ABSTRACT
Objective The nano-zirconia scaffolds we previously prepared had a good 3-dimensional ( 3D ) connectivity but did not achieve the ideal sintering rate and compressive strength .The objective of this study was to explore the enhancing effect of polyvinyl butyral ( PVB) as a dispersant on the compressive strength of 3D nano-zirconia porous scaffolds for bone tissue engineering . Methods We prepared the slurry containing different concentrations of PVB and ana-lyzed the improving effect of PVB on the mechanical properties of the scaffolds by sediment experiment , compressive strength test and scan-ning electron microscopy . Results The sediment experiment showed no significant stratification in the slurry with 0.2wt%PVB, white suspension in the upper layer and white precipitate in the lower layer , with a significantly higher compressive strength of the scaffold ([0.324 ±0.030] MPa) than that of the scaffold prepared by adding other concentrations of PVB to the slurry (P <0.01).And the compressive strength of the scaffold constructed by adding no dispersant ([0.109 ±0.021] MPa) was remarkably lower than that of the scaffold constructed by adding PVB to the slurry (P<0.05).Scanning electron microscopy demonstrated that the scaffold prepared by adding 0.2wt%PVB to the slurry had a complete porous structure with the fewest and most sparsely distributed surface cracks as compared with other PVB concentration groups . Conclusion PVB can signifi-cantly improve the stability of zirconia slurry , enhance the compressive strength of the nano-zirconia porous scaffold , and make the scaf-fold more applicable to bone tissue engineering .
ABSTRACT
Objective Nowadays, in the field of bone tissue engineering , searching for a kind of scaffold , which is both strong enough and biocompatible , is still a hot topic .Objective of this study is to evaluate the three-dimensional nano-zirconia porous scaffold′s effect on bone marrow stromal cells early attachment and proliferation . Methods Preparing nano-zirconia porous scaffold and testing its porosity and its mechanical strength .Hemolysis experiment was taken to evaluate its biosafety .Canine bone marrow stromal cells ( BM-SCs) were extracted from an adult beagle and cultured in vitro.The third generation cells were seeded onto the scaffold and induced to os-teoblasts under condition medium .Cells early attachment on scaffold was observed by SEM .MTT and ALP tests were taken to evaluate cells proliferation and osteogenesis within two weeks . Results Nano-zirconia porous scaffold in our study presented a porous structure with a well connected inner .Its porosity and compressive strength were (92.667 ±0.324)%and 4.38 MPa, respectively.Hemolysis rate was 2.19%, which was demonstrated to be safe to human body .Canine BMSCs could colonize the surface of the materials , bridge macro-poros-ity and proliferate on the scaffold .A values taken by the method of MTT showed that cells could proliferate quickly on the scaffold after 3, 6, 9, 12 d (A values were 0.604 ±0.059, 1.215 ±0.019, 1.385 ± 0.254, and 1.506 ±0.050), comparing with that on the first day (A value was 0.261 ±0.034).Statistical difference was found (P <0.01) .The ALP level raised up significantly on day 7 and 14 ( A val-ues were 0.032 ±0.002 and 0.062 ±0.007), comparing with that on the first day (A value was 0.016 ±0.003).Statistical difference was found (P<0.01). Conclusion The three-dimensional nano-ZrO2 porous scaffold in our study presented satisfactory biocompati-bility.It could offer a microenvironment for osteoblasts growth and secreted osteogenesis marker in vitro .
ABSTRACT
<p><b>OBJECTIVE</b>To construct and identify a lentiviral vector of RNA interference targeting human Notch-1 gene.</p><p><b>METHODS</b>To determine the Notch-1 gene sequences, three RNAi target sequences (shRNA1-3) were designed in accordance with the RNAi sequence design principles and cloned into the lentiviral vector pLenOR-THM by endonuclease BamH I restriction, EcoR I double digestion, and T4 DNA-ligase ligation. After the transformation into competent DH5alpha bacteria, the candidate clones were identified by Kpn I and EcoR I double digestion and DNA sequencing. The recombinant and three packaging plasmids were co-transfected into human embryonic kidney cell line 293T cells by lipofectamine to produce the lentiviral particles. The viral titer was determined. The 293T cells were infected by the lentiviral particles obtained, and transfection efficiency was assessed using a fluorescent microscope. The lentiviral vector particles were also transfected into ACC-M cells. The Notch-1 expression in the transfected cells was assayed by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and Western blot analysis.</p><p><b>RESULTS</b>The lentiviral RNAi vector pLenOR-THM-Notchl for Notch-1 gene was constructed successfully. Strong green fluorescence was observed in the 293T cells under fluorescent microscope after co-transfection of the cells with the four plasmids of lentiviral vector. The virus in the supernatant reached a titer of 5.8 x 10(8) TU x mL(-1). The transfection efficiency of the collected virus exceeded 90% in 293T cells with 1 as a multiplicity of infection. The third lentiviral vector was found to significantly inhibit the Notch-1 expression at the mRNA and protein levels.</p><p><b>CONCLUSION</b>The lentiviral RNAi vector of Notch-1 has been successfully constructed and identified.</p>