Ping Leng

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.

Repair of Large Osteochondral Defects With Mix-Mosaicplasty in a Goat Model

Osteochondral defects in weight-bearing regions must be repaired with cartilage and subchondral bone support simultaneously, as well as the integration between the 2, particularly in young, active patients. In this study, a new method called mix-mosaicplasty was used to reconstruct large osteochondral defects (6-mm diameter) in the weight-bearing region of the femoral condyle of goats. Two periosteum-bone plugs and 1 osteochondral plug harvested from the proximal tibia and intertrochlea groove were assembled to fill the defects in a mosaic mode. The goats were euthanized 16 weeks postoperatively, and the result of the repair process was assessed using macroscopy, morphologic analysis, electron microscope observation, glycosaminoglycan assay, and magnetic resonance imaging. Sixteen weeks postoperatively, the superficial surface of the defective region was covered with regenerated cartilage, and the periosteum-bone plugs were combined with each other. However, cleavage between cartilage plugs was noted. The donor site, which was filled with periosteum-bone plugs, was regenerated with fibrocartilage-like tissue. The repaired tissue was composed of small chondrocyte-like cells arranged tightly within an evenly distributed extracellular matrix containing type II collagen. Cells of the regenerated tissue in periosteum-bone plugs were smaller and distributed more densely. Electron microscopy demonstrated regular matrix fibers and abundant organelles within the repaired tissue. No significant differences of glycosaminoglycan content were observed between reconstructed tissue and normal hyaline cartilage. Magnetic resonance imaging revealed the healing process between plugs other than the control group. The new technique of mix-mosaicplasty can reconstruct full-thickness osteochondral compound defects in the weight-bearing region of the femoral condyle.

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.

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.

Simultaneous determination of AM80 (tamibarotene) and WJD-A-1 in rat plasma by ultra high-performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Abstract A compound (WJD-A-1) was previously reported as a candidate prodrug of Am80 (tamibarotene), which was approved in Japan in 2005 as a therapeutic agent for recurrent refractory acute promyelocytic leukaemia. A rapid, selective and sensitive ultra high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the first time to simultaneously determine WJD-A-1 and its major phase-I metabolites AM80 in rat plasma. After a simple sample preparation procedure by protein precipitation with methanol and acetonitrile, WJD-A-1, AM80 and the internal standard were chromatographed on an ACQUITY UPLCTM BEH C18 column. The mobile phase consisted of methanol–0.1% formic acid (80:20, v/v) and the flow rate was 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 2.6 min. The linear calibration curves for WJD-A-1 and the AM80 were obtained in the concentration range of 5.40–5.40 × 103 and 5.08–5.08 × 103 ng/mL, respectively (r ≥ 0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 8% and the accuracy (relative error, RE) was within ±6.8%, determined from quality control (QC) samples for the analytes. The method herein described was fully validated and successfully applied to pharmacokinetic study of WJD-A-1 following an intravenous administration of 300 μg/kg WJD-A-1 to rats.