Objective Allograft vasculopathy (AV), feature of chronic rejection, is a major serious long-term post-operation complication in organ transplantation. The accurate mechanisms for AV have not been definitively established, but extensive basic and clinical studies demonstrate AV is triggered by immune reaction and nonimmunologic factors, and also possibly attributed to the metabolism of branched-chain amino acids (BCAA). Methods The transplanted hearts from Lewis to Sprague-Dawely rats served as allografts and those from Lewis to Lewis rats as isografts based on Ono 's model. The differential proteins in transplanted hearts were separated by comparative proteomic technique, and some enzymes which regulated the metabolism of BCAA were identified and validated.Results All transplanted hearts at second week postoperation were characterized by lumen loss (total area-luminal area/total area) in coronary artery, but more predominant at 8th week. All samples from the left ventricles were analyzed by proteomic techniques and the subunits E1 a, E1β and E3 of branched-chain α-ketoacid dehydrogenase (BCKDH) complex were decreased in the heart allografts.Immunohistological detection also showed the expression of BCKDH was reduced not only in the cardiac muscle but also more significantly in blool vessels with cardiac allograft vasculopathy (CAV).BCAA concentrations were increased in the cardiac allografts, but there was no difference in the serum. Conclusion These findings suggest that the catabolic pathways of the BCAA may be inhibited owing to the reduced expression of BCKDH complex, and elevated intracellular concentrations of leucine. The vascular smooth muscle cell and cardiac muscle cell proliferation is stimulated via mTOR-dependent and mTOR-independent pathways, which is associated with the formation of myocardial hypertrophy and AV in the heart allografts.

BACKGROUND:Porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared previously is too thick and uneven in holes.
OBJECTIVE:To prepare the thin even porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membrane, and to evaluate the cytocompatibility and differentiation capacity.
METHODS:Porous and nonporous, thin and even poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes were prepared by phase separation method. Its thickness and weight loss rate were determined. Human bone marrow mesenchymal stem cel s were cocultured with porous and nonporous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes for 7 days. Ultrastructure of composite membranes was observed under the scanning electron microscopy. Surface markers of the bone marrow mesenchymal stem cel s on the composite membranes were analyzed using flow cytometry.
RESULTS AND CONCLUSION:The thickness of the porous and nonporous composite membranes was (0.041 ± 0.005) mm and (0.058±0.004) mm. Weight loss rates of porous and nonporous composite membranes were respectively 19.93%and 7.64%at 24 hours. Calcium metaphosphate particles were evenly distributed in porous and nonporous composite membrane. Cel s spread entirely, showing spindle shape. Calcium metaphosphate particles were evenly distributed in porous composite membrane. Pore in porous composite membranes was also uniformly distributed, and pore size was about 2-8μm. Cel s spread entirely, showing polygonal shape with multiple tentacles. The tentacles of some cel s entered into the scaffold. CD105, CD90, CD44, CD29 and CD73 expression was detected in porous and nonporous composite membranes. There was no significant difference in cel-positive rate. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared in this study has good biocompatibility and could not promote cel differentiation.

Objective To observe the pathophysiological change of right heart remodeling model of beagle dogs caused by tricuspid regurgitation. Methods Six beagle dogs were separated to TR group and sham operation group randomly and equally. The anterior and posterior leaflets of tricuspid valve of beagle dogs from TR group were cut off under direct vision on the beating heart. The echocardiography data were measured pre-operation and post-operation 3, 6, 12 months respectively and autopsy results were acquired after the sacrifice of two animals who had been raised for 18 months. Results The operation was successful and survival 100%. The weight of TR group increased, and the echocardiography showed right atrium and ventricle enlargement and right heart dysfunction. The autopsy of TR group found myocardial fibrosis , liver cirrhosis and ascites. All of the results indicated the occurrence of right heart remodeling and right heart failure. There is no significant difference in sham operation group before or after operation. Conclusion The myocardial fibrosis is one of the pathological basis of right heart remodeling.

[ ABSTRACT] AIM:To investigate the role of encapsulated protein transfected into human bone marrow mesen-chymal stem cells ( hBMSCs) by polyethyleneimine ( PEI) , and to optimize the best mole ratio of PEI-proteins.METH-ODS:6 groups of DNase I-PEI complexes were constructed and the best mole ratio was explored by laser scattering analy-sis.The appearance of complexes was presented under transmission electron microscope.Meanwhile, 4 groups of construc-ted GFP-PEI complexes were utilized to transfect into the hBMSCs, which were isolated and expand in vitro.The fluores-cence intensity of transfected cells was observed under confocal microscope.In addition, the cytotoxicity of the complexes on the cell proliferation was detected by MTT assay.The activity of the intracellular proteins was testified by aβ-galactosi-dase staining experiment.RESULTS:When the mole ratio of PEI and protein was adjusted to 4∶1, the complex transfec-tion efficiency was the best, and β-galactosidase color test turned blue.CONCLUSION:PEI has the character of encap-sulating various proteins to nano-complexes.The proteins transfected into bone marrow mesenchymal stem cells are con-firmed to have functional activity.As a protein carrier, PEI is of high efficiency and low toxicity, thus providing a new way for stem cell reprogramming.

Objective To explore the application of optical coherence tomography in vascular tissue engineering culture by dynamic monitoring its changes.Methods Human umbilical artery smooth muscle cells were isolated and culture by tissue block method.After passage culture and cell surface markers evaluation, smooth muscle cells were seeded onto polyglycolic acid scaffold and placed into the bioreactor based on Luo-Ye pump with pulsatile stress for three-dimensional culture.At 1、4、 7 、10、14、17、21 days in culture, the image data was obtained by optical coherence tomography technology.The ability of imaging TEBV via OCT was analyzed combined with histopathological observation.Results As the incubation time extended,OCT clearly showed PGA gradual degradation, decreased composite scaffold thickness and the wall structure from loose to tight.At 21 days in culture, the vessel mimics had smooth surface with extracellular matrix evenly distributed and achieved complete reconstruction in the PGA scaffold.Combining with histopathological staining, the blood vessel mimics were similar to natural blood vessels.OCT measured TEBV thickness compared with histopathological measurement had good correlation (r =0.922,P ＜ 0.05).Conclusion Optical coherence tomography could clearly image microstructures of tissue engineered blood vessels cultured in three-dimensional culture system based on Luo-Ye pump, delineate the reconstruction of TEBV-like tissue in the bioreactor and provide as a dynamic and convenient monitoring tool in vascular tissue engineering.

AIM: To screen the cytotoxicity of degradable calcium metaphosphate (dCMP) compared with hydroxyapatite (HA). The proliferation and differentiation abilities of human marrow mesenchymal stem cells (MSC) were used to exhibit the cytotoxicity. METHODS: The cell morphology of MSC was analysed after direct contact with dCMP at different time points by scanning electron microscopy analysis. The degradation products of dCMP and HA were analysed with inductively coupled plasma torch and ion chromatography. The cytotoxic effect of degradation products of dCMP was evaluated by FACS, quantitative assay of ALP and ARS, respectively. RESULTS: dCMP enhanced the proliferation of MSC, but didn't interfere the osteogenic differentiation process of MSC and its mineralization. HA inhibited the proliferation of MSC and the mineralization of osteogenic differentiated MSC, while it did not interfere the osteogenic differentiation process of MSC. CONCLUSION: dCMP had a better cytocompatibility with MSC than HA, which might allow for its use as skeleton scaffolds.

Aim To explore the anti-apoptotic function of cardiac progenitor cells(CPCs)-derived exosome in vitro.Method CPCs were isolated from mouse heart using Magnetic Cell Sorting(MACS)system.Flow Cy-tometry(FC)determine the purity of stem cell surface antigen-1 positive(Sca-1 +)CPCs.Exosome was puri-fied from conditional medium,and confirmed by West-ern blot using CD63 as a marker,Nanoparticle Traffic-king Analysis(NTA)was used to detect the diameters and concentration of exosome.Then the cells were di-vided into control groups and CPC-exosome pre-protec-tion groups.H2 O2 was added into H9c2 cells to induce oxidative stress.Western blot was adopted to determine the expression of cleaved caspase-3.Results ① Im-munofluorescence showed that CPCs isolated by MACS were positively expressing Sca-1 protein;FC analysis showed that typical purity of Sca-1 +CPCs from the first
preparations was more than 95%.② WB demonstrated that CD63 of exosome isolated from CCMwas positively expressed,and NTA results showed that the diameters of exosome were (82.33 ±3.06)nm(n =3).Micro-scope detected PKH-26 labeled exosome appeared in the cytoplasma of H9c2 cells.③ Western blot showed the CPC-exosome pre-protection groups significantly down-regulated the levels of cleaved caspase-3 com-pared to the control groups(P <0.05).Conclusion CPC can secrete exosome which carries many important cargos,which can effectively gather in H9c2 cells. CPC-exosome can protect H9c2 cells from the oxidative stress induced by H2 O2 .Our results highlight a new perspective strategy for cardiac disease.

BACKGROUND:The mechanical valves used in the adult Luo-Ye pump have a large size and great destruction to blood, which are not suitable for infant ventricular assist pump. Therefore, designing and producing a high molecular valve with smal size and low incidence of thrombosis is a research hotspot. OBJECTIVE:To design and produce a valve of 20 mL infant Luo-Ye pump, and to test its basic functions and fatigue properties. METHODS:The size and shape of valve was designed with MASTERCAM software, polyurethane valve was obtained through producing the valve model and plastic injection;the static leakage, pressure drop and fatigue resistance of polyurethane valve were tested according to the ISO5840 requirements. RESULTS AND CONCLUSION:The polyurethane trefoil valve was produced, but the failure rate of plastic injection was high;the basic function of the trefoil valve met the ISO5840 requirements bascial y;after continuously operated 1.0×107 times, stroke volume of 20 mL Luo-Ye pump was changed 5.2%, and two polyurethane valves and valve leaflets did not change and damage. Polyurethane trefoil valve was designed and produced successful y;polyurethane valves could meet the needs of 20 mL Luo-Ye pump, which already have the ability to clinical trials.

BACKGROUND:Calcium metaphosphate has excel ent biocompatibility, degradability, and cel affinity. Human bone marrow mesenchymal stem cel s can grow and proliferate in the pores of the porous calcium metaphosphate, but less is known about calcium metaphosphate nanoparticles.
OBJECTIVE:To prepare calcium metaphosphate nanoparticles, and to analyze the effect of calcium metaphosphate nanoparticles at different concentrations on apoptosis of human bone marrow mesenchymal stem cel s by flow cytometry.
METHODS:The calcium metaphosphate nanoparticles were prepared by wet bal mil ing. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the calcium metaphosphate nanoparticles, and the crystal structure of nanoparticles was analyzed by X-ray diffraction. Calcium metaphosphate nanoparticles were mixed in the CYAGON Oricel TM basal medium, and the concentrations of calcium metaphosphate nanoparticles in the medium were 10, 1, 0.1 mg/L. Human bone marrow mesenchymal stem cel s were cultured for 7 days in the above-mentioned media, and apoptosis of human bone marrow mesenchymal stem cel s was analyzed by flow cytometry.
RESULTS AND CONCLUSION:Calcium metaphosphate nanoparticles were successful y prepared by wet bal mil ing, irregular in shape, and the mean diameter was 10-30 nm. X-ray diffraction results showed the crystal structure of nonaparticles was mainlyβ-Ca(PO3)2. The cel ratio of G0/G1 phase and G2/M phase in 10 mg/L group was obviously higher than that in 1, 0.1 mg/L groups (P<0.01). The cel apoptosis rates during the early, middle, late stages in 10 mg/L group were obviously higher than those in 1, 0.1 mg/L groups (P<0.01), and the total cel apoptosis was also significantly increased in 10 mg/L group (P<0.01). These findings indicate that human bone marrow mesenchymal stem cel s proliferation can be inhibited by calcium metaphosphate nanoparticles, and apoptosis rate is increased significantly when the concentration of calcium metaphosphate nanoparticles increases from 1 mg/L to 10 mg/L.

Aim To investigate whether human umbili-cal cord mesenchymal stem cells(hUC-MSCs)exposed to inflammatory conditions could release large amounts of exosomes to induce regulatory T cells(Treg).Meth-ods hUC-MSCs were isolated by enzyme digestion method.(In vitro)interferonγ(IFN-γ)was added in-to hUC-MSCs to mimic inflammatory microenviron-ments,then exosomes were extracted from the superna-tant of normal conditional medium or IFN-γpretreated hUC-MSCs.Both sources of exosomes,Nor-hUC-exo and IFN-γ-stimulated hUC-exo, were identified by Nanoparticle Trafficking Analysis (NTA )and Western blot for the exosome-enriched protein CD63 .Next,hu-man peripheral blood mononuclear cells (PBMCs ) stimulated with PHA were respectively co-cultured with hUC-MSCs,IFN-γ-pretreated-hUC-MSCs,hUC-MSCs exosomes or IFN-γ-stimulated-hUC-MSCs exosomes for 5 days to assess the exosomes-T cells communication. The proliferation rate of PBMCs and frequency of CD4 +/CD25 +/Foxp3 + Treg were measured by flow cytometry.Results The isolated cells from human um-bilical cord tissue,which were positive for CD73, CD44,CD29,CD90 and HLA-ABC,but were nega-tive for CD31 and CD34,were mesenchymal stem cells indeed.After IFN-γtreatment,hUC-MSCs secreted nu-merous exosomes(P<0.05 ).Morerover,there was a significantly higher level of CD63 ,but no difference in diameter between Nor-hUC-exo and IFN-γ-stimulated hUC-exo.IFN-γ-stimulated hUC-exo had a superior a-bility compared with Nor-hUC-exo to suppress the pro-liferation of PHA stimulated PBMCs due to their upreg-ulation of the percentage of Treg (1 1.53 ±0.88% vs 6.60 ±0.56%,P <0.01 ).Conclusion hUC-MSCs could promote the expression of Treg to modulate im-munosuppression through exosomes,especially for IFN-γ-licenced exosomes,which might carry much immu-notherapeutic potential.