Two Different Kind Of Lysine-DOPA Compound Effect On MC3T3 Cell Adhesion And Observation About Skeletal Muscle Contraction Effect On Bone Growth Through Intravital Microscopy By Using BCI

Objective1. To detect if two different kind of synthetic Lysine-DOPA compound: LD80-20 and LD90-10 will effect on MC3T3 cell spreading, adhesion, proliferation and differentiation2. To detect if muscle contraction will effect on bone formation and microcirculation by using BCIMethods1. MC3T3 cells were seeded on cell culture plate(dish) with different surface modification(LD80-20, LD90-10,FN and PLL).At different time point,Rhodamine phalloidin-DAPI stain and Jet impingement were performanced to detect cell spreading area and cell adhesion strength on different surface. Total cell number(that was represented by total protein) and ALP activity were also measured in order to detect different surface effect on cell proliferation and differentiation. All experiments will performance both with serum and serum free.2. 30 female New Zealand white rabbits were random divided into two group: experiment group and control group. Each bone chamber was implanted medially using aseptic techniques into the right tibiae of eachrabbit. Experiment group were stimulated by TENS (4Hz,1hour/day,6day/week) from week 2. From week 3, Intravital Microscopy Observations were performanced weekly while experiment group will have stimulation at same time. Bright field images depicted bone, and were used to measure bone in-growth areas and apposition rate. Fluorescence images depicted vasculature outlined with FITC and RITC .All observation were recorded by digital image. Heart rates and blood pressures were taken during observations. At week 10, gastrocnemius were detached to measure the muscle contraction strength.. Changes in bone mineral density were evaluated at the week 3 and week 10 post-implantation with micro CT. All images were analyzed and calculated by Metamorph program.Results1. With serum free: both LD80-20 and LD90-10 can significantly increase cell spreading and cell adhesion at both lhr and 24hr,compared with control ,this effect was between specific adhesion molecule FN and non-specific adhesion molecule PLL. Also they can show cell spreading and adhesion increase in lhr group with serum condition, but not in 24hr group; Both LD80-20 and LD90-10 can increase cell proliferation and have no effect on cell ALP activity. When LD80-20 and LD90-10 were cross-linked by H2O2, they have no effect on cell spreading, adhesion, proliferation and differentiation.2. Experiment group have significantly higher bone in-grow rate and bone mineral content; Experiment group have significantly higher blood flow rate; average vessel caliber and average weekly vessel fractions have no difference between two group; capillary permeability was higher in experiment.Conclusion1. Two different kinds of Lysine-DOPA compound: LD80-20 and LD90-10can increase MC3T3 cell spreading, adhesion as well as cell proliferation, have no effect on cell ALP activity. And DOPA component is the key-component to have this effect.2. Skeletal muscle contraction can increase bone in-grow rate and bone mineral content; skeletal muscle contraction can increase blood flow rate; skeletal muscle contraction can increase capillary permeability; skeletal muscle contraction can increase has no effect on angiogenesis.