Research on the repair of acute large osteochondral defects with mosaicplasty associated with genes-enhanced tissue engineering in different proportion / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the optimum proportion of Mosaicplasty and genes-enhanced tissue engineering for the repair of acute osteochondral defects.</p><p><b>METHODS</b>Western blot test was conducted to detect the expression of hTGF-beta1, Col II and Aggrecan in 3 groups, including hTGF-beta1, transduction group, Adv-betagal transduction group and control group without transduction. Eighteen 6-month-old Shanghai male goats (weight: 22 to 25 kg) were used. BMSCs were isolated from the autologous bone marrow, and were subcultured to get the cells at passage 3. Thirty-six medial femoral condyles were used and divided into 6 groups named AR, AL, BR, BL, CR, and CL. Acute cylindrical defects (9 mm in diameter and 3 mm in depth)were created in the weight bearing area of the medial femoral condyle of hind limbs. In the single group, the autologous osteochondral mosaicplasty was performed to repair the defect; in the combination group, besides the mosaicplasty, the dead space between the cylindrical grafts and the host cartilage were injected with the suspension of hTGF-beta1, gene enhanced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels. The autologous osteochondral transplantation cover rates of group AR was 44.44% single group, AL was 44.44% combination group, BR was 33.33% single group, BL was 33.33% combination group, CR was 22.22% single group, and CL was 22.22% combination group. The goats were killed 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O'Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were also performed.</p><p><b>RESULTS</b>Western blot test showed the expression of the hTGF-beta1, Col II and the Aggrecan in the hTGF-beta1 transduction group were significantly higher than that of the Adv-betaga1 transduction and the blank control groups. The gross and histology observation revealed that each defects of six groups had different degrees of repairing. There was no significantly difference among the BL, AR, and AL groups. But the scores of the other three groups (BR, CR, and CL) were significantly poorer than the former three groups.</p><p><b>CONCLUSION</b>Mosaicplasty associated with genes enhanced tissue engineering could repair the osteochondral defects effectively. With the autologous osteochondral transplantation coverage reducing, the advantage of the combination could have a better representation.</p>

Influence of the unevenness of articular surface on the osteochondral repair / 中国骨伤

<p><b>OBJECTIVE</b>To investigate whether the unevenness of articular surface would affect the osteochondral repair.</p><p><b>METHODS</b>Eight Shanghai Chongming 6-months-old masculine goats with a mean weight of 25 kg were used in this study. Different unevenness, which were 0.5 mm, 1.0 mm, 2.0 mm protrude or concavity, were created on the weight-bearing portion of the medial femoral condyles of the goats. The goats were sacrificed 12 weeks later and were observed with the general observation, HE staining and transmission electron microscope. To evaluate the microscopic morphology, a histological grading scale described by O'Driscoll, Keeley and Salter was used.</p><p><b>RESULTS</b>The general observation and HE staining showed that the unevenness of 0.5 mm or 1.0 mm protrudes or concavity could be repaired to get the surface smooth on the whole. The transmission electron microscope showed that the reparative tissues were the same as the normal cartilage. The 2.0 mm depth couldn't be repaired satisfactorily. The transmission electron microscope showed that the fiber bundle proliferated and the chondrocytes degenerated. The scores of the 2.0 mm depth were significantly lower than that of the 0.5 mm or 1.0 mm (P < 0.05).</p><p><b>CONCLUSION</b>The unevenness could have an influence on the repair. The limited unevenness could be repaired by itself.</p>

The study of bipolar radiofrequency chondroplasty to cartilage injure of goats / 中华外科杂志

<p><b>OBJECTIVE</b>To study the evaluation of bipolar radiofrequency (RFE) chondroplasty to cartilage injure.</p><p><b>METHODS</b>Sixteen goats underwent resection of anterior cruciate and medial meniscus to create cartilage injured model. Bipolar frequency energy chondroplasty were performed on injured articular cartilage 3 months after operation. The left knee was regarded as sham-operated control. The zero time effects and later changes of radiofrequency on cartilage surface and chondrocytes were observed.</p><p><b>RESULTS</b>At zero time, the rough injured surface became smooth, and clefts were melted. Some chondrocytes were dead in the superficial layer, cells in deeper layer remained alive. Three months later, the surface of the articular was smoother than the sham-operated side. The histological modified Mankin' s score of cartilage was significantly higher than the sham-operated side (operated side 12 +/- 4; sham-operated side 14 +/- 5, P < 0.01). The result of Fluorescence-activated cell sorter (FACs) showed that there were no obvious difference of dead chondrocyte between operated side and sham-operated side, (24 +/- 10)% and (12 +/- 1)% (P > 0.05).</p><p><b>CONCLUSIONS</b>Bipolar radiofrequency energy (1 grade) does not create more chondrocyte death than the sham-operated side. Bipolar radiofrequency chondroplasty is an appropriate method to treat articular cartilage injury.</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>

Experimental research on spine fusion induced by tissue engineered bone / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a reasonable substitute for the autograft bone in vitro and transplant it back into the rabbit models to induce the spine fusion.</p><p><b>METHODS</b>The bone marrow stem cell from the seven New Zealand rabbits were cultured. Recombinant human bone morphogenetic protein-4 (rhBMP-4) that has been proved to be bioactive was obtained by the way of genetic engineering. Using the vacuum freezing machine to mix a certain quantity of rhBMP-4 into type I collagen to form a new kind of carrier. Animal model of spine facet process fusion was used. Bone marrow stem cells combined with rhBMP-4 and I type collagen were implanted between the facet process to induce the spine fusion. type I collagen and bone marrow stem cell was used in the controlled group.</p><p><b>RESULTS</b>New bone formation was obvious in the test group. The facet joint was fused very well in this side. No bone formation was present on the other side.</p><p><b>CONCLUSIONS</b>The new composite: bone marrow stem cells, rhBMP-4 and type I collagen was an ideal kind of substitute for the autograft bone.</p>