Fibroblast Seeded On The Thoroughly Acellular Human Amniotic Membrane With Three-Dimensional Adhesion In Vitro

Objective:Paxillin and a5 integrin colocalized to cell-matrix attachments parallel to fibronectin-containing extracellular fibers, These molecule structures were proposed for the term "3D-matrix adhesions." These structures in cell adhesion, migration, signaling, and cytoskeletal function is different from studies on planar 2D tissue culture substrates. We present here the human forskin fibroblasts (HFF) formed 3D-matrix adhesion complexes on the thoroughly acellular human amniotic matrix (TAHAM).Methods:TAHAM were produced by suspending digestion with trypsin. The HFF were seeded on 6 well plate, matrigel and TAHAM individually. The light microscope, scanning electronic microscope, immunohistochemistry and immunofluorescence were used to observe the micro-structures and detection the type I,111, IV, VI collagen, laminin, fibronectin, TGF-β1, TGF-β2, FGF of the TAHAM. Deoxyribonucleic acid (DNA) content of fresh HAM and TAHAM was quantified using Hoechst 33258 dye. Phase contrast microscope was engaged to observe the morphology of HFF. The timelapse CCD and the trace analysis soft ware were employed to prescribe the cell migration. The 3D adhesion foci were indentified by the laser confocal microscope. The strain of the TAHAM was tested by the universal mechanical testing instrument.Results:The fibers of the TAHAM were intact, type I, III, IV, VI collagen, laminin, fibronectin were positive, TGF-β1, TGF-β2, FGF were negative. HFF were bipolar extended, spindle shaped, form multilayer cell clusters networks and invadeded into the matrix. Hoechst 33258 dye confirmed on TAHAM surface without cells and residual debris. TAHAM was compared with fresh amniotic membrane in the transitional stress, the transitional strain, failure stress, failure strain, the difference was not statistically significant(P values> 0.05) (transitional stress:t=-1.83, P= 0.08; transitional strain:t=-1.07, P= 0.30; failure stress:t= 1.57, P= 0.13; failure strain:t =-1.81, P= 0.08). HFF seeded on the TAHAM for 24h, the cells extended to the spindle shapes and the formation of pseudopodia, a5 integrin was distribution along the cell axis, and cells also secreted large amounts of fibronectin. After a multiple layer of cells continued proliferating cell clusters, cell clusters were composed of sea star structures between the cord connected to each other, some cells in cell clumps growed into TAHAM. All of the seeded cells survived three weeks under regular culture without transfer. On TAHAM, HFF moved in a straight line with speed 12μm/h. a5 integrin (green), paxillin(red) and fibronectin(blue) colocalized to form 3D adhesion complexes(white).Conclusion:The TAHAM may also be a new candidate of 3D adhesion matrix for orthopedic tissue engineering and for universal cell study under the cell-matrix 3D adhesion in vitro.