Fibroblast-seeded collagen gels in response to dynamic equibiaxial mechanical stimuli: A biomechanical study.

The remodeling of fibroblast-seeded collagen gels in response to dynamic mechanical stimuli was investigated by using a newly developed biaxial culture system capable of cyclically stretching planar soft tissues. Fibroblast-seeded collagen gels were subjected to three distinct dynamic mechanical conditions for six days: Cyclic Equibiaxial Stretching at two constant strain magnitudes (CES-7% and CES-20%), and Cyclic Equibiaxial Stretching with incrementally Increasing stain magnitude (ICES, 7% → 15% → 20% each for two days). The frequency of cyclic stretching was set at 1 Hz. At the end of culture, mechanical properties of the gels were examined by biaxial mechanical testing and checked again upon the removal of seeded cells. Collagen microstructure within the gels was illustrated by multiphoton microscopy. The mRNA levels of collagen type I and type III and fibronectin in the cells were examined by reverse transcription PCR. The protein expression of α-smooth muscle actin was detected by immunohistochemistry. We found that the gels cultured under cyclic stretching were stiffer than those cultured under static stretching. Particularly, the stiffness appeared to be significantly enhanced when the ICES was employed. The enhancement of mechanical properties by cyclic stretching appeared to persist upon cell removal, suggesting an irreversible remodeling of extracellular matrix. Second harmonic generation images showed that collagen fibers became thicker and more compact in the gels cultured under cyclic stretching, which may explain the mechanical findings. The mRNA expression of collagen type I in the cells of the ICES was significantly greater than that of the other groups except for the CES-20%. This study suggests that when cyclic stretching is to be used in engineering soft tissues, incrementally increasing strain magnitude may prove useful in the development of the tissue.

Development of fibroblast-seeded collagen gels under planar biaxial mechanical constraints: a biomechanical study.

Prior studies indicated that mechanical loading influences cell turnover and matrix remodeling in tissues, suggesting that mechanical stimuli can play an active role in engineering artificial tissues. While most tissue culture studies focus on influence of uniaxial loading or constraints, effects of multi-axial loading or constraints on tissue development are far from clear. In this study, we examined the biaxial mechanical properties of fibroblast-seeded collagen gels cultured under four different mechanical constraints for 6 days: free-floating, equibiaxial stretching (with three different stretch ratios), strip-biaxial stretching, and uniaxial stretching. Passive mechanical behavior of the cell-seeded gels was also examined after decellularization. A continuum-based two-dimensional Fung model was used to quantify the mechanical behavior of the gel. Based on the model, the value of stored strain energy and the ratio of stiffness in the stretching directions were calculated at prescribed strains for each gel, and statistical comparisons were made among the gels cultured under the various mechanical constraints. Results showed that gels cultured under the free-floating and equibiaxial stretching conditions exhibited a nearly isotropic mechanical behavior, while gels cultured under the strip-biaxial and uniaxial stretching conditions developed a significant degree of mechanical anisotropy. In particular, gels cultured under the equibiaxial stretching condition with a greater stretch ratio appeared to be stiffer than those with a smaller stretch ratio. Also, a decellularized gel was stiffer than its non-decellularized counterpart. Finally, the retained mechanical anisotropy in gels cultured under the strip-biaxial stretching and uniaxial stretching conditions after cell removal reflected an irreversible matrix remodeling.