Objective To observe the efficiency and biological characteristics in regenerating in vitro tissue-engineered cartilage from epiphyseal chondrocyte-scaffold complex. MethodsThe first passage epiphyseal chondrocytes were collected and mixed with the biological gel-matrix, the chondrocyte-gel fluid wasdropped into the scaffold to form a complex. The complexes were in vitro cultivated. The changes of complexes in morphology and synthesis of collagens type ⅡandtypeⅠ and aggrecan were observed under the gross and the inverted and light microscopes. The sulfate GAG content in complexes was measured by the the modified dimethylmethylene blue method. Results During cultivation, thecomplexes could keep its original shape with the stable homogeneous three-dimensional distribution of chondrocytes，gradually became milk white and translucence with their rigidity increasing. In the 1st week, the chondrocytic lacunae formed in the complexes. After 2 weeks, the complex was gradually reorganized into the mature engineered cartilage with rich collagen typeⅡand aggrecan and typical cartilage histological structure, but with negative immunological staining of collagen typeⅠ. In the 4th week, the engineered cartilage resembled the nature epiphyseal plate in the characteristic of histological structure, and had over 34% of the sulfate GAG content of the natural epiphyseal plate. Conclusion Theepiphyseal chondrocyte-scaffold complex can be reorganized into typical cartilage with the epiphyseallike histological structure, and be fit for repairing the epiphyseal defect. The tissue engineered cartilage cultivated for 1-2 weeks may be a good choice for repairing epiphyseal defect.
To explore the shape and structure of calcified cartilage zone and its interface between the non-calcified articular cartilage and subchondral bone plate. Methods The normal human condyles of femur (n=20) were obtained from the tissue bank donated by the residents, 10 males and 10 females, aged 17-45 years. The longitudinal and transverse paraffin sections were prepared by the routine method. The shape and structure of calcified cartilage zone were observed with theSafranin O/fast green and von kossa stain method. The interface conjunction among zones of cartilage was researched by SEM and the 3D structural model was establ ished by serial sections and model ing technique. Results Articular bone-cartilage safranin O/fast green staining showed that cartilage was stained red and subchondral bone was stained blue. The calcified cartilage zone was located between the tidemark and cement l ine. Von kossa staining showed that calcified cartilage zone was stained black and sharpness of structure border. Upper interface gomphosised tightly with the non-calcified cartilage by the wave shaped tidemark and lower interface anchored tightly with the subchondral bone by the uneven comb shaped cement l ine. The noncalcified cartilage zone was interlocked tightly in the manner of “ravine-engomphosis” by the calcified cartilage zone as observed under SEM, and the subchondral bone was anchored tightly in the manner of “comb-anchor” by the in the calcified cartilage zone 3D reconstruction model. Conclusion The calcified cartilage zone is an important structure in the articular cartilage. The articular cartilage is fixed firmly into subchondral bone plate by the distinctive conjunct interfaces of calcified cartilage zone.
To evaluate the implantation effect of artificial vascular grafts with recombinant fibrinolytic enzyme factor II (rF II)-immobil ized lumina in animal test. Methods Four mm internal diameter (ID) polyurethane (PU) artificial vascular grafts were prepared by di pping and leaching method. The micro-pore size and morphology of the graft walls were observed by SEM. The graft lumina were immobil ized with rF II. Twenty hybrid male dogs [weighing (20 ± 1) kg] were used for animal model of carotid artery defect and were randomly divided into 3 groups: rF II -immobil ized PU group, no rF II -immobil ized PU group and expanded polytetrafluoroethylene (ePTFE) group. The vascular grafts were implanted for repairing injured segments of carotid artery in dogs. The general health state of animals was recorded. At 30 days and 60 days,the patency rate of every group was calculated. At 60 days IDs were measured, cell prol iferation in neointima was inspected by l ight microscope, morphology on neointima was observed by SEM. Results The ID of the PU vascular grafts was (3.74 ± 0.06) mm, wall thickness was 0.4-0.6 mm, the wall density was 0.25 g/cm3, the porosity was 79.8%, racical compl iance was 8.57%/100 mmHg. In the wall, micropores were well distributed and opened-pores structure was observed. Pore size was (140 ± 41) μm in the outside layer, pore size was (100 ± 3) μm in the inside layer, thickness ratio of outside / inside layers was 2 ∶ 1, the pore size was (40 ± 16) μm on the lumina surface. After operation the wounds on neck healed, all the animals survived and had no compl ication. At 30 days and 60 days after implantation, the patency rate for rF II -immobil ized PU group were 100% and 66.7%, for no rF II -immobil ized PU group were 66.7% and 33.3%, and for ePTFE group were 67.7% and 0 respectively, but at 60 days there were thrombosis at anastamotic sites of some grafts occluded. Before operation the IDs for rF II-immobil ized PU group, no rF II -immobil ized PU group and ePTFE group were (3.74 ± 0.06), (3.74 ± 0.06) and (4.00 ± 0.03) mm, at 60 days after operation the IDs were (4.51 ± 0.05), (4.31 ± 0.24) and (4.43 ± 0.12) mm respectively, showing no statistically significant differences between 3 groups (P gt; 0.05). Histological inspection indicated that at 15 days a layer of plasma protein deposited on the lumina, at 30 days some cells adhered to the lumina, at 60 days neointima could be observed on the lumina. Thickness of the neointima became larger with implantation time. At 60 days neointima thickness at proximal end, middle site and distal end ofgraft were (560 ± 22), (78 ± 5) and (323 ± 31) μm respectively for rF II -immobil ized PU group. The results of SEM showed that neointima surface consisted of flat and long cells which long axes ranged with blood flow direction and was similar to lumina morphology of carotid artery of dog. Conclusion Immobil ization of rF II to lumina of grafts could enhance fibrinolytic activity and inhibited formation of thrombo-embol ia which led to an increase in patency rate after implantation.