| Objective To investigate the feasibility of porous tantalum scaffold in repairing ofcartilage defect and promoting cartilage regeneration, which through porous tantalumproperties, cytotoxicity test, chondrocytes apoptosis and cycle, chondrocytes function andthe changes of gene MMP-13, Aggrecan and Collagen II mRNA expression in extractsincubated with tantalum, so as to provide the experimental data for cartilage tissueengineering scaffold material in the clinical application.Methods1The morphological properties of the porous tantalum were observed by theSEM. The SD rat born3weeks were selected and chondrocytes were resected、amplificated and identified in vitro. The extract fluid from porous tantalum was madeaccording to the standards of ISO10993-5/ISO10993-12. The different concentrations ofporous tantalum leaching liquor (100%,50%and25%) and porous titanium leachingliquor were prepared with medium F12-DMEM. The chondrocytes were cultured inleaching liquor of different concentrations were used as experimental groups and controlgroup were metal titanium leaching liquor and without leaching liquor. MTT assay wasapplied to detected the cell proliferation ability and cytotoxicity in the leaching liquor.2The chondrocytes were co-cultured with extract of porous tantalum in vitro and themorphology, growth, proliferation, differentiation were observed under inverted phusecontrast microscope, scanning electron microscope (SEM) and transmission electronmicroscopy (TEM).3The cell circle and apoptosis of osteoblasts were detected by flowcytometry.4Dimethylmethylene blue (DMMB) assay was applied to detected theamount of glycosaminoglycan (GAG) was measured.5The expression levels of geneMMP-13, Aggrecan and Collagen II mRNA in the chondrocytes were examined by Real-time Quantitative PCR (RT-qPCR).Results1Porous tantalum scaffolds were gray and light-proof, the surface and crosssection were evenly distributed needlepoint cellular porosity. The observed result of SEMshowed that the pore sizes of porous tantalum ranged from400μm to600μm (×500) and50μm to200μm (×1000), which had a similar three-dimensional connected holemorphology to cancellous bone.2The MTT method showed that the number of cells wasincreasing with time, the chondrocytes growing normally and having good adherent proliferation abilities. At the same time point, there were no significant difference in thecell proliferation ability between the different concentrations of leaching liquor groupsand control groups (P>0.05).3Many of chondrocytes were found adhering to the edge ofporous tantalum by inverted microscope. Cells spread well on the surface and distributedin the hole of porous tantalum, and produced some extracellular matrix by SEM.Compared with the normal chondrocytes, there were no differences in the ultrastructureof cells by TEM.4Flow cytometry showed that there were normal diploid cells andsimilar cell cycle distribution in three groups. There were no significant difference in therates of apoptosis and necrosis cells between the experimental group and control groups(P>0.05).5Quantitative determination of GAG show that chondrocytes in the poroustantalum can secret a lot of GAG.6RT-qPCR analysis shows that there was nosignificant difference in the expression of MMP-13and Collagen II mRNA between theexperimental group and control groups (P>0.05). And Aggrecan in the porous tantalumgroup was higher than control groups (P<0.05).Conclusion1The method showed that chondrocytes proliferation and growing normallyin porous tantalum leaching liquor. It was confirm that the porous tantalum has no cellulartoxicity.2The chondrocytes adhere well, proliferate adequately and extend excessively,secreting extracellular matrix on its. It proves the domestic porous tantalum has goodhistocompatibility.3The method showed that there were normal diploid cells in the poroustantalum group, the cells in S phase of cell cycle were increased and the material had noeffects on apoptosis. The domestic porous tantalum has no a marked effect on cell cycleand apoptosis. The method showed that porous tantalum has good cytocompatibility.4Theporous tantalum has no obvious inflammation reaction, and can enhance Aggrecan andCollagen II mRNA expression of chondrocytes cultured in vitro. It proves the domesticporous tantalum has good histocompatibility. |
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