Initial Research On Dynamic Construction Of Tissue-engineered Tendon In Vitro

Objective: To detect if the cyclic stretch have positive influence on the proliferation, ECM secretion, cell re-orientation , and to define a useful parameter for cyclic stretch for the construction of tissue-engineered tendon in vitro.Methods :(1)Handerson'S method was used to separate tenocytes from new born chicken tendon, and the 2-4 cell passages (were) used as the seeding cells. (2)The tensile strength and the stress-strain curve of the bio-derived tendon scaffold and normal human tendon were detected by the INSTRON biomechanical instruments, and the mechanical characteristics of the scaffold was defined. (3) After tenocyte was seeded in the bio-derive tendon scaffold and cultured for a certain period, the compatibility of the scaffold was detected by observing the configuration and the ECM secretion of the cell on the scaffold surface by scanning electron microscope (SEM) (4) After culturing of the cell-scaffold composite under different dynamic parameters, we used cell counting to find out the better parameters for the cell proliferation,. HPLC was used to detect the influence on the ECM secretion and SEM to examine cell orientation under the cyclic stretch .(5) After culturing of the cell-scaffold composite for 96 hour under dynamic and normal condition, the composite was embedded by paraffin and stained with HE method to detect if the cell can grow into the scaffold.Results: (I) The 2~4 cell passages of the tenocytes are suitable for being used as seeding cells and synthesize ECM well. ?The bio-derived tendon scaffold is suitable for being used as the scaffold for the three dimensional dynamic construction of tissue-engineered tendons. (3)The scaffold possesses good cell compatibility, and the tenocytes grow very well on the surface of the scaffold and secrete visible ECM observed under SEM . ?When the frequency of the cyclic stretch is 0.02Hz, the stretch extent is 10%,and the work time per hour is 15 minute, the proliferation(p<0.05) and the ECM secretion(p<0.01) of the experimental group are significantly better than the control group, and cell orientation of the experimental group is more orderly than the control group, the cells are tend to arrange with the direction of the stretch. (D After being embedded by paraffin and stained with HE method, the slices of the tenocyte-scaffold composite were observed under microscope 400 X, and the cell number grown into the stretch scaffold more than that grown into the non-stretch scaffold.Conclusion: ?With its excellent mechanical characteristics, the bio-derived tendon scaffold can be used as a good carrier for tenocytes to perform the research on the effect of cyclic stretch under the condition of three dimension in vitro, and also it is a nice scaffold for the construction of tissue-engineered tendons in vitro. ?With specific parameters, cyclic stretch significantly accelerates the proliferation of the tenocyte, ingrowth of the tenocyte, the secretion of the ECM, and promotes the re-orientation of the tenocyte along with the direction of the stretch.