Cyclic stretching and growth factors like TGF-Β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-Β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-Β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-Β1 samples. However, at weeks 5 and 7, -TGF-Β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-Β1 samples. The collagen was better organized in -TGF-Β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-Β1 samples, which correlated with higher collagen deposition. The TGF-Β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-Β1 samples, respectively. The role of TGF-Β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-Β1 signaling during cyclic stretching. One advantage of TGF-Β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-Β1 to obtain improved tensile properties followed by TGF-Β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-Β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.
Bibliographical noteFunding Information:
The authors like to thank Ricky Chow, Naomi Ferguson, Sandy Johnson, Justin Weinbaum, and Stephen Stephens for technical assistance. Funding was provided by NIH BRP HL71538 and R01 HL083880 to R.T.T.
- Collagen deposition
- Cyclic stretching
- Tissue engineering
- Tissue growth and remodeling