Effects Of Cyclic Tensile Strain On The Biological Characteristics Of Adipose-Derived Stem Cells-Decellularized Tendon Scaffold Complexes

Objective: This study aimed to observe the effects of cyclic tensile strain at various amplitudes on the biological characteristics of adipose-derived stem cells(ADSCs)-decellularized tendon scaffold complexes.Methods: The porcine superficial flexor tendons were processed into the decellularized tendon scaffolds using a combination of(1)freeze-thaw cycles(2)sodium dodecyl sulfate and Triton-X 100(3)DNase and RNase(4)peracetic acid.The effectiveness of decellularization was verified by histological analysis and DNA quantification.The properties of the scaffolds were evaluated by quantitative analysis of collagen,glycosaminoglycans(GAGs),α-galactosyl(α-Gal)antigen,as well as porosimetry,in vitro biocompatibility assessment and biomechanical testing.The surface markers of ADSCs were detected by flow cytometry.The proliferation and differentiation-related gene expression of ADSCs were evaluated after the short-term treatment of low air pressure.Osteogenic,adipogenic and chondrogenic differentiation of ADSCs were induced after the treatment of low air pressure,followed by alizarin red,oil red O and alcian blue staining to assess their multi-directional differentiation potential.The ADSCs-decellularized tendon scaffold complexes were constructed via cell injection assisted by low air pressure or under atmospheric pressure,respectively.The differences in cell distribution,biomechanical properties,and the total DNA content after 7 days of culture between these two groups were compared by means of histological analysis,biomechanical testing and DNA quantification.The ADSCs-decellularized tendon scaffold complexes were subjected to cyclic tensile strain at 0%,4% and 8% amplitude by a custom bioreactor.The effects of different strain amplitudes on the total DNA content in the complexes,the collagen content and the biomechanical properties of the complexes,as well as the gene expression of ADSCs,were compared after 7 days of stimulation.Results: Histologic analysis confirmed that the collagen fibers remained intact in the decellularized tendon scaffold with widened interfibrillar space,and no cellular components were found therein.Decellularization treatment resulted in a significant decrease in the contents of DNA,α-Gal and GAGs,and caused a significant increase in the porosity of the tendon.The scaffolds were cytocompatible in vitro,and did not show reduced collagen content and inferior biomechanical properties compared with the fresh deep-frozen tendons.ADSCs expressed high levels of CD29 and CD44,while low levels of CD34 and CD45.The proliferation and differentiation-related gene expression of ADSCs were not significantly affected by the treatment of low air pressure,and alizarin red,oil red O and alcian blue staining were positive after induction culture.Histological analysis showed that the assistance of low air pressure promoted the broader distribution of cells in the adjacent interfibrillar space.The biomechanical properties of the scaffolds were not significantly affected by cell injection assisted by low air pressure or under atmospheric pressure.After7 days of culture,the total DNA content in the complexes constructed under the assistance of low air pressure was greater than that under atmospheric pressure.After 7 days of culture in the bioreactor,the collagen content and biomechanical properties of the complexes decreased in varying degrees.Compared with 0% strain,4% cyclic tensile strain increases the total DNA content and ultimate tensile strength of the complexes,enhanced the tenogenic differentiation-and type Ⅰ collagen-related gene expression in ADSCs,meanwhile inhibited the osteogenic,adipogenic and chondrogenic differentiation-related gene expression;8% cyclic tensile strain reduced the collagen content,ultimate tensile strength and elastic modulus of the complexes,enhanced the osteogenic differentiation-,type Ⅲcollagen-and matrix metalloproteinases-related gene expression in ADSCs,meanwhile inhibited the type Ⅰ collagen-related gene expression.Conclusion: The sterile decellularized tendon scaffolds with low residual cellular components,intact microstructure,low immunogenicity,favorable biocompatibility and adequate mechanical properties were successfully prepared by a series of decellularization procedures.Under the assistance of low air pressure,ADSCs were successfully combined with decellularized tendon scaffold via cell injection to form a cell-scaffold complex.This construction method achieved a broader local distribution of the seeded cells without significantly affecting the biomechanical properties of the scaffolds and promoted the proliferation of the cells in the scaffold.The cyclic tensile strain at moderate amplitude promoted the proliferation and tenogenic differentiation of ADSCs in cell-scaffold complexes,while partially attenuating the loss in ultimate tensile strength of the complexes induced by bioreactor culture,which was beneficial to the activation of cell-scaffold complexes and the relative retention of their biomechanical properties.This study establishes an experimental foundation for the future clinical application of the ADSCsdecellularized tendon scaffold complexes.