| ObjectiveBenign airway stenosis is a life-threatening respiratory disease.In recent years,with the development of critical care medicine,respiratory support technology and the popularization of lung transplantation as well as proximal airway surgery,the incidence of airway stenosis has gradually increased.At present,the treatment and efficacy of benign airway stenosis are limited.The the main means of treatment include:surgical resection of the stenosis segment with end-to-end anastomosis,balloon dilatation,cryotherapy,heat techniques?Nd:YAG or CO2 laser photocoagulation treatment,electrocautery,argon plasma coagulation?,stenting and local/systemic steroid use.Surgical resection and reconstruction are considered the gold standard of therapy for post-intubation tracheal stenosis?PITS?but cannot be performed in all patients owing to the length of narrow segment and patients'cardiopulmonary function.Bronchoscopic methods have high restenosis rate and patients will need regular bronchoscopy follow-up.Acquired benign central airway stenosis shares at least some common pathological processes and overlapping phases of wound repair irrespective of the etiology.The pathogenesis of benign airway stenosis is mainly caused by airway mucosal injury and abnormal repair dominated by fibroblasts.The disease usually starts with large areas of mucosal injury?related to infection or not?,which generates edema,hemorrhage,vascular congestion with recruitment of cells and mediators,necrosis of airway wall with various degrees of inflammatory reaction,ulceration and concurrent infection.Granulation tissue then deposited from fibroblast recruitment and proliferation,inflammatory cell infiltration,and neovascularization.The final stage is airway remodeling,which include collagen deposition,scar formation and contracture with fibroblast turning into a contractive phenotype noted as myofibroblast.Migration from adjacent connective tissue,epithelial-mesenchymal transition?EMT?and peripheral blood circulating fibrocytes serve as the main sources of fibroblasts in injury repair proces.We were eager to know whether granulation tissue hyperplasia could be alleviated or averted if mucosal repair and inflammation reduction were promoted from the onset of the disease.Mesenchymal stem cells?MSCs?were considered as a perfect choice of treatment by us owing to its remarkable performance in prompting tissue repair,as well as its widely acknowledged anti-inflammation effect.MSCs are a type of adult stem cells derived from the early developmental mesoderm and have a high self-renewal and multi-directional differentiation potential.There are multiple mechanisms by which MSCs treat disease:differentiation,paracrine,exosomes,and mitochondrial metastases.Bone marrow is one of the important sources of MSCs.Our group has accumulated rich experience in the research of bone marrow-derived mesenchymal stem cells?BMMSCs?.In order to study the potential of BMMSCs in the treatment of benign airway stenosis,we established a Sprague Dawley rat model of nylon brush scraping benign tracheal stenosis and applied BMMSCs as local treatment.Treatment feasibility and possible mechanisms were also explored.MethodsFirstly,the primary culture of SD rat BMMSCs was performed by whole bone marrow adherence method.The surface markers of BMMSCs were identified by flow cytometry,and induced differentiation?osteogenesis,adipogenesis?was performed.Primary culture of tracheal-bronchial epithelial cells?TBECs?was performed by cold digestion with pronase,and cell purity was identified.After cell identification,BMMSCs and TBECs co-culture system were established by the application of transwell chamber.The effect of BMMSCs on epithelial repair was explored in vitro.All experiments were repeated with human-derived BMMSCs and normal human-derived bronchial epithelial cells?NHBE?.Secondly,220g-300g male SD rats were utilized as research objects.We established a SD rat model of benign tracheal stenosis by nylon brush scraping induced mechanical damage of the airway mucosa.Symptoms and signs of the rats after modeling were observed,survival condition were compared,and pathological changes of tracheal tissue at different time points after modeling were explored by HE staining and Masson staining.Thirdly,BMMSCs were applied locally after modeling,and the feasibility of BMMSCs in the treatment of benign airway stenosis were explored in vivo.SD rats were divided into three groups:sham operation group,stenosis model group?mechanical injury by nylon brush scraping?and BMMSCs treatment group?mechanical injury+BMMSCs topical treatment?.Tracheal tissues from naturally dead animals were collected immediately,and the remaining surviving animals were uniformly sacrificed on the 22nd day after modeling.The tracheal tissue were collected and fixed in 4%phosphate buffered paraformaldehyde.The pathological changes were observed with HE staining,the lumen sizes were measured and the stenosis index was calculated.Mechanical injury and topical BMMSCs treatment were performed in more SD rats to explore the epithelial repair circumstance at different time points after modeling and treatment by HE staining and PAS staining.In the last part,the mechanism of BMMSCs in promoting epithelial repair and preventing granulation tissue proliferation were explored.We labeled BMMSCs with fluorescent dye CFSE before treatment,rats were sacrificed on the 4th day after treatment and tracheas were dissected for frozen section.Immunofluorescent staining of pan-cytokeratin was performed to observe whether BMMSCs could differentiate into epithelium directly.SD rats were then divided into 3 groups:sham operation group,stenosis model group?mechanical injury by nylon brush scraping?and BMMSCs treatment group?mechanical injury+BMMSCs topical treatment?.On the 4th day,fresh peripheral blood and tracheal tissues were collected.Tracheal tissues underwent RNA extraction,reverse transcriptase polymerase chain reaction?RT-PCR?,and inflammatory factors expression levels were measured by real-time polymerase chain reaction?qPCR?.The percentage of circulating fibrocytes in peripheral blood of different groups was also detected by flow cytometry since fibroblasts is a main component of granulation tissue and circulating fibrocytes serve as an important source of fibroblasts.Results1.Rat-derived cells:After scratching,the cells were cultured in serum-free DMEM medium.The average migration rate of epithelial cells in the control group was?19.82±5.43??m/hr,and that in the BMMSCs co-culture group was?59.42±2.976??m/hr.The differences had statistical significance?p=0.0002?.Human-derived cells:For cells cultured with serum-free DMEM medium after scratch,healing speed of the control group was?37.91±2.79??m/hr,and was?67.60±8.51??m/hr for BMMSCs co-culture group?p<0.0001?.For cells cultured with epithelial cells complete culture medium after scratch,the average migration rate in the control group was?83.92±4.91??m/hr,and that in BMMSCs co-culture group was?124.2±5.55??m/hr?p<0.0001?.The co-culture system-scratch experiments confirmed that BMMSCs accelerate the migration/repair speed of epithelial cell.2.The SD rat model of nylon brush scraping benign tracheal stenosis was successfully established.The mechanical injury by nylon brush scraping caused serious damage of the airway epithelium,which lead to significant proliferation of granulation tissue and tracheal stenosis.On the 8th day of modeling,the survival rate in stenosis model group was 0%,and the tracheas were almost completely occluded by the granulation tissue,while the control group had no death nor occlusion.The tracheal tissues from 2,4,6,and 8 days after modeling showed pathological changes for different stages,which was similar to the tissue damage repair process reported in previous literatures.3.The stenosis index of the sham operation group was?5.55±2.71?%,that of stenosis model group was?55.87±8.76?%,and that of the BMMSCs treatment group was?11.12±4.80?%.The difference between the sham operation group and stenosis model group had statistical significance?p<0.0001?.There was no significant difference between sham operation group and BMMSCs treatment group.The difference between the stenosis model group and BMMSCs treatment group was statistically significant?p<0.0001?.The 21-day survival rate was 100%for sham operation group,0%for stenosis model group and 80%for BMMSCs treatment group.Topical application of BMMSCs after injury accelerated epithelial repair,ameliorated granulation formation,alleviated airway obstruction,greatly reduced stenosis index and improved survival rate.4.The BMMSCS?CFSE green fluorescent labeled?did not coincide with the repaired epithelium?pankeratin-stained red fluorescence?signal,suggesting that BMMSCs promoted epithelial repair without directly differentiating into epithelial cells.In sham operation group,the circulating fibrocytes from the peripheral blood were?0.85±0.34?%,and the percentage of circulating fibrocytes increased to?4.92±1.74?%after nylon brush scraping in stenosis model group.The circulating fibrocytes in the BMMSCs treatment group were lower than stenosis model group,which was?2.04±0.68?%.There were statistic significant differences between sham operation group and stenosis model group?p=0.002?,BMMSCs treatment group and stenosis model group?p=0.0037?,and there was no statistic significant difference between BMMSCs treatment group and sham operation group?p=0.2385?.qPCR results indicated that transforming growth factor?1,interleukin-1,type III Collagen,and tumor necrosis factor-?were increased on the fourth day after modeling,and significantly decreased with BMMSCs treatment.Conclusion:Airway mechanical injury by nylon brush scraping were applied in a rat model to imitate the initial pathological process of stenosis in vivo and allogeneic BMMSCs were utilized instantly as topical treatment.The capability of BMMSCs in promoting epithelial recovery and granulation amelioration were evaluated,possible therapeutic mechanisms were also investigated.Based on our data,we suggest BMMSCs as a novel and promising option or adjuvant therapy in the management of benign central airway stenosis.Besides,this simple and repeatable animal model enables further research on airway stenosis concerning pathological mechanism and therapeutic efficiency. |
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