl and drop it on a clean glass slide, press the slide, and simultaneously detect live cells (green fluorescence) and dead cells (red fluorescence) under a fluorescence microscope (IX51, Olympus, Tokyo, Japan) at 490±10 nm.

[0020] 7. In vivo magnesium patch degradation experiment All animal experiments in this study were carried out in accordance with the ethical guidelines of the Second People's Hospital of Wuxi and approved by the Ethics Committee of the Second People's Hospital of Wuxi. Randomly select 18 female SD rats with a body weight of 200 g to 250 g. Collect 2 ml of rat peripheral blood before surgery. Then randomly divide the rats into two groups and anesthetize them by intraperitoneal injection of 2% sodium pentobarbital at a dose of 1 ml / kg. After anesthesia, place the rat in the supine position to expose the abdominal skin. Remove the hair and disinfect the skin with iodophor and 75% alcohol. Make a 25-mm incision on the abdominal wall skin, implant the magnesium patch (20 mm×20 mm) subcutaneously through the incision, fix it with a suture needle, and suture the skin layer by layer. Sacrifice 3 rats in each group at 2 weeks, 4 weeks, and 8 weeks after surgery respectively. Take out the residual patch subcutaneously, wash it with distilled water, dry it at room temperature, observe the surface morphology and degradation product components of the magnesium patch with a scanning electron microscope and an energy spectrometer. Then clean it successively with chromic acid solution, acetone, and alcohol, dry it, weigh it with a balance (Shanghai Zhuojing), and conduct a weight loss experiment. Collect 2 ml of rat peripheral blood at each time point before and after surgery for each group, and check the serum magnesium ion concentration, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase concentrations. The detection instrument is an automatic blood biochemical analyzer (instrument model: Hitachi 7600-020, reagents provided by Ke's company). Collect subcutaneous tissue and muscle tissue, perform HE staining examination, and observe histopathological changes.

[0021] 8. Establish a rat abdominal wall defect model Eighteen female Sprague-Dawley rats with an initial body weight of 200 to 250 g were selected. After shaving the left and right abdominal areas, the subcutaneous superficial muscles with a diameter of 1.5 cm × 1.5 cm were removed respectively, and the peritoneum was retained to establish an abdominal wall defect model. Then, a magnesium patch group of 2.0 cm × 2.0 cm was applied to one side of the defect area, while a polypropylene patch of the same size was used for control treatment on the other side. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (0.1 ml / 100 g). 2-3 sutures were used at the abdominal wall skin incision. The differences in the effects of magnesium patches and polypropylene patches in repairing abdominal wall defects were observed by B-mode ultrasound at 2 weeks, 4 weeks, and 8 weeks after implantation respectively. After the observation, 6 rats were sacrificed, and the abdominal wall defect repair tissues were collected for histological analysis. This included: performing Masson staining on the abdominal wall defect repair tissues to observe the tissue structure. Extracting cell RNA by the Trizol method, measuring its concentration and storing it. Measuring the RNA concentration using NanoDrop to ensure the quality meets the requirements. Using the TaKaRa reverse transcription kit for RNA reverse transcription to synthesize cDNA. After the reverse transcription reaction, the obtained cDNA can be used for downstream analysis and stored in a -80°C refrigerator for later use. Using the qRT-PCR kit provided by IAGEN company and SYBR PCR Master Mix for quantitative PCR analysis to detect the expression of target genes. Extracting proteins using CSTRIPA lysis buffer. Measuring the protein concentration of the samples using the BCA protein concentration assay kit. Separating proteins using SDS-PAGE and performing Western Blot to analyze the expression level of the target protein.

[0022] 9. Statistical analysis Statistical analysis and plotting were performed using SPSS 26.0 software (SPSS Inc., Chicago, USA) and GraphPad Prism 8.0 software (San Diego, California, USA). Measurement data were first subjected to a normality test. If they conformed to a normal distribution, they were expressed as mean ± standard deviation; otherwise, they were expressed as median and interquartile range. The comparison of count data and rates was performed using the c2 test, and correlation analysis was performed using the Spearman test. For two groups of measurement data that conformed to a normal distribution, the t test was used; otherwise, the Mann-Whitney U rank sum test was used. All statistical analyses were two-sided analyses, and P < 0.05 indicated that the difference was statistically significant. Description of the drawings

[0023] Figure 1 For the physical and chemical properties and in vitro degradation of the degradable magnesium patch, in the figure: A. Appearance morphology of the degradable magnesium patch; B. Residual stress distribution of the degradable magnesium patch at the initial stage and at fracture during the tensile fracture experiment detected by finite element analysis; C. Observation of the sample surface of the degradable magnesium patch soaked in SBF solution and Hanks solution at different time points detected by scanning electron microscopy; Figure 2Effect of the leaching solution of the degradable magnesium patch on the activity of HSF cells. In the figure: A. CCK8 assay was used to detect the effect of culturing with the leaching solution of the magnesium patch and polypropylene at different times on the proliferation of HSF cells; B. Calcein-AM / PI double staining was used to observe the live and dead cells and cell morphology of HSF; C. Flow cytometry was used to analyze the cell cycle proportion; D. Flow cytometry was used to analyze the apoptosis proportion. The charts represent the mean ± standard deviation; *p < 0.05, **p < 0.01, and ***p < 0.001; Figure 3 In vivo experiment of the degradable magnesium patch. In the figure: A. Scanning electron microscopy and elemental mapping analysis were used to detect the surface morphology and elemental composition of the degradable magnesium patch at different times after implantation; B. Weight loss experiment was used to detect the remaining patch mass; C. Important blood biochemical indexes of each group at each time point before and after surgery; D. HE staining of the subcutaneous tissue at the implantation site (×200); E. HE staining of the muscle tissue at the implantation site (×200); Figure 4 Construction of the abdominal wall defect model and histological analysis. In the figure: A. Surgical steps for constructing the abdominal wall defect model; B. HE staining of the abdominal wall repair tissue (×200); C. Masson staining of the abdominal wall repair tissue (×200). D. Type B ultrasound was used to observe the implantation of the magnesium patch and polypropylene patch into the abdominal wall defect model; Figure 5 Biological effects of the degradable magnesium patch. In the figure: A. Heat map of differentially expressed proteins screened by proteomics in the abdominal wall repair tissue implanted with the magnesium patch compared with the control group; B. Volcano plot of differentially expressed proteins screened by proteomics; C. PCR was used to verify the gene expression level of the differentially expressed proteins in HSF cells cultured with the leaching solution of the magnesium patch at a concentration of 60%; D. Western Blot was used to verify the expression level of related proteins in HSF cells cultured with the leaching solution of the magnesium patch at different concentration gradients; E. KEGG enrichment pathway analysis of the differentially expressed proteins; F. GO functional pathway analysis of the differentially expressed proteins. The charts represent the mean ± standard deviation; *p < 0.05, **p < 0.01; ***p < 0.001. Detailed implementation manners Example 1

[0024] Physical and chemical properties and degradation performance of the degradable magnesium patch Figure 1 A and Table 1 show the morphological structure and related physical and chemical properties of the degradable magnesium patch under macroscopic observation. As shown in the charts, the magnesium patch has a length of 2.0 cm × 2.0 cm, a thickness of 0.3 mm, a surface area of 256 mm2, a grid length of 3 mm × 3 mm, and a porosity of 40%. The patch grid is evenly distributed, the surface is smooth, and it has a horizontally-vertically interconnected structure, which can provide sufficient support for cell proliferation and migration.

[0025] To study the mechanical properties of the degradable magnesium patch, its fracture strength, bursting properties and elongation at break were measured and recorded, and the data are shown in Table 2. In the tensile experiment, the fracture strength of the initial magnesium patch was measured to be 167.2±5.9 N / cm, and the fracture strength of the magnesium patch after four weeks of degradation in rats was 55.9±1.6 N / cm. In the ball method bursting experiment, the bursting forces of the initial magnesium patch, the magnesium patch degraded in vitro for one week and four weeks were measured to be 135.8±3.5 N, 98.3±6.8 N, and 36.8±5.0 N, respectively. The bursting performance index provided by the magnesium patch after four weeks of in vivo degradation can still meet the requirements of the hernia patch.

[0026] We used finite element analysis to simulate the fracture behavior of the degradable magnesium patch at the maximum fracture strength, as Figure 1 shown in Figure B. The finite element analysis shows that when one end of the magnesium patch is fixed and an external tensile force is applied to the other end, the residual stress distribution is uniform, and the stress is mainly concentrated in the internal grid-like structure. Under a tensile force exceeding the maximum fracture strength, the mesh structure on the tensile side of the magnesium patch fractures first, while the fixed side remains structurally stable, which indicates that the degradable magnesium patch can provide sufficient structural stability for tissue repair.

[0027] We carried out immersion tests according to the ASTM-G31-72 standard. Magnesium patches with a length and width of 20.0 mm and 0.2 mm and a weight of 300 mg were immersed in a simulated body fluid solution (SBF) at a ratio of 1 cm2:20 ml for 1, 7, and 14 days. Their masses decreased by 2.4±0.3 mg, 13.9±2.6 mg, and 14.3±2.6 mg respectively compared with the previous values. Magnesium patches with the same mass and structure were immersed in Hanks solution at a ratio of 1 cm2:20 ml for 1, 7, and 14 days. Their masses decreased by 2.0±0.3 mg, 5.9±1.9 mg, and 17.2±2.3 mg respectively compared with the previous values. The different degradation rates of the magnesium patches in SBF and Hanks solution were measured by the weight loss method, as shown in detail in Table 3. In the SBF solution, the degradation rate of the magnesium patch on the 1st day of immersion was 1.87 mm / year, and the degradation rate reached the highest value of 2.62 mm / year on the 7th day of immersion. The degradation rate decreased to 1.05 mm / year on the 14th day of immersion. In the Hanks solution, the degradation rate of the magnesium patch on the 1st day of immersion was 1.49 mm / year, and then its degradation rate slowed down. The degradation rate on the 7th day of immersion was 1.14 mm / year, and the degradation rate on the 14th day of immersion was 1.28 mm / year. Scanning electron microscopy observations were performed on the magnesium patches immersed in the above two solutions for 7 days and 14 days, as Figure 1C. The magnesium patch immersed in SBF solution for 7 days had more attached sediments and bubble-like degradation holes on its surface. As the immersion time increased, degradation cracks on the surface of the metal patch could be directly seen. The magnesium patch immersed in Hanks solution for 7 days had no obvious attached sediments. The patch showed a complete morphology and no obvious signs of degradation. Only at 14 days did bubble-like degradation holes appear, and no significant degradation cracks were seen. In contrast, the degradable magnesium patch had a faster degradation rate and more significant degradation behavior in SBF solution. During the entire immersion experiment, it was observed that the structure of the degradable magnesium patch at different time points and in different solutions was intact, and there was no visible fracture in the grid structure of the patch.

[0028] Table 1. Physical properties of degradable magnesium patch

[0029] Table 2. Mechanical properties of degradable magnesium patch Table 2.1 Breaking strength of degradable magnesium patch

[0030] Table 2.2 Bursting performance of degradable magnesium patch

[0031] Table 3. Degradation rate of degradable magnesium patch in SBF and Hanks solution Embodiment 2

[0032] In vitro biocompatibility of degradable magnesium patches Figure 2A shows the results of detecting the activity of HSF cells by CCK8 after culturing HSF cells in the leaching solutions of degradable magnesium patches and polypropylene patches with different concentration gradients for 24 h and 72 h. In the leaching solutions of magnesium patches with concentrations of 20%, 40%, 60%, 80% and 100% after culturing for 24 h, the relative proliferation rates of HSF cells are 87.3±3.1%, 92.1±2.2%, 93.4±1.2%, 78.2±3.0%, 74.4±2.1% respectively. The results show that compared with the control group, the relative proliferation rates of HSF cells in the leaching solutions of magnesium patches with concentrations of 20%, 40% and 60% decrease slightly, but the differences are not statistically significant (P≥0.05). In the leaching solutions of magnesium patches with concentrations of 80% and 100%, the relative proliferation rates of HSF cells decrease significantly, and there are significant differences between them (P<0.05). After culturing for 72 h, in the leaching solutions of magnesium patches with concentrations of 20%, 40%, 60%, 80% and 100%, the relative proliferation rates of HSF cells are 102.8±1.9%, 102.6±2.1%, 106.1±3.1%, 87.5±2.8%, 85.3±3.8% respectively. The results indicate that there is no statistical significance in the relative proliferation rates of HSF cells in the leaching solutions of magnesium patches with concentrations of 20%, 40%, 60%, 80% and 100% compared with the control group (P≥0.05). According to the ISO10993-5:2009 standard for material biocompatibility, in the leaching solutions of magnesium patches with concentrations of 80% and 100%, the relative proliferation rates of HSF cells cultured for 24 h decrease significantly, which are 78.2±3.0% and 74.4±2.1% respectively, but are still higher than 60%. The cytotoxicity ratings of the leaching solutions of magnesium patches with concentrations of 80% and 100% are grade 1, while the cytotoxicity ratings of the leaching solutions of magnesium patches with concentrations of 60%, 40% and 20% are grade 0. Therefore, we can consider that the overall safety level of the degradable magnesium patch is 0-1 grade.

[0033] Because the CCK8 results suggest that the effects of the leaching solutions of magnesium patches with concentrations of 20% and 40% on HSF cells are similar, and the effects of the leaching solutions of patches with concentrations of 80% and 100% on HSF cells are also not very different. Therefore, in the subsequent experiments, we only chose to use the leaching solutions of magnesium patches with concentrations of 20%, 60% and 100% as the experimental groups, and the leaching solution of polypropylene patches (0% concentration) as the control group.

[0034] Figure 2Figure B shows the fluorescence staining images of live and dead cells of HSF cells cultured in magnesium patch extracts at different concentrations for 24 h and 72 h. After 24 h of culture, the number of live cells in the extracts at concentrations of 20%, 60%, and 100% was less than that in the control group, while the number of dead cells was slightly more than that in the control group. After 72 h of culture, compared with the control group, there was no significant difference in the number of live and dead cells in the 20% concentration extract. In the 60% concentration extract, the number of live cells increased and the number of dead cells decreased. In the 100% concentration extract, the number of live cells was less than that in the control group and the number of dead cells was more than that in the control group, but there was no statistical significance. When cultured in magnesium patch extracts at different concentration gradients, HSF cells were round or star-shaped, with abundant cytoplasm and clear nuclear membranes. Compared with the control group, there was no obvious difference in morphology at each time point.

[0035] Next, we used flow cytometry to detect the effects of magnesium patch extracts at different concentrations on the cell cycle (G0 / 1, S, and G2 / M phases) of HSF cells after 72 h of culture. The results are shown in Figure 2 Figure C. After 3 days of culture, compared with the control group at 0% concentration, there were no obvious changes in the cell cycles of HSF cells in the extracts at 20% and 100% concentrations, and there was no statistical significance (P < 0.05). However, in the 60% concentration, the percentage of G1-phase cells decreased significantly and the percentage of S-phase cells increased significantly, and there was a statistical difference between the two (P < 0.05). This work shows that within the set concentration gradient range of the extract, an increase in the extract concentration can lead to an increase in the percentage of G1-phase cells and a decrease in the percentage of S-phase cells. Especially at the 60% concentration point, there is a statistical difference compared with the control group.

[0036] At the same time, we also detected the effects of magnesium patch extracts at different concentrations on the apoptosis of HSF cells after 72 h of culture. The results are shown in Figure 2 Figure D. Within the specified concentration gradient, as the concentration increased, the apoptosis rate of HSF cells showed a downward trend. At the 60% concentration point, the apoptosis rate of HSF cells was the lowest, and there was a statistical significance compared with the control group (P < 0.05). When the extract concentration reached the 100% end concentration, the apoptosis rate of HSF cells increased instead, but it was still lower than that of the control group, and there was no statistical difference between the two (P < 0.05). In the above experiments, we found that magnesium patch extracts below 60% concentration had little effect on HSF cells, while magnesium patch extracts above 60% concentration had an impact on HSF cells. However, even after 72 h of culture in the magnesium patch extract at the highest concentration (100%), there was no significant difference compared with the control group. These results may suggest that the 60% concentration is an important observation point for the biocompatibility experiment of degradable magnesium patches.

[0037] The above results suggest that the cytotoxicity of the magnesium patch extract is low and the effect on HSF cell proliferation, survival and apoptosis is small. The degradable magnesium patch is a relatively safe implant material with sufficient biocompatibility. Embodiment 3

[0038] In vivo degradation performance and biocompatibility of degradable magnesium patch In order to study whether the degradation behavior of the degradable magnesium patch in vivo meets the needs of the abdominal wall tissue for the patch, we conducted a rat subcutaneous implantation test to explore the biodegradability of the degradable magnesium patch. All rats survived until the completion of all experimental studies. The rats in different groups grew well, were able to move normally and complete eating behaviors independently. During the entire observation period, no adverse complications such as incision infection, patch dislocation, and wound dehiscence occurred.

[0039] In the degradable magnesium patch implantation group, 6 rats were killed at 2, 4, and 8 weeks after surgery. The magnesium patch was removed from the subcutaneous tissue of the abdominal wall, and the attached proliferative fibrous material was removed. The scanning electron microscope and elemental analysis were performed. The results were as follows: Figure 3 As shown in A. The results showed that the structure of the degradable magnesium patch was still intact 2 weeks after surgery, and the fibers inside the patch were partially degraded. Scanning electron microscopy showed that there were shallow irregular corrosion pits on the metal surface, with more deposits attached, and elemental analysis showed that the deposits were complex, including carbon, oxygen, magnesium, phosphorus, calcium and other elements. 4 weeks after surgery, the degradable magnesium patch was partially degraded, but the basic structural framework was still maintained. The deposits attached to the surface of the patch increased significantly. Scanning electron microscopy showed that the metal had obvious deep bubble-like degradation holes, and irregular cracks were visible in some areas, accompanied by obvious deposits. Elemental analysis showed that the deposit composition was similar to that before, including carbon, oxygen, magnesium, phosphorus, and calcium. 8 weeks after surgery, the degradable magnesium patch was significantly degraded and tightly integrated with the surrounding abdominal wall tissue. Obvious fibrous connective tissue and calcification deposition were visible on the patch. Scanning electron microscopy showed that the magnesium patch had deeper metal cracks. Elemental analysis of the corrosion products on the metal surface showed that it was composed of carbon, oxygen, magnesium, phosphorus, calcium and other elements, and the percentage of the components changed compared with before, mainly due to the increase in the proportion of calcium.

[0040] The magnesium patch material removed after surgery was cleaned with chromic acid solution, acetone and alcohol in turn, and then weighed after drying. The results are as follows: Figure 3As shown in Figure B. The weights of the magnesium material patches before surgery and at 2 weeks, 4 weeks, and 8 weeks after surgery were: 302.3 ± 3.0 mg, 271.3 ± 5.2 mg, 222.1 ± 10.0 mg, and 182.8 ± 5.3 mg, respectively. The weights at 2 weeks, 4 weeks, and 8 weeks after surgery were 89%, 73%, and 60% of the pre-surgery weight, respectively. Although obvious degradation behavior occurred in the magnesium patch at 8 weeks after surgery, 60% of the residual structure was still not degraded. The patch was tightly combined with the surrounding abdominal wall tissue and could still provide certain structural stability and support for tissue repair.

[0041] To further explore the in vivo biocompatibility of the degradable magnesium patch, we collected 2 ml of peripheral blood from rats in the polypropylene patch group and the magnesium patch group at set time points for detecting various blood biochemical indexes before and after surgery. The results are as Figure 3 shown in Figure C. Serum alanine aminotransferase and aspartate aminotransferase are common indexes reflecting direct liver cell damage and liver function, serum urea nitrogen is an important index reflecting renal function, and serum magnesium concentration can reflect whether there is hypermagnesemia caused by magnesium ion accumulation during the degradation process of the magnesium patch. From the line graph, we can find that although these blood biochemical indexes fluctuated at each time point, they were all within the normal range of rat normal blood biochemical indexes. There was no statistically significant difference in serum magnesium ion concentration, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase between the polypropylene patch group and the magnesium patch group of rats before surgery (P ≥ 0.05 for all). There was no statistically significant difference in various biochemical indexes between the magnesium patch group and the polypropylene patch group at the same time point after surgery (P ≥ 0.05 for all). Compared with before surgery at each time point after surgery, there was also no statistically significant difference in various biochemical indexes between the polypropylene patch group and the magnesium patch group of rats (P ≥ 0.05 for all).

[0042] Figure 3 Figures D and Figure 3 E respectively show the HE staining images of subcutaneous tissue and muscle tissue at 2 weeks, 4 weeks, and 8 weeks after implantation of the two different patches. At 2 weeks after surgery, a large number of inflammatory cells were visible around the implantation sites of both the magnesium patch and the polypropylene patch, mainly neutrophils and lymphocytes, and a small number of macrophages. The number of inflammatory cells around the implantation site of the magnesium patch was slightly less than that of the polypropylene patch group. After 4 weeks of implantation, new granulation tissue was visible around the implantation sites of both patches, accompanied by deposition of fibrous connective tissue and formation of new microvessels, and the number of inflammatory cells decreased significantly. After 8 weeks of implantation, the magnesium patch showed obvious degradation, with the edges significantly thinned and blurred, and the number of inflammatory cells at the implantation site further decreased. Compared with the polypropylene patch group, the fibrous connective tissue at the implantation site of the magnesium patch was arranged more regularly. Whether it was subcutaneous tissue or muscle tissue, except for the inflammatory reaction, no tissue damage or necrosis was observed, the surrounding tissue morphology was good, and there were no obvious adverse reactions.

[0043] Repair of abdominal wall defects with degradable magnesium patches Based on the results of the in vivo degradation experiment of the above-mentioned patch, a degradable magnesium patch with length and width dimensions of 20 mm was used to repair the abdominal wall defect of SD rats. The surgical steps are as Figure 4 shown in A. The magnesium patch was implanted on the right side of the abdominal midline of the rat, and the polypropylene patch on the left side of the abdominal midline was used as the control group. Until the final observation point, all rats survived after the patch implantation, and were able to move freely and eat independently. No signs of wound infection or rejection were observed at each time point after the operation, and no masses or bulging of intestinal contents were seen on the left and right sides of the abdominal wall of the rats.

[0044] At 2 weeks, 4 weeks, and 8 weeks after implantation, the rats were sacrificed, and the abdominal wall repair tissues at the patch implantation sites on both sides of the abdomen of the rats were taken for hematoxylin-eosin staining. The results are as Figure 4 shown in B. At 2 weeks after implantation, there were obvious inflammatory cell infiltrations in both groups of patches, mainly neutrophils and lymphocytes, accompanied by a small amount of macrophage infiltration. At the same time, a small amount of fibrous connective tissue was intertwined. At 4 weeks after implantation, the inflammatory cells at the patch implantation sites in both groups regressed compared with before. Under the microscope, fibrous connective tissue deposition was visible, accompanied by obvious microvascular angiogenesis. At 8 weeks after implantation, a small amount of chronic inflammation was still visible at the implantation site. Compared with the polypropylene patch, the abdominal wall repair tissue in the magnesium patch group had more regularly arranged fibrous connective tissue, less fibrous debris, obvious fibroblast hyperplasia, and increased extracellular matrix deposition.

[0045] Next, the abdominal wall repair tissues were subjected to Masson staining to further observe the content of collagen fibers in the repaired abdominal wall tissues. The results are as Figure 4 shown in C. The results showed that there were obvious differences between collagen fibers (dyed blue) and muscle fibers (dyed red). At 2 weeks, 4 weeks, and 8 weeks, compared with the control group on the implanted side, the collagen fibers on the implanted side of the degradable magnesium patch were arranged more neatly, intertwined better with each other, showing more hierarchical and clearer fiber bundle-like structures than the implanted side of the control group. In addition, these collagen fibers looked thicker and denser, in obvious contrast to the thin and loose reticular structure on the implanted side of the control group. The above results indicate that the degradable magnesium patch has good in vivo tissue regeneration performance.

[0046] The situation after the magnesium patch and the polypropylene patch were implanted into the abdominal wall defect model was observed using B-mode ultrasound. The results are as Figure 4 shown in D. The degradation of the magnesium patch in vivo will produce a certain amount of hydrogen gas. More black shadows generated by the gas will be observed using B-ultrasound at 2 weeks and 4 weeks. After 8 weeks, due to the absorption of hydrogen gas by the tissue and the slowdown of the degradation rate of the magnesium patch, there was no obvious difference between the magnesium patch and the polypropylene patch under B-ultrasound observation. The above results indicate that in the treatment of abdominal wall defects, the long-term effect of the degradable magnesium patch is similar to that achieved by the polypropylene patch. Example 4

[0047] Biological Effects of Degradable Magnesium Patches To obtain the differences in protein expression between the degradable magnesium patch group and the polypropylene patch group, we used TMT quantitative proteomics technology to detect the protein expression in the abdominal wall repair tissue 8 weeks after surgery. Figure 5 A The heatmap results showed that compared with the polypropylene patch on the contralateral side, the implantation of magnesium patches could cause changes in the protein expression of the abdominal wall repair tissue in rats. Significantly differentially expressed proteins could effectively separate the experimental group from the control group, indicating that magnesium patches had a biological impact on the implantation site. To display the significant differences in proteins between the comparison groups, we plotted a volcano plot of the proteins in the comparison groups. Among them, significantly downregulated proteins were marked in blue, significantly upregulated proteins were marked in red, and proteins without differences were in gray, as Figure 5 shown in B. The results showed that a total of 81 protein abundances changed significantly, including 52 upregulated and 29 downregulated proteins.

[0048] Based on the above results, we selected proteins with significantly increased expression levels among the upregulated ones (FC > 1.5 and P < 0.05) and further verified their gene expression levels in HSF cells cultured for 3 days at the key concentration point of 60% of the magnesium patch leaching solution by PCR. The results were as Figure 5 shown in C. The results showed that compared with the polypropylene patch group, the magnesium patch leaching solution could significantly increase the gene expression levels of COL5A1 and COL3A1. We re-evaluated the expression of the selected proteins by Western blot. As Figure 5 shown in D, within the set concentration gradient of the magnesium patch leaching solution, the expression levels of COL5A1 and COL3A1 in HSF cells gradually increased from 0% to 60% concentration and reached the highest level at 60% concentration. However, when reaching the extreme concentration of 100%, the expression level of HSF cells decreased, similar to that at 0% concentration.

[0049] We further performed a correlation analysis on the proteins measured by TMT quantitative proteomics. The results were as Figure 5 shown in E and Figure 5 F. The KEGG results showed that the differential proteins in the abdominal wall repair tissue treated with degradable magnesium patches and the polypropylene patch group on the contralateral side were significantly enriched in pathways such as cytokine-receptor interaction, complement and coagulation cascades. The GO functional analysis results showed that the differential proteins in the abdominal wall repair tissue of the degradable magnesium patch group and the control group were significantly enriched in biological processes such as collagen fibril organization, phagocytosis and recognition.

[0050] Discussion: The widespread use of prosthetic materials in inguinal hernia repair is one of the most common surgical procedures in the world. A mesh or screen is implanted into the defect site in the inguinal region to enhance the function of the abdominal muscles and reconstruct the mechanical structure of the abdominal wall. The mesh used for inguinal repair is made of synthetic or biological materials and has various shapes and configurations. Although there are many consumables on the market, the exploration of the ideal mesh is still ongoing. So far, no device can provide the best mechanical properties for tissue repair and recovery while minimizing postoperative complications. Biodegradable magnesium materials have been widely studied and applied in the fields of gastrointestinal surgery, cardiovascular stents, orthopedic implants, etc. due to their excellent biocompatibility, mechanical properties and suitable degradation characteristics, but there is less research on their use as patches in surgery. We explored the possibility of using biodegradable magnesium materials in abdominal wall defect repair surgery from two perspectives: in vivo and in vitro experiments, and from three aspects: mechanical properties, biocompatibility, and biological effects. Our research results preliminarily show that the biodegradable magnesium patch has relatively excellent mechanical properties and degradation behavior, high biocompatibility with fibroblast epithelial cells, and can produce certain biological effects, with prospects for clinical application and worthy of further research and exploration.

[0051] Failure to effectively control behaviors that can cause an increase in intra-abdominal pressure, such as coughing, constipation, or difficulty urinating, after surgery is the main factor for hernia recurrence. In the relevant mechanical property experiments, the breaking strength of the biodegradable magnesium patch was measured to be 167.2 ± 5.9 N / cm, and the bursting strength was 135.8 ± 3.5 N. We searched the relevant scientific literature and found that the maximum intra-abdominal pressure generated by healthy adults during coughing, jumping, and normal activities is 16 N / cm

[18] . The sufficient breaking strength and bursting strength of the biodegradable magnesium patch can withstand this pressure without early rupture. At the same time, the experiments showed that after 4 weeks of degradation, the breaking strength of the magnesium patch was 55.9 ± 1.6 N / cm, and the bursting strength was 36.8 ± 5.0 N, which can also meet the mechanical requirements of clinical hernia support materials. The time for tissue to heal after injury is 5 - 7 days. Although the breaking strength of magnesium metal is lower than the maximum intra-abdominal pressure of the human body after 3 months in vivo, the human tissue has completed the process of healing and repair 3 months after surgery.

[0052] To understand whether the degradation behavior of the degradable magnesium patch can meet the requirements of abdominal wall defect repair, we studied the in vitro and in vivo degradation properties of the degradable magnesium patch. The results of the immersion test indicated that the degradable magnesium patch degraded rapidly in the early stage in the SBF solution, but the degradation rate slowed down in the middle and late stages, and the mass loss was greater than that in the Hanks solution. Combining with the scanning electron microscope results, as the immersion time prolonged, deposits gradually adhered to the surface of the degradable magnesium patch, further blocking the contact between the immersion liquid and the patch surface, thus promoting the degradation rate of the patch to tend to be stable. The in vivo weight loss experiment showed that after 8 weeks of implantation, the remaining part of the degradable magnesium patch accounted for 60% of the original weight. This result was inconsistent with the degradation rate in the in vitro degradation experiment. We speculated that this might be due to the complex metabolic environment in the rat body and the change of residual stress distribution during activities. Witte et al. also found the characteristic that the corrosion rates of magnesium implant materials in vivo and in vitro were inconsistent, and believed that this might depend on the corrosion environment where the materials were located

[19] . Nevertheless, we still observed that at 8 weeks after surgery, the degradable magnesium patch was closely combined with the surrounding abdominal wall tissues and still could provide a certain degree of support for tissue repair.

[0053] Magnesium ion is one of the trace elements necessary to maintain the health of the body, participates in a variety of functional activities of cells, and plays an important role in basic physiological activities such as cell proliferation, protein synthesis, and DNA transcription. According to the ISO10993-5:2009 biological material safety performance evaluation standard, the safety rating of the degradable magnesium patch is grade 0-1. These results might indicate that the 60% concentration is an important observation concentration point in the biocompatibility experiment of the degradable magnesium patch. In the in vivo biocompatibility study, we found that after the degradable magnesium patch was implanted subcutaneously in rats, it did not cause significant changes in important biochemical indicators, and its degradation did not cause magnesium ion accumulation. These results suggest that the degradable magnesium patch has low cytotoxicity to HSF cells and is a relatively safe patch implant material.

[0054] The tissue repair of the abdominal wall defect site has always been a research hotspot in the field of hernia patches. Relevant studies have shown that the implantation of magnesium materials promotes the expression of collagen and increases the content of vascular endothelial growth factor, thereby promoting tissue repair and the generation of new blood vessels. Our experimental results show that there are significant differences in protein expression in the repaired tissues at the implantation sites of magnesium patches and polypropylene patches. These differential proteins are mainly enriched in signal pathways such as collagen fibril tissue, complement, and coagulation cascade. In the verification of cell lines, the expression levels of COL5A1 and COL3A1 in HSF cells in the magnesium patch extract at a concentration of 60% increased. COL5A1 is an extracellular matrix protein that participates in the synthesis of type V collagen. It may be a secondary participant in structure, but it has a prominent function in the collagen hierarchy, and type V collagen plays an important role in regulating fiber diameter and collagen fiber assembly. COL3A1 is an extracellular matrix protein that participates in the synthesis of type III collagen. It can be synthesized and secreted by fibroblasts and participates in the formation of collagen fibers in fibrous connective tissues. It is the main structural component of hollow organs such as large blood vessels, uterus, and intestine. Other functions include its interaction with platelets in the coagulation cascade and it is also an important signaling molecule for wound healing. The above results may suggest that the degradable magnesium patch promotes the repair of abdominal wall defect tissues mainly by promoting the synthesis of collagen fiber proteins, which is consistent with what we observed in the Masson staining - the magnesium patch group has more collagen fiber content and is arranged more neatly. This indicates that compared with the commonly used traditional polypropylene patch, the degradable magnesium patch has good healing - promoting ability.

[0055] This article mainly studied the biocompatibility and bioactivity of the degradable magnesium material patch on abdominal wall defect tissues, providing a theoretical basis for the use of degradable magnesium patches in the field of hernia surgery. The degradable magnesium patch has high biological safety in the body. Its degradation behavior can basically meet the requirements of tissue repair and has certain biological activity.

[0056] In summary, we first reported the biological efficacy (including biological safety and bioactivity) of the degradable magnesium patch in the repair of abdominal wall defect tissues and found that the degradable magnesium patch has excellent biocompatibility, degradation performance, and certain biological activity. In terms of biocompatibility, the safety rating of the degradable magnesium patch is 0 - 1 level, with little impact on cell proliferation and apoptosis. At certain specific concentrations (60%), it can even promote proliferation and reduce apoptosis. In terms of mechanical properties and degradation performance, with appropriate breaking strength and degradation rate, the degradable magnesium patch can provide sufficient stress support for tissue repair and degrade at the appropriate time. In terms of bioactivity, the degradable magnesium patch can increase the expression of healing - related proteins COL3A1 and COL5A1 in abdominal wall defect tissues, thereby promoting the generation of collagen and fibrous connective tissues and contributing to the repair of defect tissues.

Claims

1. A method for testing the biological efficacy of pure magnesium materials. Select a pure magnesium plate with a thickness of 0.2 - 0.3 mm, and make a magnesium patch with a thickness of 0.3 mm, a 3 mm × 3 mm grid, and a porosity of 40%. Test its in vitro biocompatibility, in vivo biomechanics, and in vivo bioactivity. It is characterized in that, To evaluate the in vitro biocompatibility of the magnesium patch, the magnesium patch extract is co - cultured with human dermal fibroblasts in vitro. The cytotoxicity is evaluated by CCK8, the cell morphology is detected by Calcein - AM / PI double staining, and the cell cycle and apoptosis are detected by flow cytometry; To evaluate the in vivo biomechanics of the magnesium patch, a subcutaneous implantation test in rats is carried out to explore the biodegradability of the degradable magnesium patch; To evaluate the in vivo biological effects of the magnesium patch, a rat abdominal wall defect model is established, repaired with the magnesium patch, and TMT protein quantification detection, differential protein correlation analysis, PCR, and Western blot are performed.

2. The method for testing the biological efficacy of pure magnesium materials according to claim 1, characterized in that, When evaluating the in vitro biocompatibility of the magnesium patch, the following steps are included: 1) Preparation of extraction liquid: According to the ISO10993 - 5 standard, the magnesium patch and the polypropylene patch are immersed in the iCell primary fibroblast medium for 24 hours, the supernatant is collected, diluted to different concentrations, 20%, 40%, 60%, 80%, 100%, and stored; 2) Immersion test: The immersion liquid of the experimental group is simulated body fluid solution (SBF), and the immersion liquid of the control group is Hanks solution. According to the ASTM G31 - 72 standard, the experimental operation is carried out. According to the ratio of surface area to liquid of 1 cm2 to 20 ml, the magnesium patch is placed in the research group and the control group. On the 1st, 7th, and 14th days of the experiment, the magnesium patch is removed, washed successively in chromic acid solution, acetone, and alcohol, dried, and weighed on a balance. The corrosion rate is calculated according to the formula CR=(K×W)÷(A×T×D). At the same time, the magnesium patch on the 7th day and the 14th day is examined by scanning electron microscopy; 3) Cell treatment: Cell culture All human dermal fibroblast cell lines involved in this study are cultured in a complete medium prepared with the iCell primary fibroblast culture system and placed in a constant temperature incubator at 5% CO2 and 37°C; 4) CCK8 experiment: The HSF cells in the logarithmic phase are digested and resuspended, inoculated into a 96 - well plate at a density of 1000 cells / well and a content of 100 μl per well. After standing in the incubator for 24 hours, the old medium is removed, and the polypropylene patch extract and magnesium patch extracts with concentration gradients, including 20%, 40%, 60%, 80%, 100%, 5 concentration gradients, 100 μl per well, and three replicates in each group, are added. After 24 h and 72 h of culture, the old medium is discarded, 10 μl of CCK - 8 working solution and 90 μl of complete medium are added to each well, and then incubated in the incubator for 2 hours, and the absorbance at 460 nm is detected. The relative proliferation rate is calculated according to the following formula: Relative proliferation rate = experimental group / control group × 100%; 5) Live / dead cell double staining experiment: HSF cells were cultured for 1 day and 3 days under the conditions of concentration gradient magnesium patch extracts, including 20%, 60%, 100% and polypropylene extracts, and then digested. The cells were collected, washed with 1× Assay Buffer, resuspended to make the cell suspension count 1×105-6 cells / ml, 1-2 μl of Calcein-AM was added to each 1 ml and pipetted gently to mix evenly, incubated at 37°C in the dark for 20-25 min, 3-5 μl of PI stock solution was added, incubated at room temperature in the dark for 5 min, centrifuged at 450 g for 50 min to remove the staining solution, the cells were resuspended with PBS, 3-5 μl was taken and dropped on a clean glass slide, and after pressing the slide, the live cells and dead cells were simultaneously detected under a fluorescence microscope at 490±10 nm.

3. A method for testing the biological efficacy of a pure magnesium material according to claim 1, wherein when evaluating the in vivo biomechanics of the magnesium patch, the following steps are included: Randomly select 18 female SD rats with a body weight of 200 g to 250 g. Collect 2 ml of peripheral blood from the rats before surgery. Then the rats are randomly divided into two groups, anesthetized by intraperitoneal injection of 2% pentobarbital sodium at a dose of 1 ml / kg. After anesthesia, place the rats in the supine position to expose the abdominal skin. After removing the hair and exposing the skin, disinfect with iodophor and 75% alcohol. Make a 25-mm incision on the abdominal wall skin, retain the peritoneum, establish an abdominal wall defect model, implant the magnesium patch subcutaneously through the incision, and fix it with suture needles. Suture the skin layer by layer. At 2 weeks, 4 weeks, and 8 weeks after surgery, 3 rats in each group were sacrificed. The residual patch was taken out subcutaneously, rinsed with distilled water, and dried at room temperature. The surface morphology and degradation product components of the magnesium patch were observed with a scanning electron microscope and an energy spectrometer. Then it was cleaned successively with chromic acid solution, acetone, and alcohol, dried, weighed with a balance to perform a weight loss experiment. Collect 2 ml of peripheral blood from each group of rats at each time point before and after surgery, check the serum magnesium ion concentration, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase concentrations. Collect subcutaneous tissue and muscle tissue, perform HE staining examination, and observe the histopathological changes.

4. A method for testing the biological efficacy of a pure magnesium material according to claim 1, wherein when evaluating the in vivo biological effect of the magnesium patch, the following steps are included: Eighteen female SD rats with an initial body weight of 200 to 250 g were selected. After shaving the left and right abdominal areas, the subcutaneous superficial muscles with a diameter of 1.5 cm × 1.5 cm were removed respectively, and the peritoneum was retained to establish an abdominal wall defect model. Then, a magnesium patch group of 2.0 cm × 2.0 cm was applied to one side of the defect area, while the other side was treated with a polypropylene patch of the same size as a control. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (0.1 ml / 100 g). Two to three sutures were used at the abdominal skin incision. The differences in the effects of magnesium patches and polypropylene patches in repairing abdominal wall defects were observed by B-mode ultrasound at 2 weeks, 4 weeks, and 8 weeks after implantation. After the observation, six rats were sacrificed, and the abdominal wall defect repair tissues were collected for histological analysis, including: Masson staining was performed on the abdominal wall defect repair tissues to observe the tissue structure, cell RNA was extracted by the Trizol method, its concentration was measured and stored, the RNA concentration was measured using NanoDrop to ensure that the quality met the requirements, RNA reverse transcription was performed using the TaKaRa reverse transcription kit to synthesize cDNA. After the reverse transcription reaction, the obtained cDNA could be used for downstream analysis and was stored in a -80°C refrigerator for later use. Quantitative PCR analysis was performed using SYBR PCR Master Mix to detect the expression of target genes. Proteins were extracted using CSTRIPA lysis buffer, the protein concentration of the samples was measured using a BCA protein concentration assay kit, the proteins were separated using SDS-PAGE, and Western Blot analysis was performed to detect the expression level of the target protein.

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