目的 觀察糖皮質激素對胎盤組織促腎上腺皮質激素釋放激素（CRH）的分泌水平的影響。 方法 收集2006年1月－3月住院分娩的正常妊娠婦女的胎盤組織與妊娠肝內膽汁淤積癥（ICP）患者胎盤及其血清各10例。分3組進行胎盤組織培養，即正常胎盤組、ICP胎盤組，正常胎盤組織加ICP患者血清組，分別用放射免疫法測定各組加與不加地塞米松胎盤組織培養液中CRH的水平。 結果 正常組與正常胎盤加地塞米松組培養24、48、72、96 h其CRH分泌水平分別為：（74.81 ± 27.92）、（63.71 ± 24.72）、（91.87 ± 41.64）、（98.90 ± 42.52） pg/mL；（66.94 ± 29.62）、（77.39 ± 31.84）、（61.89 ± 33.94）、（75.13 ± 36.98） pg/mL，兩組比較差異有統計學意義（P＞0.05）。ICP組與ICP加地塞米松組培養上清液中CRH水平在24、48、72、96 h其CRH分泌水平分別為：（48.28 ± 16.56）、（60.20 ± 29.97）、（72.92 ± 31.65）、（69.22 ± 29.33）pg/mL；（41.81 ± 25.00）、（57.36 ± 39.75）、（57.72 ± 23.29）、（61.43 ± 20.77）pg/mL, 兩組比較差異有統計學意義（P＞0.05）；正常胎盤加ICP血清培養組與正常胎盤加ICP血清加地塞米松培養組上清液中CRH水平在24、48、72、96 h其CRH分泌水平分別為：（84.9 ± 34.98）、（74.5 ± 29.93）、（71.1 ± 27.26）、（81.0 ± 37.18）pg/mL；（76.29 ± 33.11）、（63.70 ± 24.20）、（64.85 ± 28.39）、（67.65 ± 33.20）pg/mL，兩組比較差異有統計學意義（P＞0.05）。3組加入地塞米松培養的胎盤組織，CRH分泌水平并無明顯改變。 結論 地塞米松不影響體外培養胎盤組織CRH分泌。
Objective To compare the characteristic differences between apoptotic extracellular vesicle (ApoEV) and non-apoptotic cell-derived extracellular vesicle (NCEV) from bone marrow mesenchymal stem cells (BMSCs), and to explore their therapeutic effects on inflammatory macrophages and cisplatin-induced acute kidney injury (AKI) in mice. Methods Staurosporine (STS) was used to induce apoptosis in BMSCs, and the apoptotic model was verified by cell morphology observation, Annexin V/propidium iodide double-staining flow cytometry, and Western blot. NCEV and ApoEV were isolated via differential centrifugation combined with ultracentrifugation, and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. The particle yield and protein yield of extracellular vesicles (EV) per unit donor cell were compared between the two groups. EVs were labeled with DiD, and the uptake of EV by RAW264.7 macrophages was detected using laser scanning confocal microscopy and flow cytometry. Lipopolysaccharide (LPS) was applied to stimulate RAW264.7 cells to establish an in vitro inflammatory cell model. The mRNA expression levels of interleukin 10 (IL-10), arginase 1 (Arg-1), IL-1β, IL-6, IL-18, and tumor necrosis factor α (TNF-α) were determined by real-time fluorescence quantitative PCR. The intracellular reactive oxygen species (ROS) level was measured using 2’, 7’-dichlorodihydrofluorescein diacetate fluorescent probe with laser scanning confocal microscopy and flow cytometry. EVs were labeled with cyanine 7 N-hydroxysuccinimide ester to observe their distribution in major organs and renal tissues of normal mice. A cisplatin-induced AKI mouse model was established, followed by intervention with NCEV or ApoEV. Serum creatinine (Scr), blood urea nitrogen (BUN), renal histopathological injury, kidney injury molecule 1 (Kim-1), cell apoptosis, macrophage infiltration, and DNA damage-related indicators were detected. Based on previously published proteomic data of EV derived from mouse BMSCs, differentially expressed proteins were screened and gene ontology enrichment analysis was performed. Results After STS treatment, BMSCs exhibited typical apoptotic morphology with increased early and late apoptotic rates, alongside upregulated expression of Cleaved Caspase-3 and Bcl-2-associated X protein (Bax). Both NCEV and ApoEV presented spherical or quasi-spherical structures enclosed by a lipid bilayer membrane, with similar particle sizes and Zeta potentials. Bax and tumor susceptibility gene 101 protein were detected in both vesicles, while Cleaved Caspase-3 and Bax were detectable in ApoEV. The particle yield and protein yield of ApoEV were (5.626±0.302)-fold and (2.972±0.124)-fold higher than those of NCEV, respectively (P<0.05). Compared with NCEV, RAW264.7 cells displayed significantly elevated uptake ratio and mean fluorescence intensity for ApoEV (P<0.05). Under LPS stimulation, ApoEV markedly upregulated the mRNA expressions of IL-10 and Arg-1 to levels higher than those in the NCEV treatment group (P<0.05), whereas NCEV significantly suppressed the mRNA expressions of IL-1β and IL-6. Both NCEV and ApoEV reduced intracellular ROS levels, and flow cytometry revealed that the mean 2’, 7’-dichlorofluorescein fluorescence intensity in the ApoEV group was significantly lower than that in the NCEV group (P<0.05). Ex vivo organ imaging demonstrated that the strongest fluorescent signals of both types of EVs appeared in the liver, and obvious signals were also observed in the lung, spleen, and kidney. No significant difference in relative radiation efficiency of each organ was found between the two groups (P>0.05). In cisplatin-induced AKI mice, NCEV significantly reduced serum Scr and BUN levels, while ApoEV significantly decreased serum Scr levels. Both EV preparations alleviated renal tubular histopathological damage and lowered the levels of Kim-1, Bax, TUNEL-positive cells, F4/80-positive macrophages, and serine 139-phosphorylated histone H2AX. No significant intergroup differences in the above renal injury indicators were observed between the two EV intervention groups (P>0.05). Proteomic analysis identified 50 significantly upregulated proteins and 35 significantly downregulated proteins in ApoEV relative to NCEV. The upregulated proteins in ApoEV were mainly enriched in biological processes including extracellular matrix organization, classical complement pathway activation, proteolysis, and angiogenesis. Conclusion ApoEV possess higher production yield, enhanced macrophage uptake capacity and superior anti-inflammatory regulatory activity compared with NCEV. Both ApoEV and NCEV can alleviate renal injury in cisplatin-induced AKI, providing experimental evidence supporting the translational application of ApoEV for AKI treatment.
ObjectiveTo summarize the expression and role of CD146 in mesenchymal stem cells (MSCs).MethodsThe literature related to CD146 at home and abroad were extensively consulted, and the CD146 expression in MSCs and its function were summarized and analyzed.ResultsCD146 is a transmembrane protein that mediates the adhesion of cells to cells and extracellular matrix, and is expressed on the surface of various MSCs. More and more studies have shown that CD146+ MSCs have superior cell properties such as greater proliferation, differentiation, migration, and immune regulation abilities than CD146- or unsorted MSCs, and the application of CD146+ MSCs in the treatment of specific diseases has also achieved better results. CD146 is also involved in mediating a variety of cellular signaling pathways, but whether it plays the same role in MSCs remains to be demonstrated by further experiments.ConclusionThe utilization of CD146+ MSCs for tissue regeneration will be conducive to improving the therapeutic effect of MSCs.
Objective To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.
ObjectiveTo understand the obstacles in the practice of pulmonary rehabilitation between doctors and patients.MethodsMedical staff and patients with chronic obstructive pulmonary disease (COPD) in public hospitals in this region were randomly sampled, and a questionnaire survey was conducted on possible obstacles to the practice of pulmonary rehabilitation.ResultsTotal of 265 medical staff and 120 COPD patients were recruited in this survey. The obstacles of pulmonary rehabilitation practice of medical staff in clinical work are poor cooperation of patients and their families (84.2%), medical staff’s insufficient awareness of pulmonary rehabilitation (82.3%), and lack of practice and guidelines (78.9%), lack of objective conditions such as site, equipment and equipment (75.1%), lack of multidisciplinary teams (74.3%), pulmonary rehabilitation has fallen by the wayside (73.6%) etc. The main obstacles for COPD patients in pulmonary rehabilitation are lack of access to relevant knowledge (52.4%), insufficient knowledge (36.5%), inconvenient transportation, economic problems and other objective conditions (33.3%). After multiple correspondence analysis, there are differences in the degree of correlation between obstacle factors and groups with different characteristics.ConclusionsThere are many factors hindering the development of pulmonary rehabilitation and there are certain differences among different populations, but the lack of understanding of pulmonary rehabilitation between doctors and patients is the primary problem. It is necessary to improve the cognition of both doctors and patients on pulmonary rehabilitation, and then to solve the obstacles in the implementation of pulmonary rehabilitation.
Objective To construct a recombinant adenovirus vector pAdxsi-GFP-NELL1 that co-expressing green fluorescent protein (GFP) and homo sapiens NEL-l ike 1 (NELL1) protein (a protein bly expressed in neural tissue encoding epidermal growth factor l ike domain), to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for further study on osteogenesis of NELL1 protein. Methods From pcDNA3.1-NELL1, NELL1 gene sequence was obtained, then NELL1 gene was subcloned into pShuttle-GFP-CMV (-)TEMP vector which was subsequently digested with enzyme and insterted into pAdxsi vector to package the recombinant adenovirus vector (pAdxsi-GFP-NELL1). After verified by enzyme cutting and gel electrophoresis, pAdxsi-GFPNELL1 was ampl ified in HEK293 cells and purified by CsCl2 gradient purification, titrated using 50% tissue culture infective dose (TCID50) assay. The rat BMSCs were cultured and identified by flow cytometry and directional induction, then were infected with adenoviruses (pAdxsi-GFP-NELL1 and pAdxsi-GFP). NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope. Cell counting kit-8 (CCK-8) was used for investigate the influence of vectors on the prol iferation of rat BMSCs. Results Recombinant adenoviral vector pAdxsi-GFP-NELL1, which encodes a fusion protein of human NELL1, was successfully constructed and ampl ified with titer of 1 × 1011 pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis, osteogenic and adipogenic induction, then were used for adenoviral transfection efficiency and cell toxicity tests. An multipl icity of infection of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transfection with 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP, over 60% BMSCs showed green fluorescent by fluorescence microscopy. Imunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFPNELL1 at 200 pfu/cell, whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The prol iferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstratedno significant difference among BMSCs transfected with pAdxsi-GFP-NELL1, pAdxsi-GFP, and no treatment control at 7 days (P gt; 0.05). Conclusion Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
ObjectiveTo review the research progress regarding the effects of the local microenvironment after meniscal injury and immunomodulatory strategies for meniscal regeneration. MethodsRelevant literature on meniscal regeneration, macrophage polarization, and tissue engineering was retrieved. The mechanisms underlying immune microenvironment imbalance after injury were summarized, and immunomodulatory interventions were analyzed from four perspectives: drugs and bioactive molecules, stem cells and exosomes, biomaterials, and external physical stimuli. ResultsThe highly inflammatory microenvironment dominated by M1 macrophages after injury significantly hinders tissue repair. Current studies mainly promote functional meniscal regeneration through immunomodulatory interventions in four aspects: drugs and bioactive molecules inhibit inflammatory cascades by blocking inflammatory receptors and related signaling pathways; stem cells and exosomes modulate the local immune microenvironment through paracrine effects and enhance the functions of repair-related cells; functionalized biomaterials achieve synergy between immunomodulation and mechanical support through active component delivery, physicochemical property regulation, and structural design; and external physical stimuli, such as acoustic, electrical, and magnetic stimulation, serve as non-invasive auxiliary approaches for inflammation regulation and tissue repair. ConclusionRemodeling the immune microenvironment is critical for functional meniscal regeneration. Smart scaffolds with both mechanical support and immunomodulatory functions should be further developed.
Objective To manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix (DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. Methods PLGA scaffolds were prepared by low temperature deposition 3D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods. DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques. PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test. Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cell-scaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8 (CCK-8) was used to detect cell proliferation. Results General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores; the surface of the DACECM oriented scaffold was rough, which was a 3D structure with loose pores and interconnected; and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds (P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold (P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance (A) value between the groups at each time point (P>0.05). Conclusion The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.
ObjectiveTo manufacture a polycaprolactone (PCL)/type Ⅰ collagen (COL Ⅰ) tissue engineered meniscus scaffold (hereinafter referred to as PCL/COL Ⅰ meniscus scaffold) by three-dimensional (3D) printing with low temperature deposition technique and to study its physicochemical properties.MethodsFirst, the 15% PCL/4% COLⅠ composite solution and 15% PCL simple solution were prepared. Then, 15% PCL/4% COL Ⅰmeniscus scaffold and 15% PCL meniscal scaffold were prepared by using 3D printing with low temperature deposition techniques. The morphology and microstructure of the scaffolds were observed by gross observation and scanning electron microscope. The compression modulus and tensile modulus of the scaffolds were measured by biomechanical test. The components of the scaffolds were analyzed by Fourier transform infrared spectroscopy (FTIR). The contact angle of the scaffold surface was measured. The meniscus cells of rabbits were cultured with the two scaffold extracts and scaffolds, respectively. After cultured, the cell proliferations were detected by cell counting kit 8 (CCK-8), and the normal cultured cells were used as controls. Cell adhesion and growth of scaffold-cell complex were observed by scanning electron microscope.ResultsAccording to the gross and scanning electron microscope observations, two scaffolds had orientated 3D microstructures and pores, but the surface of the PCL/COLⅠ meniscus scaffold was rougher than the PCL meniscus scaffold. Biomechanical analysis showed that the tensile modulus and compression modulus of the PCL/COL Ⅰ meniscus scaffold were not significantly different from those of the PCL meniscus scaffold (P>0.05). FTIR analysis results showed that COL Ⅰ and PCL were successful mixed in PCL/ COL Ⅰ meniscus scaffolds. The contact angle of PCL/COLⅠ meniscus scaffold [(83.19±7.49)°] was significantly lower than that of PCL meniscus scaffold [(111.13±5.70)°] (t=6.638, P=0.000). The results of the CCK-8 assay indicated that with time, the number of cells cultured in two scaffold extracts showed an increasing trend, and there was no significant difference when compared with the control group (P>0.05). Scanning electron microscope observation showed that the cells attached on the PCL/ COL Ⅰ meniscus scaffold more than that on the PCL scaffold.ConclusionPCL/COLⅠmeniscus scaffolds are prepared by 3D printing with low temperature deposition technique, which has excellent physicochemical properties without cytotoxicity. PCL/COLⅠmeniscus scaffold is expected to be used as the material for meniscus tissue engineering.