Establishment of the Optimized AFLP Analysis System for Alnus

The optimized AFLP analysis system was established for Alnus. A. glutinosa was used to comparatively study and analyze key factors influencing the results of AFLP, including quality and concentration of extracted DNA, enzyme digestion time and amplification system that influenced the results of the AFLP. The results suggested an amount of DNA template of 300 ng; reaction time for enzyme digestion of 5 h; ligation time of 16 h; a 10 times dilution of ligation products for pre-amplification ; and a 20 times dilution of pre-amplification products for selective amplification. The AFLP-PCR reaction system （20 μl） for Alnus was determined as follows ： DNA template 300 ng, 10 x Taq DNA polymerase Buffer （ containing Mg2＋ ） 2.0μl, dNTPs 0. 3 μl, primers 1.0 μl, Taq DNA polymerase 0. 6 U, and ddH2O in balance. Seven pairs of primer combinations were selected with many clear bands and high polymorphism. By using the AFLP reaction system, primers suitable for Alnus were obtained. Those results provided certain basis for further molecular studies on Alnus using AFLP markers.

Cell osteogenic bioactivity mediated precisely by varying scaled micro-pits on ordered micro/nano hierarchical structures of titanium

Hierarchical surface structures with micro–nano scale play a crucial role in regulation of cell proliferation and osteogenic differentiation.It has been proven that cells are extremely sensitive to the nanoscaled structure and show multifarious phenotypes.Though a vital function of microstructure on osseointegration has been confirmed,the cell performances response to different microscaled structure is needed to be further dissected and in depth understood.In this work,the ordered micro–nano hierarchical structures with varying micro-scaled pits were precisely fabricated on titanium successfully by the combination of electrochemical,chemical etching and anodization as well.In vitro systematical assessments indicated that the micro–nano multilevel structures on titanium exhibited excellent cells adhesion and spreading ability,as well as steerable proliferation and osteogenic differentiation behaviors.It is shown that smaller micro-pits and lower roughness of the hierarchical structures enabled faster cell propagation.Despite cell growth was delayed on micro–nano titanium with relatively larger cell-match-size micro-pits and roughness,osteogenic-specific genes were significantly elevated.Furthermore,the alkaline phosphatase activity,collagen secretion and extracellular matrix mineralization of MC3T3-E1 on multiscaled titanium were suppressed by a large margin after adding IWP-2(an inhibitor of Wnt/b-catenin signal pathway),indicating this pathway played a crucial part in cell osteogenic differentiation modulated by micro–nano structures.