Effect of Acellular Amnion With Increased TGF-β and bFGF Levels on the Biological Behavior of Tenocytes

Abstract

The human amniotic membrane has been a subject for clinical and basic research for nearly 100 years, but weak rejection has been reported. The purpose of this research is to remove the cellular components of the amnion for eliminating its immune-inducing activity to the utmost extent. The amniotic membrane treated by acid removed the epithelial cell, fibroblast, and sponge layers and retained only the basal and dense layers. In vitro, biological effects of the new material on tenocytes were evaluated. The levels of transforming growth factor (TGF-β1), fibroblast growth factor (bFGF) proteins were measured. In vivo, the tendon injury model of chickens was constructed to observe effects on tendon adhesion and healing. The acellular amniotic membrane effectively removed the cell components of the amnion while retaining the fibrous reticular structure. Abundant collagen fibers enhanced the tensile strength of amnion, and a 3D porous structure provided enough 3D space structure for tenocyte growth. In vitro, acellular amnion resulted in the fast proliferation trend for tenocytes with relatively static properties by releasing TGF-β1 and bFGF. In vivo, the experiment revealed the mechanism of acellular amnion in promoting endogenous healing and barrier exogenous healing by evaluating tendon adhesion, biomechanical testing, and labeling fibroblasts/tendon cells and monocytes/macrophages with vimentin and CD68. The acellular amnion promotes endogenous healing and barrier exogenous healing by releasing the growth factors such as TGF-β1 and bFGF, thereby providing a new direction for the prevention and treatment of tendon adhesion.

Keywords: amnion; collagen; growth factor; tendon adhesions; tenocytes.

FIGURE 1

The appearance of fresh amniotic membranes (A). The epithelial cell layer of fresh amniotic membranes (B) and the histoarchitecture of the acellular amnion (C) observed by SEM. The cell structure was not detected and the intimal surface was rougher than that of the native amnion. (D) The remaining cellular DNA in the amnion and acellular amnion was measured using Quant-iT PicoGreen dsDNA Assay Kit. The total DNA content (351.5 ± 9.23 ug/mL) decreased significantly after the decellularization treatment when compared to the acellular amnion (15.66 ± 0.82 ug/mL). The epithelial cell layer of native amniotic membranes (E). Hematoxylin-eosin (H&E) staining confirmed complete removal of epithelial cells (F). ***p < 0.001.

FIGURE 2

Fluorescence images of tenocytes after 5 days of culture on the surface of a culture plate (control group) and acellular amniotic membrane (amnion group). Tenocytes presented a clear cytoskeleton, good biocompatibility with acellular amniotic membrane, and even distribution on the surface of the materials, showing better growth activity than the control group (A,B). Cell viability was measured by CCK-8, and the proliferation curve of tenocytes in the control and amniotic membrane groups was drawn (C). Western blot assay for collagen I, fibronectin, TGF-β1, and bFGF expression in the tenocytes of the control and amnion groups for 1 week (D,E). *p < 0.05; **p < 0.01; and ***p < 0.001.

FIGURE 3

Representative immunofluorescence images of collagen I. Fluorescent micrographs of tenocytes after 2 and 5 days of culture on the surface of a culture plate and acellular amniotic membrane. Tenocyte nucleus shape observed under a fluorescence microscope (A,D,G,J). Collagen I presented positive after the fluorescent FITC mark was observed under a fluorescence microscope (B,E,H,K). Tenocyte nucleus and collagen I merging (C,F,I,L). The corresponding semi quantitative analysis of collagen fluorescence intensity in panels (M,N) (scale bar = 50 um, n = 5, *P < 0.05 and **P < 0.01).

FIGURE 4

Representative immunofluorescence images of fibronectin. Fluorescent micrographs of tenocytes after 2 and 5 days of culture on the surface of a culture plate and acellular amniotic membrane. Tenocyte nucleus shape observed under a fluorescence microscope (A,D,G,J). Fibronectin presented positive after the fluorescent FITC mark was observed under a fluorescence microscope (B,E,H,K). Tenocyte nucleus and fibronectin merging (C,F,I,L). The corresponding semi quantitative analysis of fibronectin fluorescence intensity in panels (M,N) (scale bar = 50 um, n = 5, *P < 0.05).

FIGURE 5

(A) Operative technique for the application of the acellular amnion allograft. Establishment of flexor digitorum profundus tendon model in chickens. The tendon has been sutured, and the membrane is placed between the flexor tendons. The membrane is wrapped around the FDP tendon and fixed to the remaining tendon sheath. General observation of the control group (B) and amnion group (C) on the 4th week after the operation. (D) The macroscopic and microscopic evaluation of adhesion on the 2nd, 4th, and 6th week after the operation. Scheme of the total flexion angle measurement (E) and the results of the total flexion angle (F). Scheme of the sliding distance, the maximal tensile strength (G) and the results of the sliding distance (H), the maximal tensile strength (I) between the two groups on the 2nd, 4th, and 6th week after the operation. *p < 0.05; **p < 0.01; and ***p < 0.001.

FIGURE 6

Exemplary images of inflammatory cell and fibroblast infiltration visualized by immunohistological CD68 (monocytes/macrophages) (A–C) and vimentin (fibroblasts/tenocytes) (E–G) staining in ruptured tendons between the control group and amnion group at the 2nd week after operation. The CD68 and vimentin of the acellular amniotic membrane group were higher than that of the control group (D,H). High expression of CD68 and vimentin in the amnion group meaned that a large number of monocytes/macrophages infiltrated, neovascularization increased, and fibroblasts/tenocytes proliferated rapidly. *p < 0.05.

FIGURE 7

Mechanism of the acellular amniotic membrane that promotes endogenous healing of tendon and prevents exogenous adhesion. The acellular amnion contains several active factors, such as TGF-β1, bFGF. These factors are released to the tendon repair area, which promotes the proliferation of tenocytes and the synthesis of collagen. The acellular amnion also functions as a barrier to fibroblasts and other exogenous tissue invasion.