Objective To investigate the effects of scoparone(Sco)on proliferation and invasion of colon cancer cell line HCT116,and its effect on the expression of epidermal growth factor receptor(EGFR).Methods 1)HCT116 cells were divided into control group,50Sco group,100Sco group and 200Sco group.The cells in con-trol group were incubated with culture medium for 48 hrs.The 50Sco group,100Sco group and 200Sco group were incubated with 50,100 and 200 μmol/L scoparone for 48 hrs respectively.2)HCT116 cells were divided into con-trol group,NC-200Sco group,NC-LV+200Sco group and EGFR-LV+200Sco group.The control group was incuba-ted with normal culture medium for 48 hrs.NC-200Sco group was incubated with 200 μmol/L scoparone for 48 hrs.NC-LV and EGFR-LV were infected into HCT116 cells in NC-LV+200Sco group and EGFR-LV+200Sco group,then incubated with 200 μmol/L scoparone for 48 hrs.Cell proliferation was detected by MTT assay and EdU stai-ning,cell apoptosis was detected by flow cytometry and cell invasion was detected by Transwell assay.EGFR mRNA was detected by RT-qPCR,the level of EGFR,Bcl-2,Bax,matrix metalloproteinase(MMP)-2 and MMP-9 protein was detected by Western blot.Results Compared to the control group,the cell viability,proportion of EdU positive cells and counting number of invasive cells in 50Sco group,100Sco group and 200Sco group all decreased(P＜0.05).Cell apoptosis rate and Bax protein expression increased(P＜0.05),the protein expression of Bcl-2,MMP-2 and MMP-9 decreased(P＜0.05).mRNA and protein expression of EGFR were de-creased(P＜0.05).Compared with NC-200 Sco group and NC-LV+200Sco group,the expression level of mRNA and protein of EGFR in EGFR-LV+200Sco group was increased(P＜0.05).Cell viability,proportion of EdU posi-tive cells and counting number of invasive cells all increased(P＜0.05).The cell apoptosis rate and Bax protein expression level were decreased(P＜0.05).The protein expression of Bcl-2,MMP-2 and MMP-9 was increased(P＜0.05).Conclusions Scoparone has anti-colon cancer cell activity and inhibits proliferation as well as invasion of colon cancer cells through inhibition of EGFR.

Objective:To analyze the effect and safety of vibrating mesh atomization inhalation of pulmonary surfactant (PS) combined with nasal continuous positive airway pressure (NCPAP) in the treatment of neonatal respiratory distress syndrome (NRDS).Methods:150 Children with NRDS in department of pediatrics of the Second Affiliated Hospital of Air Force Medical University from May 2021 to May 2024 were enrolled as study subjects for a prospective cohort study. According to the propensity score matching, children who adopted traditional intratracheal infusion of PS combined with NCPAP were selected as infusion group (75 cases), and children who received vibrating mesh atomization inhalation of PS combined with NCPAP treatment were included in atomization group (75 cases). The therapeutic effect, breathing conditions, blood gas changes [pH value, arterial partial pressure of oxygen (PaO 2), arterial partial pressure of carbon dioxide (PaCO 2)] and NCPAP parameters changes [fraction of inspired oxygen (FiO 2), oxygenation index (OI), positive end-expiratory pressure (PEEP)] before and after treatment and incidence rates of adverse reactions were compared between groups. Normally distributed quantitative data were expressed as xˉ± s, and t test was used for inter-group comparison. Counting data were expressed as case(%), and χ2 test was used for inter-group comparison. Comparison of repeated measurement indicators at different time points using repeated meausurement analysis of variance. Results:There was no statistical significance in the total effective rate of treatment between groups ( P>0.05). The NCPAP treatment time and oxygen therapy time were (4.69±0.61) d and (14.52±1.16) d in atomization group, significantly shorter than (5.08±0.80) d and (15.30±1.28) d in infusion group ( t=3.36, 3.91, P=0.001, <0.001). The success rate of one-time weaning in atomization group was 88.00% (66/75), significantly higher than 73.33% (55/75) in infusion group ( χ2=5.17, P=0.023). After 3 and 7 days of treatment, the pH [(3 d : 7.31±0.04, 7 d: 7.34±0.03) and (3 d: 7.35±0.03, 7 d: 7.38±0.02)], PaO 2 [(3 d: (58.55±6.51) mmHg (1 mmHg=0.133 kPa), 7 d: (68.19±7.58) mmHg ) and (3 d: (65.16±7.24) mmHg, 7 d: (75.57±8.40) mmHg)] in infusion group and atomization group increased obviously over time, and the pH and PaO 2 were significantly higher in atomization group than those in infusion group ( t=6.93, 8.68, 5.88, 5.65, all P<0.001). The PaCO 2 in infusion group and atomization group (3 d: (48.45±5.38 ) mmHg, 7 d: (43.64±4.85 ) mmHg) and (3 d: (45.41±5.02) mmHg, 7 d: (40.35±4.59) mmHg) decreased obviously over time at 3 and 7 days of treatment, and the PaCO 2 in atomization group was significantly lower than that in infusion group ( t=3.58, 4.27, both P<0.001). After 3 days of treatment, FiO 2 [(41.06±4.67)% and (38.52±4.21)%] and PEEP [(4.39±0.19) cmH 2O (1 cmH 2O=0.098 kPa) and (4.28±0.13) cmH 2O] in infusion group and atomization group decreased obviously, and the indicators in atomization group were significantly lower than those in infusion group ( t=3.50, 4.14, both P<0.001). After 3 days of treatment, oxygenation index (OI) in infusion group and atomization group [(278.35±30.48) mmHg and (296.67±32.96) mmHg] increased obviously, and the OI in atomization group was significantly higher than that in infusion group ( t=3.53, P<0.001). The incidence of adverse reactions in atomization group was 4.00% (3/75), significantly lower than 13.34% (10/75) in infusion group ( χ2=4.13, P=0.042). Conclusion:Both vibrating mesh atomization inhalation of PS or traditional infusion of PS combined with NCPAP have similar efficacy in the treatment of NRDS, but vibrating mesh atomization inhalation of PS combined with NCPAP can more effectively improve the breathing conditions, blood gas indicators and NCPAP parameters of children, and has higher safety.

Objective To explore and verify the active components of Dachengqi decoction in regulating autophagy and its mechanism of protecting the intestinal mucosal barrier through network pharmacology and animal experiments.Methods The chemical components and autophagy-related target points of Dachengqi decoction were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database Analysis Platform(TCMSP)and GeneCards databases.The intersection of the drug target points and disease target points was taken and analyzed.The Cytoscape 3.10.2 software's Network Analyzer tool was used to analyze the drug components and target points,and the core target points were screened out to construct a traditional Chinese medicine compound regulatory network.The drug active component target point-disease network model and protein-protein interaction(PPI)network were visualized.Then,30 C57BL/6J mice were randomly divided into the Dachengqi decoction group,the intestinal infection group,and the control group,with 10 mice in each group.The intestinal infection group was given 200 μL/d of Klebsiella pneumoniae strain by gavage for 5 consecutive days,with a colony count of 109 CFU/mL,to create an intestinal infection model.The control group was given 200 μL/d of sterile normal saline by gavage.The Dachengqi decoction group(drug composition:Rhubarb 12 g,Aurantii Fructus 12 g,Magnolia Officinalis 24 g,Mirabilite 9 g,the drugs were dissolved in boiling distilled water to make a 1 kg/L solution)was given by gavage at a dose of 8 g·kg-1·d-1 for 3 consecutive days,and then given Klebsiella pneumoniae by gavage for 5 consecutive days on the 4th day.Detection indicators and methods:after the experiment,the mice were sacrificed and the terminal ileum tissues were collected.The tissues were stained with hematoxylin-eosin(HE),and the pathological changes of the intestinal mucosa were observed under a light microscope;immunofluorescence staining was used to observe the positive expressions of junction proteins ZO-1,Claudin-2,light chain 3-Ⅱ(LC3-Ⅱ),and Beclin-1 and the intestinal mucosal autophagy;the mRNA expression levels of autophagy genes were determined by polymerase chain reaction(PCR).Results The intersection of the obtained drug targets and disease targets yielded 111 potential autophagy-related targets for drug treatment of diseases.Key targets included β2-adrenergic receptor(ADRB2),heme oxygenase-1(HO-1),etc.,and the signaling pathways involved included AMP-activated protein kinase(AMPK)pathway,mammalian target of rapamycin(mTOR)pathway,etc.Animal experiments confirmed that the intestinal mucosal barrier function in the Dachengqi decoction group was better than that in the intestinal infection group,and the positive expression of microtubule-associated protein 1 lingt chain 3-Ⅱ(LC3-Ⅱ)and autophagy gene Beclin1 was significantly higher than that in the intestinal infection group.Transcriptome sequencing results showed that the key genes associated with autophagy and oxidative stress included ADRB2,HO-1,etc.The mRNA expression levels of ADRB2 and HO-1 in the Dachengqi decoction group were significantly higher than those in the intestinal infection group[HO-1 mRNA expression(FPKM):11.20±0.80 vs.6.63±0.53,ADRB2 mRNA expression(FPKM):6.98±0.54 vs.3.98±0.32,both P<0.01],verifying some of the predictions from network pharmacology.Conclusions Dachengqi decoction regulates autophagy through multiple components,multiple targets and multiple pathways,protecting the intestinal mucosal barrier function and reducing the translocation of intestinal microbiota.This lays a certain foundation for further in-depth research on the mechanism of reducing intestinal bacterial translocation by Dachengqi decoction.

The paper summarizes Professor YANG Jun's thoughts on clinical treatment with acupuncture and moxibustion. Professor YANG Jun puts forward the "refined mode for diagnosis and treatment of diseases with acupuncture and moxibustion", aiming to improve the capacity of diagnosis and treatment in clinical practice. He advocates that the diagnosis and treatment should be guided by the identification of etiologies, syndromes and meridians; in accordance with regulating the shape/form, balancing yin and yang, and harmonizing the mind; and by means of skillful techniques of acupuncture and moxibustion, simplified selection of acupoints and delicate manipulations. Besides, he stresses on the combination of multiple techniques of acupuncture (such as penetrating technique with long needle, stuck needling by lifting and pulling, and micro-acupuncture systems) with moxibustion techniques (moxibustion for resolving stasis and unblocking collaterals, pressing moxibustion, borneol moxibustion, moxibustion with medicinal plaster) in clinical practice, so as to enhance the therapeutic effects.
Moxibustion/methods* ; Acupuncture Therapy/methods* ; Humans ; China ; Acupuncture Points

Cardiovascular diseases(CVD)are characterized by high morbidity,disability,and mortality rates,making them one of the leading causes of human death worldwide.Vascular remodeling refers to changes in the structure and function of blood vessels under pathological or physiological conditions,typically occurring during processes such as tissue damage repair and disease progression.Investigating the mechanisms of vascular remodeling helps in understanding the progression of CVD,thereby developing more effective early diagnosis and treatment plans,and providing new insights for the prevention and treatment of CVD.The modeling methods of vascular remodeling are the foundation for studying vascular remodeling.Significant progress has been made in vascular remodeling models for studying multiple disease mechanisms,and they are particularly important in the fields of atherosclerosis,hypertension,and vascular remodeling.Common animal models for vascular remodeling include rats,mice,pigs,and other species.Research methods cover mechanical injury,drug intervention,genetic modification,and so on.Different types of animal models have their own advantages.For example,mouse and rat models are suitable for gene studies and high-throughput screening,while rabbit and monkey models,due to their closer resemblance to human pathology,are helpful for simulating vascular remodeling under clinical conditions.Among them,rat models are widely used as frontline models in medical research due to their cost-effectiveness and ease of operation.Current vascular remodeling models mainly rely on classical methods(such as carotid artery balloon injury method,ligation method,and arterial clamping)and are combined with emerging dietary methods(such as high-fat diet,high-salt diet)for construction.Different rat modeling methods are selected according to different experimental needs.The combination of these methods can effectively simulate different mechanisms of vascular remodeling and provide reliable animal models for CVD research.In addition,these rat models can reflect vascular responses under different pathological conditions,offering an important experimental basis for drug development and the formulation of disease treatment strategies.Although these rat models provide valuable tools for vascular remodeling research,challenges such as large model variability,poor reproducibility,and differences from clinical manifestations remain.Future research should focus on improving the accuracy and reliability of existing models to develop new animal models.This article uses rats as examples to summarize the current research progress,model types,and applications of vascular remodeling models,particularly their value in CVD and vascular remodeling research,and provides a theoretical reference for future vascular remodeling-related research by reviewing the advantages and disadvantages of different rat vascular remodeling models.

Cardiovascular diseases(CVD)are characterized by high morbidity,disability,and mortality rates,making them one of the leading causes of human death worldwide.Vascular remodeling refers to changes in the structure and function of blood vessels under pathological or physiological conditions,typically occurring during processes such as tissue damage repair and disease progression.Investigating the mechanisms of vascular remodeling helps in understanding the progression of CVD,thereby developing more effective early diagnosis and treatment plans,and providing new insights for the prevention and treatment of CVD.The modeling methods of vascular remodeling are the foundation for studying vascular remodeling.Significant progress has been made in vascular remodeling models for studying multiple disease mechanisms,and they are particularly important in the fields of atherosclerosis,hypertension,and vascular remodeling.Common animal models for vascular remodeling include rats,mice,pigs,and other species.Research methods cover mechanical injury,drug intervention,genetic modification,and so on.Different types of animal models have their own advantages.For example,mouse and rat models are suitable for gene studies and high-throughput screening,while rabbit and monkey models,due to their closer resemblance to human pathology,are helpful for simulating vascular remodeling under clinical conditions.Among them,rat models are widely used as frontline models in medical research due to their cost-effectiveness and ease of operation.Current vascular remodeling models mainly rely on classical methods(such as carotid artery balloon injury method,ligation method,and arterial clamping)and are combined with emerging dietary methods(such as high-fat diet,high-salt diet)for construction.Different rat modeling methods are selected according to different experimental needs.The combination of these methods can effectively simulate different mechanisms of vascular remodeling and provide reliable animal models for CVD research.In addition,these rat models can reflect vascular responses under different pathological conditions,offering an important experimental basis for drug development and the formulation of disease treatment strategies.Although these rat models provide valuable tools for vascular remodeling research,challenges such as large model variability,poor reproducibility,and differences from clinical manifestations remain.Future research should focus on improving the accuracy and reliability of existing models to develop new animal models.This article uses rats as examples to summarize the current research progress,model types,and applications of vascular remodeling models,particularly their value in CVD and vascular remodeling research,and provides a theoretical reference for future vascular remodeling-related research by reviewing the advantages and disadvantages of different rat vascular remodeling models.

Objective:To analyze the effect and safety of vibrating mesh atomization inhalation of pulmonary surfactant (PS) combined with nasal continuous positive airway pressure (NCPAP) in the treatment of neonatal respiratory distress syndrome (NRDS).Methods:150 Children with NRDS in department of pediatrics of the Second Affiliated Hospital of Air Force Medical University from May 2021 to May 2024 were enrolled as study subjects for a prospective cohort study. According to the propensity score matching, children who adopted traditional intratracheal infusion of PS combined with NCPAP were selected as infusion group (75 cases), and children who received vibrating mesh atomization inhalation of PS combined with NCPAP treatment were included in atomization group (75 cases). The therapeutic effect, breathing conditions, blood gas changes [pH value, arterial partial pressure of oxygen (PaO 2), arterial partial pressure of carbon dioxide (PaCO 2)] and NCPAP parameters changes [fraction of inspired oxygen (FiO 2), oxygenation index (OI), positive end-expiratory pressure (PEEP)] before and after treatment and incidence rates of adverse reactions were compared between groups. Normally distributed quantitative data were expressed as xˉ± s, and t test was used for inter-group comparison. Counting data were expressed as case(%), and χ2 test was used for inter-group comparison. Comparison of repeated measurement indicators at different time points using repeated meausurement analysis of variance. Results:There was no statistical significance in the total effective rate of treatment between groups ( P>0.05). The NCPAP treatment time and oxygen therapy time were (4.69±0.61) d and (14.52±1.16) d in atomization group, significantly shorter than (5.08±0.80) d and (15.30±1.28) d in infusion group ( t=3.36, 3.91, P=0.001, <0.001). The success rate of one-time weaning in atomization group was 88.00% (66/75), significantly higher than 73.33% (55/75) in infusion group ( χ2=5.17, P=0.023). After 3 and 7 days of treatment, the pH [(3 d : 7.31±0.04, 7 d: 7.34±0.03) and (3 d: 7.35±0.03, 7 d: 7.38±0.02)], PaO 2 [(3 d: (58.55±6.51) mmHg (1 mmHg=0.133 kPa), 7 d: (68.19±7.58) mmHg ) and (3 d: (65.16±7.24) mmHg, 7 d: (75.57±8.40) mmHg)] in infusion group and atomization group increased obviously over time, and the pH and PaO 2 were significantly higher in atomization group than those in infusion group ( t=6.93, 8.68, 5.88, 5.65, all P<0.001). The PaCO 2 in infusion group and atomization group (3 d: (48.45±5.38 ) mmHg, 7 d: (43.64±4.85 ) mmHg) and (3 d: (45.41±5.02) mmHg, 7 d: (40.35±4.59) mmHg) decreased obviously over time at 3 and 7 days of treatment, and the PaCO 2 in atomization group was significantly lower than that in infusion group ( t=3.58, 4.27, both P<0.001). After 3 days of treatment, FiO 2 [(41.06±4.67)% and (38.52±4.21)%] and PEEP [(4.39±0.19) cmH 2O (1 cmH 2O=0.098 kPa) and (4.28±0.13) cmH 2O] in infusion group and atomization group decreased obviously, and the indicators in atomization group were significantly lower than those in infusion group ( t=3.50, 4.14, both P<0.001). After 3 days of treatment, oxygenation index (OI) in infusion group and atomization group [(278.35±30.48) mmHg and (296.67±32.96) mmHg] increased obviously, and the OI in atomization group was significantly higher than that in infusion group ( t=3.53, P<0.001). The incidence of adverse reactions in atomization group was 4.00% (3/75), significantly lower than 13.34% (10/75) in infusion group ( χ2=4.13, P=0.042). Conclusion:Both vibrating mesh atomization inhalation of PS or traditional infusion of PS combined with NCPAP have similar efficacy in the treatment of NRDS, but vibrating mesh atomization inhalation of PS combined with NCPAP can more effectively improve the breathing conditions, blood gas indicators and NCPAP parameters of children, and has higher safety.

Objective To explore and verify the active components of Dachengqi decoction in regulating autophagy and its mechanism of protecting the intestinal mucosal barrier through network pharmacology and animal experiments.Methods The chemical components and autophagy-related target points of Dachengqi decoction were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database Analysis Platform(TCMSP)and GeneCards databases.The intersection of the drug target points and disease target points was taken and analyzed.The Cytoscape 3.10.2 software's Network Analyzer tool was used to analyze the drug components and target points,and the core target points were screened out to construct a traditional Chinese medicine compound regulatory network.The drug active component target point-disease network model and protein-protein interaction(PPI)network were visualized.Then,30 C57BL/6J mice were randomly divided into the Dachengqi decoction group,the intestinal infection group,and the control group,with 10 mice in each group.The intestinal infection group was given 200 μL/d of Klebsiella pneumoniae strain by gavage for 5 consecutive days,with a colony count of 109 CFU/mL,to create an intestinal infection model.The control group was given 200 μL/d of sterile normal saline by gavage.The Dachengqi decoction group(drug composition:Rhubarb 12 g,Aurantii Fructus 12 g,Magnolia Officinalis 24 g,Mirabilite 9 g,the drugs were dissolved in boiling distilled water to make a 1 kg/L solution)was given by gavage at a dose of 8 g·kg-1·d-1 for 3 consecutive days,and then given Klebsiella pneumoniae by gavage for 5 consecutive days on the 4th day.Detection indicators and methods:after the experiment,the mice were sacrificed and the terminal ileum tissues were collected.The tissues were stained with hematoxylin-eosin(HE),and the pathological changes of the intestinal mucosa were observed under a light microscope;immunofluorescence staining was used to observe the positive expressions of junction proteins ZO-1,Claudin-2,light chain 3-Ⅱ(LC3-Ⅱ),and Beclin-1 and the intestinal mucosal autophagy;the mRNA expression levels of autophagy genes were determined by polymerase chain reaction(PCR).Results The intersection of the obtained drug targets and disease targets yielded 111 potential autophagy-related targets for drug treatment of diseases.Key targets included β2-adrenergic receptor(ADRB2),heme oxygenase-1(HO-1),etc.,and the signaling pathways involved included AMP-activated protein kinase(AMPK)pathway,mammalian target of rapamycin(mTOR)pathway,etc.Animal experiments confirmed that the intestinal mucosal barrier function in the Dachengqi decoction group was better than that in the intestinal infection group,and the positive expression of microtubule-associated protein 1 lingt chain 3-Ⅱ(LC3-Ⅱ)and autophagy gene Beclin1 was significantly higher than that in the intestinal infection group.Transcriptome sequencing results showed that the key genes associated with autophagy and oxidative stress included ADRB2,HO-1,etc.The mRNA expression levels of ADRB2 and HO-1 in the Dachengqi decoction group were significantly higher than those in the intestinal infection group[HO-1 mRNA expression(FPKM):11.20±0.80 vs.6.63±0.53,ADRB2 mRNA expression(FPKM):6.98±0.54 vs.3.98±0.32,both P<0.01],verifying some of the predictions from network pharmacology.Conclusions Dachengqi decoction regulates autophagy through multiple components,multiple targets and multiple pathways,protecting the intestinal mucosal barrier function and reducing the translocation of intestinal microbiota.This lays a certain foundation for further in-depth research on the mechanism of reducing intestinal bacterial translocation by Dachengqi decoction.

Objective:To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix (ECM) microcarriers.Methods:Fresh porcine articular cartilage was taken. The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size, which were prepared by the acellular method of combining physical centrifugation and chemical extraction, were set as microcarrier group. Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells (hUCMSCs) and human chondrocytes (hCho) with a ratio of 3∶1 with microcarriers through a rotating bioreactor. The organoids with different induction times were divided into 0-, 7-, 14-, and 21-day induction groups. The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′, 6-diaminidine 2-phenylindole (DAPI) fluorescence staining and DNA quantitative analysis. The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings, and the collagen and glycosaminoglycan (GAGs) levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue (DMMB) method. The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy. The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with fetal bovine serum (FBS) in a volume fraction of 10% was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group. Subgroups of hUCMSCs cultured at 3 time points: 1, 3 and 5 days were set up in the two groups separately. Cell Counting Kit 8 (CCK-8) was used to detect the biocompatibility of the two groups. The cellular activity of the organoids of the 0-, 7-, 14-, and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining. The organoids of the 7-, 14-, and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan (ACAN), type II collagen (COL2A1), SRY-related high mobility group-box gene-9 (SOX9), cartilage hypertrophy-and mineralization-related marker type I collagen (COL1A1), Runt-related transcription factor-2 (RUNX2), and osteocalcin (OCN). Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-, 7-, 14-, and 21-day induction groups to secrete collagen and GAGs.Results:The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei. The DNA content of the microcarrier group was (7.8±1.8)ng/mg, which was significantly lower than that of the natural cartilage group [(526.7±14.7)ng/mg] ( P<0.01). Saffranin O and toluidine blue staining showed that the microcarrier was dark- and uniform-colored and it kept a lot of cartilage ECM components. The collagen and GAGs contents of the microcarrier group were (252.9±1.4)μg/mg and (173.4±0.8)μg/mg, which were significantly lower than those of the natural cartilage group [(311.9±2.2)μg/mg and (241.3±0.7)μg/mg] ( P<0.01). Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network. The results of energy spectrum analysis showed that elements C, O and N were evenly distributed in the microcarriers, indicating that the composition of the microcarrier was uniform. The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture ( P>0.05). After 5 days of culture, the A value of the experimental group was 0.53±0.02, which was better than that of the control group (0.44±0.03) ( P<0.05). In the 0-, 7-, 14-, and 21-day induction groups, hUCMSCs and hCho were attached to the surface of the microcarriers, with good cellular activity, and the live/death rates were (70.6±1.1)%, (80.5±0.6)%, (94.5±0.9)%, and (90.8±0.5)% respectively ( P<0.01). There were a large number of Ki67 positive cells in cartilage organoids. RT-PCR showed that the expression levels of ACAN, COL2A1, SOX9, COL1A1, RUNX2 and OCN were 1.00±0.09, 1.00±0.24, 1.00±0.18, 1.00±0.03, 1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group; 4.16±0.28, 5.09±1.25, 5.65±1.05, 0.47±0.01, 1.68±0.02 and 0.21±0.06 respectively in the 14-day induction group; 13.42±0.92, 3.07±0.21, 1.84±1.08, 2.72±0.17, 2.91±0.18 and 3.32±1.20 respectively in the 21-day induction group. Compared with the 7-day induction group, the expression levels of ACAN, COL2A1, SOX9 and RUNX2 in the 14-day group were increased ( P<0.05), but COL1A1 expression level was decreased ( P<0.05), with no significant difference in OCN expression level ( P>0.05). Compared with the 7-day induction group, the expression levels of ACAN, COL1A1 and RUNX2 in the 21-day induction group were significantly increased ( P<0.01), with no significant differences in the expression levels of COL2A1, SOX9 and OCN ( P>0.05). Compared with the 14-day induction group, the expression levels of ACAN, COL1A1, RUNX2 and OCN in the 21-day group were increased ( P<0.05 or 0.01), with no significant difference in the expression level of COL2A1 ( P>0.05), but the expression level of SOX9 was decreased ( P<0.05). The contents of collagen in 0-, 7-, 14-and 21-day induction groups were (219.15±0.48)μg/mg, (264.07±1.58)μg/mg, (270.83±0.84)μg/mg and (280.01±0.48)μg/mg respectively. The GAGs contents were (171.18±1.09)μg/mg, (184.06±1.37)μg/mg, (241.08±0.84)μg/mg and (201.14±0.17)μg/mg respectively. Compared with the 0-day induction group, the contents of collagen and GAGs in 7-, 14-, and 21-day induction groups were significantly increased ( P<0.01), among which the content of collagen was the lowest in 7-day induction group ( P<0.01) but the highest in the 21-day induced group ( P<0.01); the content of GAGs was the lowest in the 7-day induced group ( P<0.01) but the highest in the 14-day induction group ( P<0.01). Conclusions:The microcarriers prepared by combining physical and chemical methods are decellularized successfully, with more matrix retention, uniform composition and on cytotoxicity. By loading microcarriers with hUCMSCs and hCho, cartilage organoids are successfully constructed in vitro, which are characterized by good cell activity, self-renewal ability, strong expression of genes related to chondrogenesis and secretion of collagen and GAGs. The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.

BACKGROUND:Compared with traditional two-dimensional culture,three-dimensional microtissue culture can show greater advantages.However,more favorable cultivation methods in three-dimensional culture still need to be further explored. OBJECTIVE:To evaluate the cell behavior of microtissue and its ability to promote cartilage formation under two three-dimensional culture methods. METHODS:Cartilage-derived microcarriers were prepared by chemical decellularization and tissue crushing.DNA quantification and nuclear staining were used to verify the success of decellularization,and histological staining was used to observe the matrix retention before and after decellularization.The microcarriers were characterized by scanning electron microscopy and CCK-8 assay.Cartilage-derived microtissues were constructed by combining cartilage-derived microcarriers with human adipose mesenchymal stem cells through three-dimensional static culture and three-dimensional dynamic culture methods.The cell viability and chondrogenic ability of the two groups of microtissues were detected by scanning electron microscopy,live and dead staining,and RT-qPCR. RESULTS AND CONCLUSION:(1)Cartilage-derived microcarriers were successfully prepared.Compared with before decellularization,the DNA content significantly decreased after decellularization(P<0.001).Scanning electron microscope observation showed that the surface of the microcarrier was surrounded by collagen,maintaining the characteristics of the natural extracellular matrix of cartilage cells.CCK-8 assay indicated that microcarriers had no cytotoxicity and could promote cell proliferation.(2)Scanning electron microscopy and live and dead staining results showed that compared with the three-dimensional static group,the three-dimensional dynamic group had a more extended morphology of microtissue cells,and extensive connections between cells and cells,between cells and matrix,and between matrix.(3)The results of RT-qPCR showed that the expressions of SOX9,proteoglycan,and type Ⅱ collagen in microtissues of both groups were increased at 7 or 14 days.The relative expression levels of each gene in the three-dimensional dynamic group were significantly higher than those in the three-dimensional static group at 14 days(P<0.05).At 21 days,the three-dimensional static group had significantly higher gene expression compared with the three-diomensional dynamic group(P<0.001).(4)The results showed that compared with three-dimensional static culture microtissue,three-dimensional dynamic culture microtissue could achieve higher expression of chondrogen-related genes in a shorter time,showing better cell viability and chondrogenic ability.