ObjectiveTo construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity. MethodsThe bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG). ResultsBright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05). ConclusionA transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.
ObjectiveTo analyze the reason and prevention of late bioprosthetic heart valve thrombosis (LBVT). MethodsBioprosthetic heart valves were implanted in 580 patients between January 2001 and July 2013 in Changhai Hospital, and only found one case of LBVT (0.2%). Reoperation was performed for a 67-year-old male patient 3 years after bioprosthetic aortic valve replacement due to severe aortic valve stenosis. Retrospectively analyzed the clinical data and reviewed the literature between January 1989 and December 2013 in Pubmed. ResultsNo risk factor for thrombosis was revealed in this patient. Pathology revealed valve thrombosis and collagen and elastic fibers fragmentation and disruption in valve leaflets. In literature review, 20 articles and 47 cases were brought in. The morbidity of LBVT was 0.2%-1.0%. At least 18 patients had high risk factors, and 15 of the 18 patients discontinued anticoagulation after 3 months. However, at least 10 patients had no high risk factors. Forty-four of the 47 patients received stented porcine valve replacement, only one patient received stented bovine pericardial valve replacement. ConclusionsLBVT is a rare but serious complication after bioprosthetic valve replacement, the causes of which include the feature of the patients and the bioprosthetic valves. Bovine pericardial valves could be superior to porcine valves in preventing LBVT. Postoperative long-time aspirin therapy is recommended for patients without high risk factors. Patients with any high risk factors should prolong anticoagulation.