1.Finite element analysis of stress distribution of anchors at different implantation depths under different bone density conditions in rotator cuff tears
Meng WANG ; Tan LU ; Minjie LI ; Zhicheng LIU ; Xiaoyong GUO
Chinese Journal of Tissue Engineering Research 2026;30(3):561-569
BACKGROUND:Arthroscopic anchor repair has become the main treatment method for rotator cuff tears at present.Among them,the insertion status of the anchor is a key factor in the success or failure of the operation.However,currently,the impact of the insertion depth of the anchor on the stress of the bone tunnel and the anchor under different bone density conditions remains unclear.OBJECTIVE:To explore the stress distribution of the bone tunnel and the anchor when the insertion depth of the anchor varies under different bone density conditions by using three-dimensional finite element analysis technology.METHODS:The CT image data of the humerus of volunteers were collected,and the models of the humerus and the anchor were constructed by using Mimics,3-Matic,and Solidworks software.In 3-Matic,holes with distances of 0,2,4,6,and 8 mm from the surface of the humerus were respectively created at the same position of the humerus and assembled with the anchor.In Mimics,values were assigned based on the CT gray value to obtain a model with normal bone mass(T value ≥-1.0).The parameters were changed to construct models with reduced bone mass(-2.5<T value<-1.0)and osteoporosis(T value<-2.5).In each model,a 70 N pulling force was applied to the anchor along the direction tangent to the inner edge of the bone tunnel.The stress distribution and magnitude of the bone tunnel and the anchor when inserted at different depths under different bone density conditions were observed.RESULTS AND CONCLUSION:(1)When the insertion depth was the same,as the bone density decreased,the maximum equivalent stress of the anchor increased,while the maximum equivalent stress of the bone tunnel decreased.(2)When the bone density was the same,as the insertion depth of the anchor increased,the maximum equivalent stress of the anchor decreased.When the insertion depth was 4 mm,the stress of the bone tunnel was the smallest and the distribution was relatively uniform.The stress of the anchor was mainly distributed around the lower anchor hole and the proximal thread,and the stress of the bone tunnel was mainly at the part in contact with the proximal thread.The increase in the insertion depth would change the uniformity and pattern of the stress distribution,while the bone density had a relatively small impact on the stress distribution pattern.(3)It is concluded that the bone density of the humerus is crucial for the anchor repair of rotator cuff tears.It is recommended that clinicians measure the bone density of the greater tuberosity of the humerus before the operation.Excessive insertion depth of the anchor does not significantly increase its stability.Clinicians can conduct personalized preoperative assessments by using the finite element analysis method in combination with the actual situation of patients to achieve the best surgical results.
2.Finite element analysis of stress distribution of anchors at different implantation depths under different bone density conditions in rotator cuff tears
Meng WANG ; Tan LU ; Minjie LI ; Zhicheng LIU ; Xiaoyong GUO
Chinese Journal of Tissue Engineering Research 2026;30(3):561-569
BACKGROUND:Arthroscopic anchor repair has become the main treatment method for rotator cuff tears at present.Among them,the insertion status of the anchor is a key factor in the success or failure of the operation.However,currently,the impact of the insertion depth of the anchor on the stress of the bone tunnel and the anchor under different bone density conditions remains unclear.OBJECTIVE:To explore the stress distribution of the bone tunnel and the anchor when the insertion depth of the anchor varies under different bone density conditions by using three-dimensional finite element analysis technology.METHODS:The CT image data of the humerus of volunteers were collected,and the models of the humerus and the anchor were constructed by using Mimics,3-Matic,and Solidworks software.In 3-Matic,holes with distances of 0,2,4,6,and 8 mm from the surface of the humerus were respectively created at the same position of the humerus and assembled with the anchor.In Mimics,values were assigned based on the CT gray value to obtain a model with normal bone mass(T value ≥-1.0).The parameters were changed to construct models with reduced bone mass(-2.5<T value<-1.0)and osteoporosis(T value<-2.5).In each model,a 70 N pulling force was applied to the anchor along the direction tangent to the inner edge of the bone tunnel.The stress distribution and magnitude of the bone tunnel and the anchor when inserted at different depths under different bone density conditions were observed.RESULTS AND CONCLUSION:(1)When the insertion depth was the same,as the bone density decreased,the maximum equivalent stress of the anchor increased,while the maximum equivalent stress of the bone tunnel decreased.(2)When the bone density was the same,as the insertion depth of the anchor increased,the maximum equivalent stress of the anchor decreased.When the insertion depth was 4 mm,the stress of the bone tunnel was the smallest and the distribution was relatively uniform.The stress of the anchor was mainly distributed around the lower anchor hole and the proximal thread,and the stress of the bone tunnel was mainly at the part in contact with the proximal thread.The increase in the insertion depth would change the uniformity and pattern of the stress distribution,while the bone density had a relatively small impact on the stress distribution pattern.(3)It is concluded that the bone density of the humerus is crucial for the anchor repair of rotator cuff tears.It is recommended that clinicians measure the bone density of the greater tuberosity of the humerus before the operation.Excessive insertion depth of the anchor does not significantly increase its stability.Clinicians can conduct personalized preoperative assessments by using the finite element analysis method in combination with the actual situation of patients to achieve the best surgical results.
3.Study of the occurrence of metabolic dysfunction-associated fatty liver disease in obstructive sleep apnea hypopnea syndrome and its risk factors
Yanlan GUO ; Daiyi ZHANG ; Shenjie XU ; Minjie ZHANG ; Jie LI
Chinese Journal of Internal Medicine 2025;64(2):128-133
Objective:To analyze the occurrence of metabolic dysfunction-associated fatty liver disease (MAFLD) and related inflammatory indicators in obstructive sleep apnea hypopnea syndrome (OSAHS) and explore the risk factors of MAFLD.Methods:A cross-sectional study. From January 2022 to October 2024,172 patients with sleep disorders were enrolled in the First Affiliated Hospital of Soochow University,including 38 patients with non-OSAHS,53 patients with mild OSAHS,37 patients with moderate OSAHS,and 44 patients with severe OSAHS. The occurrence of MAFLD was comprehensively judged from three aspects: metabolic dysfunction-associated fatty liver (MAFL),elevated liver enzymes,and liver fibrosis. The situation of MAFLD and the level of related inflammatory markers were compared among the four groups. Binary logistic regression was used to analyze the risk factors for MAFLD in OSAHS.Results:There were significant differences in the prevalence of MAFL,the percentage of elevated liver enzymes,and interleukin-6 and tumor necrosis factor-alpha levels among the four groups ( P<0.05). The differences of fibrosis-4 index and C-reactive protein among the four groups were not statistically significant ( P>0.05). Binary logistic regression showed that BMI,triglycerides,longest time of sleep apnea and tumor necrosis factor-alpha were the risk factors for MAFL ( P<0.05). BMI,glucose,and apnea-hypopnea index were the risk factors for elevated liver enzymes ( P<0.05). Conclusions:OSAHS is strongly associated with MAFLD,and the involvement of OSAHS in the occurrence and development of MAFLD may be related to obesity,lipid metabolism disorders,insulin resistance,inflammatory responses,and intermittent hypoxia.
4.Study on the Protective Effects of the Mongolian Prescription Jiruhen Gurigumu-7 and Guangzao Sanwei Tang on Myocardial Ischemia-Reperfusion Mice
Zhongyue ZHANG ; Shuhong ZHOU ; Qian GAO ; Xiaoxia SONG ; Xiaoru ZHANG ; Lingze YU ; Yulu DU ; Na GUO ; Minjie WANG
World Science and Technology-Modernization of Traditional Chinese Medicine 2025;27(8):2331-2339
Objective To explore the protective effects of pretreatment with the Mongolian medicine Jiruhen Gurigumu-7(JG-7)and Guangzao Sanwei Tang(GZ-3)on myocardial ischemia-reperfusion injury(MIRI)in mice.Methods 60 male C57BL/6J mice were randomly divided into sham operation(Sham)group,model(Model)group,compound danshen drip pill(CDDP)positive control group,JG-7 group,GZ-3 group,and 12 mice in each group to establish the MIRI model,and the H9C2 cells were randomly divided into Control(normoxic)group,H/R(hypoxia 6 h reoxygenation 14 h)group,H/R+JG-7 group,H/R+GZ-3 group.The mice in each group were tested for cardiac function indexes after 30 min of ischemia,24 h and 7 d of reperfusion,TTC staining to detect infarct area after 24 h of MIRI,HE staining to detect myocardial tissue structure and cellular morphology after 24 h of MIRI,TUNEL apoptosis kit to detect apoptosis of myocardial cells after 24 h of MIRI,Masson staining to detect myocardial fibrosis after 7 d of MIRI.Blood was taken from the abdominal aorta,serum was separated,and the indexes after oxidative stress of MIRI were detected in each group of mice,and the survival rate of H9C2 cells after H/R was detected in each group by CCK-8 method.Results The results of TTC showed that JG-7 and GZ-3 reduced the infarct area after 24 h of MIRI in mice.ELISA and kit assays proved that JG-7 and GZ-3 reduced creatine phosphokinase isoenzyme(Creatinekinase-MB,CK-MB),Lactic dehydrogenase(LDH),malondialdehyde(MDA)levels,and increased superoxide dismutase(SOD)levels.HE staining showed that JG-7 and GZ-3 improved myocardial pathology after MIRI 24 h.The results of TUNEL apoptosis assay showed that JG-7 and GZ-3 improved apoptosis in myocardial tissues 24 h after MIRI.Masson staining results showed that JG-7 and GZ-3 could reduce the area of myocardial tissue fibrosis after MIRI 7 d.CCK-8 assay results showed that JG-7 and GZ-3 could improve the cell survival rate after H/R in H9C2 cells.Conclusion Pre-treatment with Mongolian medicine Jiruhen Gurigumu-7 and Guangzao Sanwei Tang can reduce the damage caused after ischemia-reperfusion(I/R),decrease the area of myocardial infarction and fibrosis after I/R in mice,and protect the heart.
5.Study on the Protective Effects of the Mongolian Prescription Jiruhen Gurigumu-7 and Guangzao Sanwei Tang on Myocardial Ischemia-Reperfusion Mice
Zhongyue ZHANG ; Shuhong ZHOU ; Qian GAO ; Xiaoxia SONG ; Xiaoru ZHANG ; Lingze YU ; Yulu DU ; Na GUO ; Minjie WANG
World Science and Technology-Modernization of Traditional Chinese Medicine 2025;27(8):2331-2339
Objective To explore the protective effects of pretreatment with the Mongolian medicine Jiruhen Gurigumu-7(JG-7)and Guangzao Sanwei Tang(GZ-3)on myocardial ischemia-reperfusion injury(MIRI)in mice.Methods 60 male C57BL/6J mice were randomly divided into sham operation(Sham)group,model(Model)group,compound danshen drip pill(CDDP)positive control group,JG-7 group,GZ-3 group,and 12 mice in each group to establish the MIRI model,and the H9C2 cells were randomly divided into Control(normoxic)group,H/R(hypoxia 6 h reoxygenation 14 h)group,H/R+JG-7 group,H/R+GZ-3 group.The mice in each group were tested for cardiac function indexes after 30 min of ischemia,24 h and 7 d of reperfusion,TTC staining to detect infarct area after 24 h of MIRI,HE staining to detect myocardial tissue structure and cellular morphology after 24 h of MIRI,TUNEL apoptosis kit to detect apoptosis of myocardial cells after 24 h of MIRI,Masson staining to detect myocardial fibrosis after 7 d of MIRI.Blood was taken from the abdominal aorta,serum was separated,and the indexes after oxidative stress of MIRI were detected in each group of mice,and the survival rate of H9C2 cells after H/R was detected in each group by CCK-8 method.Results The results of TTC showed that JG-7 and GZ-3 reduced the infarct area after 24 h of MIRI in mice.ELISA and kit assays proved that JG-7 and GZ-3 reduced creatine phosphokinase isoenzyme(Creatinekinase-MB,CK-MB),Lactic dehydrogenase(LDH),malondialdehyde(MDA)levels,and increased superoxide dismutase(SOD)levels.HE staining showed that JG-7 and GZ-3 improved myocardial pathology after MIRI 24 h.The results of TUNEL apoptosis assay showed that JG-7 and GZ-3 improved apoptosis in myocardial tissues 24 h after MIRI.Masson staining results showed that JG-7 and GZ-3 could reduce the area of myocardial tissue fibrosis after MIRI 7 d.CCK-8 assay results showed that JG-7 and GZ-3 could improve the cell survival rate after H/R in H9C2 cells.Conclusion Pre-treatment with Mongolian medicine Jiruhen Gurigumu-7 and Guangzao Sanwei Tang can reduce the damage caused after ischemia-reperfusion(I/R),decrease the area of myocardial infarction and fibrosis after I/R in mice,and protect the heart.
6.Study of the occurrence of metabolic dysfunction-associated fatty liver disease in obstructive sleep apnea hypopnea syndrome and its risk factors
Yanlan GUO ; Daiyi ZHANG ; Shenjie XU ; Minjie ZHANG ; Jie LI
Chinese Journal of Internal Medicine 2025;64(2):128-133
Objective:To analyze the occurrence of metabolic dysfunction-associated fatty liver disease (MAFLD) and related inflammatory indicators in obstructive sleep apnea hypopnea syndrome (OSAHS) and explore the risk factors of MAFLD.Methods:A cross-sectional study. From January 2022 to October 2024,172 patients with sleep disorders were enrolled in the First Affiliated Hospital of Soochow University,including 38 patients with non-OSAHS,53 patients with mild OSAHS,37 patients with moderate OSAHS,and 44 patients with severe OSAHS. The occurrence of MAFLD was comprehensively judged from three aspects: metabolic dysfunction-associated fatty liver (MAFL),elevated liver enzymes,and liver fibrosis. The situation of MAFLD and the level of related inflammatory markers were compared among the four groups. Binary logistic regression was used to analyze the risk factors for MAFLD in OSAHS.Results:There were significant differences in the prevalence of MAFL,the percentage of elevated liver enzymes,and interleukin-6 and tumor necrosis factor-alpha levels among the four groups ( P<0.05). The differences of fibrosis-4 index and C-reactive protein among the four groups were not statistically significant ( P>0.05). Binary logistic regression showed that BMI,triglycerides,longest time of sleep apnea and tumor necrosis factor-alpha were the risk factors for MAFL ( P<0.05). BMI,glucose,and apnea-hypopnea index were the risk factors for elevated liver enzymes ( P<0.05). Conclusions:OSAHS is strongly associated with MAFLD,and the involvement of OSAHS in the occurrence and development of MAFLD may be related to obesity,lipid metabolism disorders,insulin resistance,inflammatory responses,and intermittent hypoxia.
7.Experts consensus on standard items of the cohort construction and quality control of temporomandibular joint diseases (2024)
Min HU ; Chi YANG ; Huawei LIU ; Haixia LU ; Chen YAO ; Qiufei XIE ; Yongjin CHEN ; Kaiyuan FU ; Bing FANG ; Songsong ZHU ; Qing ZHOU ; Zhiye CHEN ; Yaomin ZHU ; Qingbin ZHANG ; Ying YAN ; Xing LONG ; Zhiyong LI ; Yehua GAN ; Shibin YU ; Yuxing BAI ; Yi ZHANG ; Yanyi WANG ; Jie LEI ; Yong CHENG ; Changkui LIU ; Ye CAO ; Dongmei HE ; Ning WEN ; Shanyong ZHANG ; Minjie CHEN ; Guoliang JIAO ; Xinhua LIU ; Hua JIANG ; Yang HE ; Pei SHEN ; Haitao HUANG ; Yongfeng LI ; Jisi ZHENG ; Jing GUO ; Lisheng ZHAO ; Laiqing XU
Chinese Journal of Stomatology 2024;59(10):977-987
Temporomandibular joint (TMJ) diseases are common clinical conditions. The number of patients with TMJ diseases is large, and the etiology, epidemiology, disease spectrum, and treatment of the disease remain controversial and unknown. To understand and master the current situation of the occurrence, development and prevention of TMJ diseases, as well as to identify the patterns in etiology, incidence, drug sensitivity, and prognosis is crucial for alleviating patients′suffering.This will facilitate in-depth medical research, effective disease prevention measures, and the formulation of corresponding health policies. Cohort construction and research has an irreplaceable role in precise disease prevention and significant improvement in diagnosis and treatment levels. Large-scale cohort studies are needed to explore the relationship between potential risk factors and outcomes of TMJ diseases, and to observe disease prognoses through long-term follw-ups. The consensus aims to establish a standard conceptual frame work for a cohort study on patients with TMJ disease while providing ideas for cohort data standards to this condition. TMJ disease cohort data consists of both common data standards applicable to all specific disease cohorts as well as disease-specific data standards. Common data were available for each specific disease cohort. By integrating different cohort research resources, standard problems or study variables can be unified. Long-term follow-up can be performed using consistent definitions and criteria across different projects for better core data collection. It is hoped that this consensus will be facilitate the development cohort studies of TMJ diseases.
8.Application of scaffold-based and scaffold-free strategy for treatment of growth plate cartilage regeneration
Ruoyi GUO ; Hanjie ZHUANG ; Xiuning CHEN ; Yulong BEN ; Minjie FAN ; Yiwei WANG ; Pengfei ZHENG
Chinese Journal of Tissue Engineering Research 2024;28(15):2437-2444
BACKGROUND:Tissue engineering is considered an ideal treatment for growth plate regeneration.However,most of the current research on regenerative tissue engineering is the traditional scaffold-based strategy.As the limitations of traditional scaffolds are gradually revealed,the research direction is gradually diversifying. OBJECTIVE:To summarize the application of scaffold-based and scaffold-free strategies in the treatment of growth plate cartilage regeneration and their respective advantages and disadvantages. METHODS:The relevant articles were searched from PubMed,Wiley,and Elsevier.The search terms were"growth plate injury,regeneration,tissue engineering,scaffold,scaffold-free,biomimetic,cartilage"in English.The time was limited from 1990 to 2023.Finally,104 articles were included for review. RESULTS AND CONCLUSION:The biomimetic strategy is to reduce the cell composition,biological signals and unique mechanical properties of each region to the greatest extent by simulating the unique organizational structure of the growth plate,so as to build a biomimetic microenvironment that can promote tissue regeneration.Therefore,the design of a biomimetic scaffold is to simulate the original growth plate as far as possible in terms of composition,structure and mechanical properties.Although some results have been achieved,there is still the problem of the unstable regeneration effect.The scaffold-free strategy believes that the limitations of scaffolds will have adverse effects on regenerative therapy.Therefore,the design of scaffold-free constructs relies as much as possible on the ability of cells to generate and maintain extracellular matrix without interfering with cell-cell signals or introducing exogenous substances.However,there are some problems,such as poor stability,low mechanical strength and greater difficulty in operation.Biomimetic strategy and scaffold-free strategy have different emphases,advantages and disadvantages,but they both have positive effects on growth plate cartilage regeneration.Therefore,subsequent studies,whether adopting a biomimetic strategy or a scaffold-free strategy,will focus on the continuous optimization of existing technologies in order to achieve effective growth plate cartilage regeneration therapy.
9.Long-term effect of modified Morrow surgery on hypertrophic obstructive cardiomyopathy in children: A retrospective study in a single center
Xiaoyi LI ; Hongxiang WU ; Ruobing WANG ; Haozhong LIU ; Xiaodong ZENG ; Ying ZENG ; Shengwen WANG ; Minjie HUANG ; Haiyun YUAN ; Jian LIU ; Huiming GUO
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2024;31(07):985-990
Objective To analyze the long-term outcome of modified Morrow surgery (interventricular septal cardiomyectomy) in the treatment of hypertrophic obstructive cardiomyopathy (HOCM) in children. Methods The clinical data of the children with HOCM (aged≤14 years) who underwent modified Morrow surgery from January 2010 to August 2022 in Guangdong Provincial People's Hospital were retrospectively analyzed, including changes in hospitalization status, perioperative period, and long-term 15-lead electrocardiogram and echocardiography. Results A total of 29 patients were collected, including 22 males and 7 females, aged 10.00 (5.00, 12.00) years. Five (17.9%) patients had New York Heart Association (NYHA) heart function grade Ⅲ or Ⅳ. Ventricular septal cardiomyectomy was performed in all patients. All 29 patients survived and their cardiac function recovered after operation. Before discharge, right bundle branch block was observed in 2 patients and left bundle branch block in 6 patients. After surgery, in the left ventricular septal cardiomyectomy, the left atrial diameter decreased (P<0.001), left ventricular end-systolic diameter increased (P=0.009), the peak pressure gradient of left ventricular outflow tract decreased (P<0.001), and the thickness of ventricular septum decreased (P<0.001). The systolic anterior motion of mitral valve disappeared and mitral regurgitent jet area decreased (P<0.001). The flow velocity and peak pressure gradient of right ventricular outflow tract also decreased in the patients who underwent right ventricular septal cardiomyectomy. The average follow-up of the patients was 69.03±10.60 months. All the patients survived with their NYHA cardiac function grading Ⅰ or Ⅱ. No new-onset arrythmia event was found. Echocardiography indicated that the peak pressure gradient of the left ventricular outflow tract remained low (P<0.001). Moderate mitral regurgitation occurred in 2 patients, and left ventricular outflow tract obstruction with moderate mitral regurgitation occurred in 1 patient after simple right ventricular septal cardiomyectomy. Conclusion Right ventricular or biventricular obstruction is frequent in the children with HOCM and they usually have more symptoms before surgery. Modified Morrow surgery can effectively relieve outflow tract obstruction and improve their cardiac function. The long-term outcome is satisfactory. However, the posterior wall of the left ventricle remains hypertrophic. Also, there is an increased risk of a conduction block.
10.Toxic effects of sodium hypochlorite as disinfectants on human bronchial epithelial cells
Jiawei LI ; Jiangzheng LIU ; Xiaojie GUO ; Yongmei TU ; Minjie SHI ; Wenli LI ; Jinsuo LU
Journal of Environmental and Occupational Medicine 2024;41(7):822-827
Background There are a variety of microorganisms in ambient air, and susceptible people can be infected once contact with pathogenic microorganisms in the environment. In order to avoid the spread of pathogenic bacteria, disinfection is the simplest and most effective way of killing pathogenic bacteria in the environment to block the contact between pathogenic bacteria and humans. Sodium hypochlorite (NaClO) is the most widely used disinfectant, but its safety in ambient air disinfection is not clear yet. Objective To establish a model of bronchial epithelial cell (BEAS-2B) injury induced by NaClO, and to explore the mechanism of the toxic effect of NaClO disinfectants on BEAS-2B. Methods Cells were treated with concentration gradients of 0, 25, 50,100, 200, and 400 μmol·L−1 of the diluted NaClO (100 mmol·L−1) standard solution, respectively, and cell activity was measured by cell counting kit-8 (CCK-8) assay after 15 and 30 min. Cells treated with 0, 25, and 50 μmol·L−1 NaClO were selected to observe the cell morphology under an inverted microscope, apoptosis was determined by flow cytometry Annexin V FITC / PI double staining to determine the final experimental concentration. The morphology of organelles such as mitochondria was observed under a transmission electron microscope. Mitochondrial membrane potential of the cells was detected by JC-1 staining. Intracellular Ca2+ concentration was measured with a Fluo-4 AM fluorescent probe. Total cellular reactive oxygen species (ROS) was detected with a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, cell mitochondrial ROS with a dihydroethidium (DHE) fluorescent probe, and lipid peroxidation intermediate malondialdehyde (MDA) with a commercial kit. Results Compared with 0 μmol·L−1, NaClO treatment group, cell morphology did not change a lot after 25 μmol·L−1 NaClO treatment for 30 min, and the cells began to wrinkle and become round after 30 min treatment with 50 μmol·L−1 NaClO, showing about 70% of normal cell viability (P<0.01). So 30 min 50 μmol·L−1 NaClO treatment was selected for the subsequent experiment. The experimental results found that compared with the 0 μmol·L−1 NaClO treatment group, the number of apoptotic cells increased (P<0.05), the mitochondrial membrane potential decreased (P<0.01), the intracellular Ca2+ concentration increased (P<0.05), the cellular ROS level increased (P<0.05), the mitochondrial ROS level increased (P<0.01), and the MDA content increased (P<0.01) in the NaClO treatment group.. Conclusion The study has successfully established a model of BEAS-2B injury induced by NaClO, and found that NaClO can lead to cell damage by inducing apoptosis and oxidative stress in BEAS-2B cells. According to the results, there are two possible reasons. First, NaClO solves in water to form hypochlorous acid (HClO) which is oxidative and increases the intracellular ROS level after entering cells, leading to cellular oxidative stress. Second, HClO enters cells to directly attack the mitochondrial membrane, resulting in the imbalance of potential inside and outside the mitochondrial membrane, and apoptosis caused by Ca2+ efflux.

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