Preliminary Study On Constructing The Tissue Engineered Tendon At Hypoxia Environment

Objective:To construct tissue engineered tendon by using pig tendon cells in hypoxia environment.The tissue engineering tendon under the low oxygen environment and conventional oxygen environment were compared through the aspect of the general observation,histology,polarizing microscope and biomechanics.engineering tendon was analyzed under the low oxygen environment in order to make the preliminary exploration for following up study.Methods:Tenocytes were isolated using tissue explant method and expand in vitro.Tenocytes (20×106) at the second passage were collected and then seeded onto PGA unwoven fibers to form cell-scaffold construct in a shape of tendon.The cell-scaffold construct were then cultured under hypoxia environment(5%O2) generated by a u-shape spring(n=3),which served as experimental group.or cultured conventional oxygen environmen(21%O2)(n=3),which served as control group. The first generation and the second generation of tendon cell was observed morphology by phase contrast microscopy.When tendon cells in two group were cultured in PGA for48hours and7days, Tenocytes-scaffold were observed whether tenocytes adhesion are good or not on the scaffold. We make the general observation for knowing the difference at2weeks and8weeks.Tendon cells inoculated on PGA were observed by histologic and polarized light microscopy in order to knowing PGA material degradation and collagen alignment.Engineering Tendon were tested by biomechanical instrument and were statistical analyzed using SPSS11.5software package. The data using T test for statistical processing of experimental results, P<0.05had significant difference, P<0.01have more significant difference.By comparison the difference between the two groups,we make the preliminary study in constructing tissue engineered tendon by the static under the hypoxia condition.Results:No obvious difference were observed between the first and second generation of tendon cells under normal oxygen environment and hypoxia environment.Tendon cells cultured in PGA for48hours and7days found that the adhesion ability of two groups tenocytes on the scaffolds is better. Tendon cells culture in PGA take general observation at2weeks and8weeks and found that the control group and the experimental group had no obvious difference in appearance at2weeks.The control group manifested in the shape of stripe newborn tissue and the experimental group show that new tissue is close to the normal tendon and is significantly thinner.Tendon cells inoculated on PGA were observed by histologic and polarized light microscopy and found that cells composites showed large amounts of PGA undegraded in the control group and experimental group at2weeks.Cells composites Can still see a lot of undegraded PGA at2weeks.At the same time,we can see a small amount of collagen fibers between undegraded PGA and PGA fibers in two groups.HE staining showed that the cells of control group arranged disorderly than the experimental group and there is a lot of undegraded PGA at6weeks,while there is a collagen formation between PGA fiber.HE staining in the control group showed that the collagen fiber is more disorder than the experimental group at8weeks.HE staining in the control group and the experimental showed that collagen fibers along the direction of the stress axis are arranged neatly and tendon tissue is the basic formation. HE staining of control group showed that the cells and collagen arrangement is still disorder. collagen content is not much and appear hollow phenomenon at14weeks.through two groups Biomechanical data of tendon analyzed, T test showed that the experimental group after6weeks tensile strength was significant than of the control group, p<0.05.However,There was no significant difference between8weeks and later time points of the experiment group and the control group, p>0.05.Conclusion:Cells in a hypoxia environment have the viability of survival proliferation and differentiation.Under constructing engineering tendon in hypoxia environment engineering tendon was formated by increased in the degration of PGA.Through the preliminary simulated hypoxic environment construction of tissue engineered tendon from experiment, cytological, morphological, histological, polarized light microscopy, detection of biomechanical performance, the feasibility of constructing static tension engineered tendon was proved in hypoxia environment.