BACKGROUND:Exosomes derived from mesenchymal stem cells play pivotal roles in cell communication and epigenetic regulation due to their low immunogenicity and targeted delivery effects,and have been clinically applied in the treatment of various diseases.OBJECTIVE:To review the isolation,purification,identification methods,and application progress of mesenchymal stem cell-derived exosomes,and to facilitate the development of large-scale preparation techniques and clinical translation of mesenchymal stem cell-derived exosomes.METHODS:The Chinese search terms"exosome,mesenchymal stem cells,isolation,purification,characterization,clinical application"and the English search terms"exosome,extracellular vesicles,mesenchymal stem cells,isolation,characterization,application"were used to search the literature published before September 2024 in CNKI,PubMed,and Web of Science databases.Articles with poor relevance to the topic,outdated,or duplicated content were excluded,and finally,109 articles were included for review.RESULTS AND CONCLUSION:(1)This paper reviews recent methods for isolating and purifying exosomes,comparing the characteristics of ultracentrifugation,ultrafiltration,size-exclusion chromatography,polymer precipitation,immunoaffinity,microfluidic methods,and other novel approaches based on their underlying principles.(2)Methods for identifying exosomes can be categorized into physical and biochemical analyses,characterizing exosomes based on their shape,size,and characteristic proteins.(3)Mesenchymal stem cell-derived exosomes have broad applications in multiple fields such as medical aesthetics,wound repair,and cancer treatment,due to their immune-regulatory properties and ability to cross biological barriers.(4)The clinical translation of exosomes faces challenges due to their complex structure,lack of universal isolation techniques,and poor stability,making it difficult to achieve in a short period of time.

BACKGROUND:Exosomes derived from mesenchymal stem cells play pivotal roles in cell communication and epigenetic regulation due to their low immunogenicity and targeted delivery effects,and have been clinically applied in the treatment of various diseases.OBJECTIVE:To review the isolation,purification,identification methods,and application progress of mesenchymal stem cell-derived exosomes,and to facilitate the development of large-scale preparation techniques and clinical translation of mesenchymal stem cell-derived exosomes.METHODS:The Chinese search terms"exosome,mesenchymal stem cells,isolation,purification,characterization,clinical application"and the English search terms"exosome,extracellular vesicles,mesenchymal stem cells,isolation,characterization,application"were used to search the literature published before September 2024 in CNKI,PubMed,and Web of Science databases.Articles with poor relevance to the topic,outdated,or duplicated content were excluded,and finally,109 articles were included for review.RESULTS AND CONCLUSION:(1)This paper reviews recent methods for isolating and purifying exosomes,comparing the characteristics of ultracentrifugation,ultrafiltration,size-exclusion chromatography,polymer precipitation,immunoaffinity,microfluidic methods,and other novel approaches based on their underlying principles.(2)Methods for identifying exosomes can be categorized into physical and biochemical analyses,characterizing exosomes based on their shape,size,and characteristic proteins.(3)Mesenchymal stem cell-derived exosomes have broad applications in multiple fields such as medical aesthetics,wound repair,and cancer treatment,due to their immune-regulatory properties and ability to cross biological barriers.(4)The clinical translation of exosomes faces challenges due to their complex structure,lack of universal isolation techniques,and poor stability,making it difficult to achieve in a short period of time.

Objective:To investigate the clinical efficacy of liquid nitrogen cryopreservation and reimplantation versus allograft reconstruction in patients underwent resection of primary malignant bone tumors of the long bones of the extremities.Methods:A retrospective analysis was conducted on 144 patients who underwent resection of primary malignant bone tumors of the long bones of the extremities followed by either liquid nitrogen cryopreservation and reimplantation or massive allografts reconstruction at the Beijing Jishuitan Hospital affiliated with Capital Medical University from January 2012 to July 2023. The study included 82 males and 62 females, with an average age of 23.8±12.3 years (range, 6-64 years). Patients were divided into two groups based on the reconstruction method: the cryopreservation and reimplantation group (72 cases) and the allograft group (72 cases). The following outcomes were recorded during follow-up: local tumor recurrence, bone union, union time, graft survival, and reasons for graft removal. Graft-related complications were recorded using the modified Henderson classification system of the International Limb Salvage Association. Limb function was assessed at the last follow-up using the Musculoskeletal Tumor Society score (MSTS-93).Results:All patients completed surgery and were followed up for a mean of 60.2±32.1 months (range, 12-149 months). At the last follow-up, 24 patients were dead from the tumor, 16 patients survived with the tumor (2 cases of local recurrence and 14 cases of distant metastasis), and 104 patients survived without the tumor. The bone union rate and union time in cryopreservation and reimplantation group were 90% (65/72) and 9.6±4.8 months, respectively, which was significantly superior to those in allograft group [68% (49/72) and 15.9±6.7 months, P<0.05]. The 5-year overall graft survival rate was 86.8% [95% CI (80.1%, 95.7%)] in cryopreservation and reimplantation group, higher than 73.2% [95% CI(68.4%, 84.5%)] in allograft group significantly (χ 2=7.122, P=0.017). The rates of graft removal due to non-union and infection in the cryopreservation and reimplantation group were 0% (0/72) and 1.4% (1/72), respectively, which were significantly lower than those in the allograft group [5.6% (4/72) and 9.7% (7/72), P<0.05]. Overall, 48.6% (70/144) of patients experienced at least one graft-related complication, with a complication rate of 33.3% in cryopreservation and reimplantation group, lower than the 61.1% in allograft group significantly (χ 2=11.146, P<0.001). The complications with the highest incidence rate were nonunion (20.8%, 30/144), followed by structural failure (17.4%, 25/144), tumor progression (10.4%, 15/144), infection (10.4%, 15/144), and soft tissue failure (5.6%, 8/144). The incidence rates of the atrophic non-union and the structural failure of grafts were 9.7% (7/72) and 1.4% (1/72) respectively in the cryopreservation and reimplantation group, which were significantly lower compared to the allograft group [29.2% (21/72) and 13.9% (10/72), P<0.05]. At the last follow-up, the MSTS-93 score was 89.7%±8.3% in the cryopreservation and reimplantation group, and 87.6%±7.5% in the allograft group, with no statistically significant difference ( t=0.326, P=0.542). Conclusion:Compared with allograft reconstruction, autologous inactivated bone grafting demonstrated superior bone union efficiency and fewer complications, it may be considered for reconstruction in cases whose tumor bone is not severely osteolytic or pathologically fractured.

Elderly insomnia refers to a type of insomnia that is specific to individuals over the age of 60,characterized by prolonged sleep latency,low sleep efficiency,and reduced total sleep time.It can be categorized under TCM as"insomnia","inability to close the eyes",and"inability to sleep".This article combined the physiological and pathological characteristics of the elderly population,based on the theory of"nutritious and defensive levels"and the theory of"five zang-organs governing spirits".It is believed that the harmony of nutritious and defensive levels and the balance of the five spirits are the physiological basis of elderly sleep.The disharmony between nutritious and defensive levels and the internal disturbance of pathogenic factors are the initial stages of insomnia in the elderly,and the imbalance of the five zang-organs,the imbalance of the five spirits,and the loss of mental health are the key pathological mechanisms of abnormal sleep rhythms.In clinical treatment,the fundamental approach should be to nourish nutritious and defensive levels,balance the five spirits,and calm the heart and soothe the spirit.Guizao Longmu Decoction created based on Guizhi Longgu Muli Decoction combined with Ganmai Dazao Decoction,follows the principles of nutritious and defensive levels circulation and the coordination of the functions of five zang-organs.It aims to nourish nutritious and defensive levels to adjust nutritious and defensive levels,balance the five zang-organs and calm the five spirits,and calm the heart and soothe the spirit to aid sleep and stabilize the mind,achieving a peaceful rest.The goal is to provide insights and methods from TCM for the clinical diagnosis and treatment of insomnia in the elderly.One medical case was attached to support evidence.

Objective:To investigate the effect of progressive resistance training on inflammatory markers, motor function and quality of life in the elderly after total knee arthroplasty (TKA).Methods:This study was a randomized controlled trial. A total of 46 elderly patients aged≥60 years who underwent total knee arthroplasty in the Affiliated Hospital 6 of Nantong University from January 2023 to June 2024 were selected and divided into experimental group and control group by computer random number method (23 cases in each group). The control group received routine rehabilitation management intervention, progressive resistance training was added to the experimental group on the basis of the control group, and all patients were intervened for 4 weeks. Interleukin-6 (IL-6), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), 30-Second Chair Stand Test (30sCST), Knee Society Score (KSS), and World Health Organization Quality of Life Scale-Brief Form Questionnaire (WHOQOL-BREF) scores were measured at 1 day before operation, 2 weeks and 4 weeks after operation, and were compared respectively.Results:At 2 and 4 weeks after operation, the levels of IL-6 [(22.44±2.17) and (9.91±1.41) pg/ml], CRP[(19.61±2.20) and (3.17±0.40) mg/L] and ESR[(44.85±3.78) and (28.28±3.31) mm/1 h] in the experimental group were significantly lower than those in control group [IL-6: (24.65±1.77) and (11.35±1.67) pg/ml, CRP: (23.24±2.69) and (4.15±0.45) mg/L and ESR: (48.54±3.66) and (34.60±2.98) mm/1 h](all P<0.05). At 2 and 4 weeks after operation, the 30sCST[(9.87±0.92) and (11.83±1.03) times], clinical scores of KSS[(48.44±3.13) and (71.09±3.30) points], functional scores of KSS[(40.44±3.96) and (69.35±4.07) points] in the experimental group were significantly higher than those in control group [30sCST: (9.30±0.70) and (10.52±0.79) times, clinical scores of KSS: (46.17±2.86) and (67.00±2.89) points, functional scores of KSS: (38.91±3.68) and (66.30±5.05) points](all P<0.05). At 2 weeks after operation, the scores of physical health, mental health and social relations in the WHOQOL-BREF of the experimental group [(16.96±1.02), (17.96±1.46) and (6.74±0.62) points], which were significantly higher than those in the control group [(16.09±1.08), (17.14±1.12), (6.44±0.51) points](all P<0.05). There was no significant difference in the environmental condition score between the two groups. At 4 weeks after operation, the scores of physical health, mental health, social relations and environmental conditions in WHOQOL-BREF of the experimental group [(22.09±1.81), (22.17±2.19), (12.09±1.28) and (33.91±2.26) points] were significantly higher than those in the control group [(19.65±1.80), (20.39±1.95), (10.17±1.30), (31.96±2.51) points] (all P<0.05). Conclusion:Progressive resistance training can effectively reduce the inflammatory response in the elderly after total knee arthroplasty, enhance lower limb muscle strength and knee joint function, and improve the quality of life.

Trigeminal neuralgia is characterized by intense pain in the sensory distribution area of the trigeminal nerve. It can be triggered by non-noxious stimuli such as brushing teeth and washing face. At present, the treatment of trigeminal neuralgia mainly includes oral drugs and surgical treatments. A 92-year-old patient with trigeminal neuralgia was reported. The pain could not be alleviated because the patient was unable to tolerate the side effects of drugs and surgical treatment. Taking into account the onset time and the duration of the curative effect, botulinum toxin type A was combined with cobrotoxin for the treatment of the patient. As a result, the pain symptoms were rapidly alleviated and remained in a relieved state for 8 months. The clinical characteristics of this patient were summarized in this article, and the possible synergistic mechanisms of action of the 2 drugs were discussed. The ultimate objective is to furnish a broader spectrum of alternatives and references for clinical practice.

Objective To explore the role of sevoflurane(SEV)in sepsis-induced acute lung injury(ALI)and observe its impact on the Wnt/β-catenin signaling pathway.Methods Forty C57 mice were randomly divided into 4 groups(n=10 each):Sham,CLP,SEV,and SEV+XAV(β-catenin inhibitor).A sepsis model was established via cecal ligation and puncture.Lung injury was evaluated using HE staining,lung wet/dry weight ratio,and TUNEL staining.Levels of inflammatory factors(TNF-α,IL-1β,IL-6)were detected by ELISA.Oxidative stress indices(SOD,MDA,ROS)were measured by colorimetry and flow cytometry.Hindlimb blood perfusion and oxygenation were assessed with laser speckle flowmetry.Expressions of key Wnt pathway molecules and down-stream target genes(c-Myc,Cyclin D1)were detected by RT-qPCR and Western blot.Co-localization of β-catenin and SP-C(a marker of type Ⅱ alveolar epithelial cells)in lung tissues was determined by immunofluorescence staining.Results Compared with the Sham group,the CLP group exhibited significant increases in sepsis severity,lung pathological damage including alveolar structure destruction,inflammatory infiltration,and apoptosis,elevation in pro-inflammatory cytokine levels,and significant decrease in SOD and increase in MDA and ROS.Additionally,lower limb blood flow and oxygenation levels were significantly reduced,while the expression of β-catenin and its downstream target genes,as well as the co-localization signal and fluorescence intensity of β-catenin with SP-C,were significantly downregulated(all P<0.05).Compared with the CLP group,the SEV group showed significant improvements in all these indicators.However,compared with the SEV group,the SEV+XAV group demon-strated a reversed protective effect,with all indicators approaching the levels observed in the CLP group(all P<0.05).Conclusion Sevoflurane alleviates sepsis-induced ALI by activating Wnt/β-catenin signaling pathway,exerting anti-inflammatory and antioxidant effects,and enhancing the expression and localization of β-catenin in type Ⅱ alveolar epithelial cells.

Objective:To investigate the clinical efficacy of liquid nitrogen cryopreservation and reimplantation versus allograft reconstruction in patients underwent resection of primary malignant bone tumors of the long bones of the extremities.Methods:A retrospective analysis was conducted on 144 patients who underwent resection of primary malignant bone tumors of the long bones of the extremities followed by either liquid nitrogen cryopreservation and reimplantation or massive allografts reconstruction at the Beijing Jishuitan Hospital affiliated with Capital Medical University from January 2012 to July 2023. The study included 82 males and 62 females, with an average age of 23.8±12.3 years (range, 6-64 years). Patients were divided into two groups based on the reconstruction method: the cryopreservation and reimplantation group (72 cases) and the allograft group (72 cases). The following outcomes were recorded during follow-up: local tumor recurrence, bone union, union time, graft survival, and reasons for graft removal. Graft-related complications were recorded using the modified Henderson classification system of the International Limb Salvage Association. Limb function was assessed at the last follow-up using the Musculoskeletal Tumor Society score (MSTS-93).Results:All patients completed surgery and were followed up for a mean of 60.2±32.1 months (range, 12-149 months). At the last follow-up, 24 patients were dead from the tumor, 16 patients survived with the tumor (2 cases of local recurrence and 14 cases of distant metastasis), and 104 patients survived without the tumor. The bone union rate and union time in cryopreservation and reimplantation group were 90% (65/72) and 9.6±4.8 months, respectively, which was significantly superior to those in allograft group [68% (49/72) and 15.9±6.7 months, P<0.05]. The 5-year overall graft survival rate was 86.8% [95% CI (80.1%, 95.7%)] in cryopreservation and reimplantation group, higher than 73.2% [95% CI(68.4%, 84.5%)] in allograft group significantly (χ 2=7.122, P=0.017). The rates of graft removal due to non-union and infection in the cryopreservation and reimplantation group were 0% (0/72) and 1.4% (1/72), respectively, which were significantly lower than those in the allograft group [5.6% (4/72) and 9.7% (7/72), P<0.05]. Overall, 48.6% (70/144) of patients experienced at least one graft-related complication, with a complication rate of 33.3% in cryopreservation and reimplantation group, lower than the 61.1% in allograft group significantly (χ 2=11.146, P<0.001). The complications with the highest incidence rate were nonunion (20.8%, 30/144), followed by structural failure (17.4%, 25/144), tumor progression (10.4%, 15/144), infection (10.4%, 15/144), and soft tissue failure (5.6%, 8/144). The incidence rates of the atrophic non-union and the structural failure of grafts were 9.7% (7/72) and 1.4% (1/72) respectively in the cryopreservation and reimplantation group, which were significantly lower compared to the allograft group [29.2% (21/72) and 13.9% (10/72), P<0.05]. At the last follow-up, the MSTS-93 score was 89.7%±8.3% in the cryopreservation and reimplantation group, and 87.6%±7.5% in the allograft group, with no statistically significant difference ( t=0.326, P=0.542). Conclusion:Compared with allograft reconstruction, autologous inactivated bone grafting demonstrated superior bone union efficiency and fewer complications, it may be considered for reconstruction in cases whose tumor bone is not severely osteolytic or pathologically fractured.

Objective To explore the role of sevoflurane(SEV)in sepsis-induced acute lung injury(ALI)and observe its impact on the Wnt/β-catenin signaling pathway.Methods Forty C57 mice were randomly divided into 4 groups(n=10 each):Sham,CLP,SEV,and SEV+XAV(β-catenin inhibitor).A sepsis model was established via cecal ligation and puncture.Lung injury was evaluated using HE staining,lung wet/dry weight ratio,and TUNEL staining.Levels of inflammatory factors(TNF-α,IL-1β,IL-6)were detected by ELISA.Oxidative stress indices(SOD,MDA,ROS)were measured by colorimetry and flow cytometry.Hindlimb blood perfusion and oxygenation were assessed with laser speckle flowmetry.Expressions of key Wnt pathway molecules and down-stream target genes(c-Myc,Cyclin D1)were detected by RT-qPCR and Western blot.Co-localization of β-catenin and SP-C(a marker of type Ⅱ alveolar epithelial cells)in lung tissues was determined by immunofluorescence staining.Results Compared with the Sham group,the CLP group exhibited significant increases in sepsis severity,lung pathological damage including alveolar structure destruction,inflammatory infiltration,and apoptosis,elevation in pro-inflammatory cytokine levels,and significant decrease in SOD and increase in MDA and ROS.Additionally,lower limb blood flow and oxygenation levels were significantly reduced,while the expression of β-catenin and its downstream target genes,as well as the co-localization signal and fluorescence intensity of β-catenin with SP-C,were significantly downregulated(all P<0.05).Compared with the CLP group,the SEV group showed significant improvements in all these indicators.However,compared with the SEV group,the SEV+XAV group demon-strated a reversed protective effect,with all indicators approaching the levels observed in the CLP group(all P<0.05).Conclusion Sevoflurane alleviates sepsis-induced ALI by activating Wnt/β-catenin signaling pathway,exerting anti-inflammatory and antioxidant effects,and enhancing the expression and localization of β-catenin in type Ⅱ alveolar epithelial cells.

Elderly insomnia refers to a type of insomnia that is specific to individuals over the age of 60,characterized by prolonged sleep latency,low sleep efficiency,and reduced total sleep time.It can be categorized under TCM as"insomnia","inability to close the eyes",and"inability to sleep".This article combined the physiological and pathological characteristics of the elderly population,based on the theory of"nutritious and defensive levels"and the theory of"five zang-organs governing spirits".It is believed that the harmony of nutritious and defensive levels and the balance of the five spirits are the physiological basis of elderly sleep.The disharmony between nutritious and defensive levels and the internal disturbance of pathogenic factors are the initial stages of insomnia in the elderly,and the imbalance of the five zang-organs,the imbalance of the five spirits,and the loss of mental health are the key pathological mechanisms of abnormal sleep rhythms.In clinical treatment,the fundamental approach should be to nourish nutritious and defensive levels,balance the five spirits,and calm the heart and soothe the spirit.Guizao Longmu Decoction created based on Guizhi Longgu Muli Decoction combined with Ganmai Dazao Decoction,follows the principles of nutritious and defensive levels circulation and the coordination of the functions of five zang-organs.It aims to nourish nutritious and defensive levels to adjust nutritious and defensive levels,balance the five zang-organs and calm the five spirits,and calm the heart and soothe the spirit to aid sleep and stabilize the mind,achieving a peaceful rest.The goal is to provide insights and methods from TCM for the clinical diagnosis and treatment of insomnia in the elderly.One medical case was attached to support evidence.