Fibroblast seeded on the thoroughly acellular human amniotic membrane with three-dimensional adhesion in vitro
10.3760/cma.j.issn.0253-2352.2011.02.014
- VernacularTitle:成纤维细胞与完全脱细胞人羊膜的三维黏附
- Author:
Xuya LU
;
Yuan XUE
;
Pei WANG
;
Chengliang CUI
;
Wei LIU
;
Yang LI
- Publication Type:Journal Article
- Keywords:
Amnion;
Cell adhesion;
Integrins
- From:
Chinese Journal of Orthopaedics
2011;31(2):184-190
- CountryChina
- Language:Chinese
-
Abstract:
Objective Three-dimension (3D) cell matrix adhesion in vivo is fundamentally important for a wide variety of cellular physiological and pathological phenomena, however, the cell-matrix 3D adhesion is hardly observed in vitro. We present the human foreskin fibroblasts (HFF) formed 3D 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 detect the type Ⅰ , Ⅲ, Ⅳ, Ⅵ collagen, laminin, fibronectin, TGF-β1, TGF-β2, FGF of the TAHAM. Phase contrast microscope was engaged to observe the morphology of HFF. The time-lapse CCD and the trace analysis software were employed to prescribe the cell migration. The 3D adhesion foci were identified 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 had a bipolar extension to form multilayer cell clusters networks and grew into the matrix. All of the seeded cells survived three weeks under regular culture without transfer. On TAHAM, HFF moved in a straight line with a speed of 12 μm/h. α5 integrin (green), paxillin (red) and fibronectin (blue) co localized to form 3D adhesion complexes (white). Conclusion The main molecular components and biomechanical properties is preserved in TAHAM. HFF forms 3D adhesion complexes on TAHAM. Cell morphology and migration of HFF on TAHAM correspond to that under 3D adhesion behavior.
