Effect of the human insulin-like growth factor 1 gene transfection to human umbilical cord blood mesenchymal stem cells

To observe the effect of transfecting the gene human insulin-like growth factor [hIGF]-1 into human umbilical cord blood mesenchymal stem cells [hUCB-MSCs] via non-viral vector. This study was performed in the Affiliated Hospital of Qingdao University, Qingdao, China from June 2012 to May 2013. Twelve hUCB samples were harvested, and isolated in lymphocyte separation medium, and then cultured. Surface antigen expression in MSCs was detected by flow cytometry. Recombinant plasmid pIRES2-enhanced green fluorescent protein [EGFP]-hIGF-1 was transfected into MSCs by X-treme GENE HP DNA transfection reagent. Then, EGFP was observed with reverse fluorescent microscope at different time points. Enzyme-linked immunosorbent assay was used to determine the hIGF-1 protein concentration in supernatants. Immunofluorescence microscopy and reverse transcription polymerase chain reaction were used to detect the expression of hIGF-1 in the hUCB-MSCs. Expression of type II collagen was detected by immunohistochemistry staining. Transfection efficiency was 28.74 +/- 7.31%. The cluster of differentiation [CD]90, CD105, and CD146 expression increased CD34, CD45, and anti-HLA-DR expression decreased. Results of immunofluorescence microscopy and RT-PCR confirmed expression of the hIGF-1 gene. The hIGF-1 protein concentration in the supernatants showed a peak level at 34.63 +/- 1.61 ng/ml 48 hours after transfection. Immunohistochemical analysis of transfected hUCB-MSCs proved that type II collagen could be expressed positively. Human IGF-1 gene can be transfected into hUCB-MSCs, and expressed at a high level with subsequent expression of type II collagen

The efficacy of open-wedge high tibial osteotomy for varus knee / 中华外科杂志

<p><b>OBJECTIVE</b>To introduce the biplanar opening high tibial osteotomy with rigid fixation for adult varus knee.</p><p><b>METHODS</b>Twelve patients with varus knee and degeneration of medial compartment received the biplanar opening high tibial osteotomy between June 2001 and July 2008. The pre-operative deformity was about 11.5 degrees without osteoarthritis changes in other compartments. The average range of motion was more than 90 degrees . One of the patients had ruptures of LCL and ACL, who received osteotomy after reconstruction of the ligaments. The main symptom before the operation was pain in medial compartment. The range of motion, Lysholm score and subjective satisfactory examinations were assessed before and after the osteotomy.</p><p><b>RESULTS</b>All of the osteotomy sites were healed at twelve to sixteen weeks after operation. No complications such as plate broken or injury of nerve or blood vessel had occurred. The mean correct angle was 9.5 degrees . No degenerative changes had developed in other compartments of the knee. The mechanical axis of the lower extremities was maintained during the follow-up. The overall satisfactory rate was 83.3%. Statistically significant changes exist in the Lysholm score and varus degree.</p><p><b>CONCLUSION</b>The open-wedge high tibial osteotomy is suitable for the symptomatic genu varum in younger patients with good short-term and mid-term results.</p>

Repair full-thickness meniscal defects with injectable tissue engineering technique / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effectiveness of injectable tissue engineering to repair full-thickness meniscal defects.</p><p><b>METHODS</b>From June 2008 to February 2009 full-thickness of meniscal defects were created in the anterior corner of goats, which with no blood supply, in a diameter of 2 mm. Then bone marrow stem cells (BMSCs) was mixed with injectable calcium alginate gel to fill the defects. Other groups include the calcium alginate gel and empty group were served as control groups. At different time points, the animals were sacrificed and macroscopy, microscopy determination, electroscopy and MRI detection were performed to assess the outcomes of repairing.</p><p><b>RESULTS</b>The meniscal defects had been filled thoroughly in 16 weeks after operation with white, tough and elastic repair tissue similar to normal meniscal fibrocartilage in the tissue engineering groups. The repair tissue was mainly fibrochondrocytes in line with the calcium alginate fiber. Thick matrix secreted by the cells crammed the space between fibers. The view under electroscopy demonstrated that the microstructure of the repair tissue was normal and cells were in a fibrocartilage phenotype.</p><p><b>CONCLUSION</b>The full-thickness meniscal defects in regions without blood supply can be reconstructed effectively with injectable tissue engineering.</p>

Uninduced adipose-derived stem cells repair the defect of full-thickness hyaline cartilage / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects.</p><p><b>METHODS</b>Adipose-derived stem cells (ADSCs) were derived from adipose tissue and cultured in vitro. Twenty-seven New Zealand white rabbits were divided into three groups randomly. The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint, and the defects repaired with gel or without treatment served as control groups. After 4, 8 and 12 weeks, the reconstructed tissue was evaluated macroscopically and microscopically. Histological analysis and qualitative scoring were also performed to detect the outcome.</p><p><b>RESULTS</b>Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived tissue. The result was better in ADSCs group than the control ones. The microstructure of reconstructed tissue with ADSCs was similar to that of hyaline cartilage and contained more cells and regular matrix fibers, being better than other groups. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Statistical analysis revealed a significant difference in comparison with other groups at each time point (t equal to 4.360, P less than 0.01).</p><p><b>CONCLUSION</b>These results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects.</p>

Experimental research on human insulin-like growth factor I gene transfect the cultured bone marrow mesenchymal stem cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effectiveness of human insulin-like growth factor I (hIGF-I) gene transferred into the cultured goat bone marrow mesenchymal stem cells with liposome, and find a new method of cell-mediated gene therapy.</p><p><b>METHODS</b>Bone marrow was extracted from adult goats and cultured in vitro by monolayer. Then the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF was transfected into cells by FuGene 6. After transfection, the marker gene coding enhanced green fluorescent protein (EGFP) was observed at different time points. The hIGF concentration in the supernatant fluids was measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry stain of hIGF was performed to detect the target protein. Besides, reverse transcription polymerase chain reaction and flow cytometry were also adopted in order to find out the changes of cells after transfection.</p><p><b>RESULTS</b>Bone marrow stem cells were all in the long shuttle-like shape and adhered to the disk. The expression of EGFP was first found at 4 h after transfection. The amount and intensity of EGFP increased gradually during the period of detection and got to the peak degree at 72 h, after that decreased slowly. EGFP was also seen in the second generation cells, but the intensity was relatively faint. The IGF-I concentration secreted into the supernatant was in accordance with the EGFP observed with the peak concentration at 34.75 ng/ml. The outcome of RT-PCR and immunohistochemistry was positive. The morphology of many stem cells was changed into triangular or irregular forms under the circumstance of the secreted hIGF-I. Percentage of stem cells in the S stage increased after transfection.</p><p><b>CONCLUSION</b>The hIGF-I gene can be transfected efficiently and safely into BMSCs by FuGene 6, and the hIGF-I protein can be secreted into the supernatant in a relatively high level during a long period, therefore accelerate the proliferation and differentiation of the transfected cells.</p>