Ying-zhen Wang

Reconstruct large osteochondral defects of the knee with hIGF-1 gene enhanced Mosaicplasty

OBJECTIVE To investigate a compound technique including gene therapy, injectable tissue engineering and Mosaicplasty to reconstruct large osteochondral defect. METHODS Plasmid vector containing hIGF-1 cDNA was created and transfected into BMSCs in vitro with FuGene6. After gene expression determination, cells were mixed with calcium alginate gel. Osteochondral defects were created on the femoral condyle of goats in a diameter of 6mm. Osteochondral plugs were harvested from the intertrochlea groove and pressed into the recipient sites in a mosaic mode. Gene modified BMSCs-scaffold complex was applied to fill the residual defects. Control groups were also set up. At 4 and 16 weeks, specimens were investigated in gross and under microscopy, electromicroscopy and MRI detection. RESULTS hIGF-I gene was expressed effectively with the peak concentration at 34.75 ng/ml. Subchondral bone and cartilage were integrated well in gene enhanced Mosaicplasty group. The reconstructed tissue filled up the gaps between columns, which appeared better than other groups. The regenerated cartilage was integrated with neighbor tightly in regular arrange. Extracellular matrix distributed evenly and deeply stained by alcian blue. Quantitative histologic assessments showed higher score in gene enhanced Mosaicplasty group. Glycosaminoglycan assay revealed no difference between groups involving Mosaicplasty. MRI analysis demonstrated the healing process between the subchondral bone other than control groups. CONCLUSIONS hIGF-I gene enhanced tissue engineering can modify the outcome of Mosaicplasty to reconstruct large osteochondral defects in weight-bearing region.

Modified biplanar open-wedge high tibial osteotomy with rigid locking plate to treat varus knee

ObjectiveTo introduce and characterize the modified biplanar opening high tibial osteotomy with rigid fixation to treat varus knee in young and active patients.MethodsBetween June 2001 to July 2008, 18 patients with monocompartmental degeneration of the knee combined with a varus malalignment of the leg had the modified biplanar opening high tibial osteotomy and the osteotomy was fixed with the locking plates (Locking Compression Plate System). The mean varus deformity before operation was 11.5° (5°∼19°) and no degenerative changes were found in other departments. Stability of the knee was normal in 15 patients, but ruptures in anterior cruciate ligaments or lateral collateral ligament were presented in the remaining 3 patients. Preoperative symptom was mainly limited in the pain of medial compartment. The preoperative and follow-up data for the range of motion and Lysholm score were determined. Subjective satisfactory examination was also applied to the patients for the operation they selected.ResultsAll of the patients were followed up with an average of 32.5 months (12∼82 months). There was no ununion or delayed union in this group during the follow-up period. No complications like broken plate, nerve injury, or blood vessel injury occurred. The postoperative average corrected degree was 9.5° (5.5°∼18°). No degenerations developed in the three departments of the knee. The Lysholm scores before and after surgery were 42.5 and 77.5, respectively (P<0.01). The overall fineness rate was 83.3%. The subjective satisfactory survey demonstrated that about 83.3% patients showed satisfactory on the operation. There was no obvious difference in the range of motion before and after operation, but significant changes were found in the Lysholm score and varus degree from preoperative to follow-up.ConclusionProximal opening high tibial osteotomy performed in conjunction with the special rigid locking plate yielded good results for symptomatic genu varum. This new classic technique can be effectively applied to the medial compartment degeneration of the knee in active young patients.

In Vivo Tracking of Novel SPIO-Molday ION Rhodamine-B™-Labeled Human Bone Marrow-Derived Mesenchymal Stem Cells After Lentivirus- Mediated COX-2 Silencing: A Preliminary Study

PURPOSE Magnetic resonance imaging (MRI) has been used to track magnetically labeled human bone marrow-derived mesenchymal stem cells (hBMSCs) in vivo after COX-2 silencing and transplantation into nude rats via tail vein injection. METHODS In the present study, we knocked down COX-2 expression in hBMSCs through lentivirus transduction. The COX-2 knockdown was confirmed by real-time PCR and Western blotting analyses. Subsequently, we labeled cells with the novel reagent SPIO-Molday ION Rhodamine-B™ (MIRB). The viability, proliferation and differentiation of these cells were assessed in vitro. Labeled lenti-shCOX2 hBMSCs, unlabeled hBMSCs and phosphate-buffered saline (PBS) were individually injected into the tail veins of nude rat models, forming three treatment groups. All nude rats underwent GRE T2*-weighted MRI at 1 h, 7 days and 14 days post-injection. After MRI examination, the animals were sacrificed, and the brain and liver were examined by fluorescence microscopy and Prussian Blue staining. RESULTS Our results confirmed the successful down-regulation of COX-2 at the mRNA and protein levels in hBMSCs by lentivirus transduction. The viability and differentiation of hBMSCs were not affected by MIRB labeling. After 7 days, hypointense signal void areas in the rat livers were observed on MRI. After 14 days, iron particles were detected in the blood vessels, sinusoids, interlobular septum and capsule tissues of the liver. CONCLUSION The MIRB-labeled lenti-shCOX2 hBMSCs transplanted into nude rat models via tail vein injection can be detected and monitored in vivo using 3.0 T clinical MRI for up to 14 days after cell transplantation.

Outbreak of Serratia marcescens postoperative infection traced to barbers and razors

BACKGROUND Fourteen postoperative infections caused by Serratia marcescens were detected in patients on the neurosurgical wards and spinal surgery ward of a 2640-bed hospital between 26th December 2012 and 5th June 2013. AIM To investigate the source of the outbreak, identify risk factors and implement infection control measures. METHODS Cultures were collected from healthcare workers and potential environmental sources. S. marcescens isolates were characterized by antibiotic susceptibility testing and pulsed-field gel electrophoresis (PFGE). A retrospective case-control study was performed to identify the risk factors. FINDINGS The outbreak involved 14 patients, five of whom required more than one surgical procedure. S. marcescens was isolated from cerebrospinal fluid, brain tissue, sputum and other secretions. S. marcescens was also cultured from samples taken from the hands of two barbers and their razors. Exposure to the two barbers [odds ratio (OR) 78.0, P < 0.0001] and wound drainage (OR 4.889, P = 0.028) were risk factors. Pre-operative shaving by the barbers was the only independent risk factor (OR 78.0, P < 0.0001). Isolates of S. marcescens from patients, barbers and razors were indistinguishable by PFGE and antibiotic susceptibility pattern. The outbreak ended after removal of the implicated barbers, extensive re-inforcement of infection control procedures and re-education. CONCLUSION These results underscore the risk of postoperative infection associated with pre-operative wet shaving.

Comparison of hIGF-1 Gene Transfection to the hBMSCs and Human Meniscal Fibrochondrocytes

Background Treatment strategies for meniscal injury are shifting from meniscectomy to repair, especially cell-based therapy. Delivering selected genes to donor cells can modify differentiation and proliferation. Efficiency of gene transfection and expression may relate to cell type. Material/Methods Full-length hIGF-1 cDNA was cloned into eukaryotic expression vector by PCR. Human BMSCs and meniscal fibrochondrocytes were isolated and cultured in vitro and hIGF-1 gene was transfected by FuGene 6. Expression of EGFP and hIGF-1 were determined. Biological activity of the hIGF-1 in medium was assessed by MTT chromatometry. Real-time quantitative PCR and Western blot were used to assess the expression of exogenous genes. Efficacy of gene transfection was detected by immunohistochemistry staining and flow cytometry. Results Sequences of hIGF-1 were verified by sequence analysis. Expression of EGFP increased gradually and reached peak intensity 48 h after transfection. Transfection efficiency of BMSCs was higher than meniscal fibrochondrocytes. The population doubling time was decreased in both cell types. Peak concentration of hIGF-1 in the medium of BMSCs and meniscal cells was 32.5±4.8 ng/ml and 24.5±4.6 ng/ml, respectively. Secreted hIGF-1 possessed the ability to enhance proliferation of the cell line. Results of qPCR and Western blot confirmed the expression of hIGF-1. Type II collagen appeared within the cells, and percentage of cells in S stage was increased in both cell types after transfection. Conclusions hIGF-1 cDNA can be transfected into BMSCs and meniscal fibrochondrocytes, resulting in gene expression. Expression efficiency in BMSCs was higher than that in fibrochondrocytes.

Micrometer-Sized Titanium Particles Induce Aseptic Loosening in Rabbit Knee

Wear debris induced aseptic loosening is the leading cause of total knee arthroplasty (TKA) failure. The complex mechanism of aseptic loosening has been a major issue for introducing effective prevention and treatment methods, so a simplified yet efficient rabbit model was established to address this concern with the use of micrometer-sized titanium particles. 20 New Zealand white rabbits were selected and divided into two groups (control = 10, study = 10). A TKA surgery was then performed for each of them, with implantation of a titanium rod prosthesis which was coated evenly with micrometer-sized titanium in the study group and nothing in the control group, into right femoral medullary cavity. After 12 weeks, all the animals were euthanized and X-ray analyses, H&E staining, Goldner Masson trichrome staining, Von Kossa staining, PCR, and Western blotting of some specific mRNAs and proteins in the interface membrane tissues around the prosthesis were carried out. The implantation of a titanium rod prosthesis coated with 20 μm titanium particles into the femoral medullary cavity of rabbits caused continuous titanium particle stimulation around the prosthesis, effectively inducing osteolysis and aseptic loosening. Titanium particle-induced macrophages produce multiple inflammatory factors able to activate osteoclast differentiation through the OPG/RANKL/RANK signaling pathway, resulting in osteolysis while suppressing the function of osteoblasts and reducing bone ingrowth around the prosthesis. This model simulated the implantation and loosening process of an artificial prosthesis, which is an ideal etiological model to study the aseptic prosthetic loosening.

Effects of human cyclooxygenase-2 gene silencing on synovial cells of rheumatoid arthritis mediated by lentivirus

Abstract The aim of the study is to screen the effective shRNA sequence which can silence the human COX-2 expression level in synovial cells of rheumatoid arthritis (RA) patient transfected by the lentivirus. Four pairs of hCOX-2 shRNA were designed and inserted into lentivirus to form pGPHI/GFP/Neo-shRNA vector. The reconstructed virus was transfected into synovial cells derived from RA patients, and then the expression level of hCOX-2 mRNA and the protein of the inflammatory factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), interleukin-1β (IL-1β) and tumour necrosis factor alpha (TNF-α) in the supernatants were examined with real-time PCR and ELISA, respectively. There was no obvious negative influence on cell growth and morphology after hCOX-2 shRNA gene transfection mediated by lentivirus. The hCOX-2 mRNA expression level, as well as the concentration of PGE2, VEGF, IL-1β and TNF-α, decreased significantly (p < .05). RNAi mediated by lentivirus can significantly inhibit hCOX-2 mRNA expression level in synovial cells of RA patients, so as to reduce the expression of inflammatory cytokines.

A Novel Method for Pathway Identification Based on Attractor and Crosstalk in Polyarticular Juvenile Idiopathic Arthritis.

Background Juvenile idiopathic arthritis (JIA) is one of the most common inflammatory disorders of unknown etiology. We introduced a novel method to identify dysregulated pathways associated with polyarticular JIA (pJIA). Material/Methods Gene expression profiling of 61 children with pJIA and 59 healthy controls were collected from E-GEOD-13849; 300 pathways were obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG) database and 787,896 protein-protein interaction sets were gathered from the Retrieval of Interacting Genes. Attractor and crosstalk were designed to complement each other to increase the integrity of pathways assessment. Then, impact factor was used to assess the interactions inter-pathways, and RP-value was used to evaluate the comprehensive influential ability of attractors. Results There were seven attractors with p<0.01 and 14 pathways with RP<0.01. Finally, two significantly dysfunctional pathways were found, which were related to pJIA progression: p53 signaling pathway (KEGG ID: 04115) and non-alcoholic fatty liver disease (NAFLD) (KEGG ID: 04932). Conclusions A novel approach that identified the dysregulated pathways in pJIA was constructed based on attractor and crosstalk. The new process is expected to be efficient in the upcoming era of medicine.